Conjugates and small molecules, interacting with cd16a receptor

FIELD: chemistry.

SUBSTANCE: compounds can be applied for treatment of oncologic and autoimmune diseases. Invention also characterises method of obtaining conjugates, pharmaceutical composition and medication, which contains modified proteins. In general formulae 1 or 2 , R1 is selected from the group representing (CH3)2N-,

R2 is selected from the group representing where R3 as terminal substituent represents -NH2, or and R4 represents H or C1-C3alkyl.

EFFECT: novel compounds possess affinity for CD16a receptor.

18 cl, 20 dwg, 3 tbl, 19 ex

 

The invention relates to medicine, in particular to Oncology and immunology, new compounds that bind to the CD 16A receptor, and modified their proteins (conjugates)used for the induction of antibody-dependent cellular cytotoxicity and removal thus from the body of a certain target group of cells, for example, cancer cells or autoimmune lymphocytes. The invention relates also to a method for producing conjugates, pharmaceutical compositions and medicines containing modified proteins (conjugates), for the treatment of cancer and autoimmune diseases.

The FcγIIIa receptor (CD16) belongs to a group of receptors responsible for binding of the Fc fragment of antibodies. CD 16A is expressed on the surface of NK-cells (killer cells) and macrophages and is responsible for the induction of antibody-dependent cellular cytotoxicity (ADCC), interacting with the Fc-fragment associated with the cell antibodies. ADCC, along with complementability cytotoxicity (CDC) and apoptosis, is one of the main mechanisms for the destruction of cancer cells from the body. It is also the reason mediated by autoantibodies of autoimmune diseases, such as autoimmune polyendocrinopathy of the first type, autoimmune hemolytic anemia, idiopathic thrombocytopenia, hemolytic disease of the newborn is so That is, antibody-dependent cellular cytotoxicity may play both positive and negative role in the development of pathological processes in the human body.

Autoimmune disease is a group of diseases that develop due to the generation of an immune response against healthy tissues of the body and leading to damage to these tissues. Currently, for the treatment of autoimmune diseases are immunosupressant that suppress the immune system in General. Selective suppression of the autoimmune response would significantly reduce the frequency of side effects of treatment.

Antibodies sufficiently long been used for the targeted destruction of cancer cells. Examples include rituximab, trastuzumab, cetuximab, and many other antibodies which target on the surface of cancer cells and acting through complementability cytotoxicity and antibody-dependent cellular cytotoxicity. This allows you to use data unconjugated monoclonal antibodies as drugs for the treatment of cancer, for example, rituximab for the treatment of CD20-positive b-cell, nizkozameshhennoj or follicular non-Hodgkin's lymphoma, trastuzumab for the treatment of advanced breast cancer. Successful application of these products is due to not only the efficiency, but they are very good safety profiles (Grillo-Lopez A. - J et al. Semin. Oncol., 26, 1999, pp.66-73).

They all brought the possibility of a new kind of therapy. But, despite the pronounced efficacy, approximately half of patients do not respond to therapy with rituximab, and up to 60% become resistant after repeated application. In light of the successes associated with these drugs, there is currently a great need for achieving higher specific activity of antibodies compared with one that normally is provided by treatment with unconjugated antibodies.

This was the reason for the gain directional therapeutic effect of antibodies to surface antigens. The first direction modification of antibodies was the development of modified proteins (conjugates) antibody - drug (ADC) for local delivery of cytotoxic or cytostatic funds, i.e. drugs that are already used for the destruction or inhibition of tumor cells in the treatment of malignant tumors [Payne, G. (2003) Cancer Cell 3:207-212; Trail et al. (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; US 4,975,278]. Such conjugates provide the directed delivery of medicinal substance to tumors and their accumulation inside the cells, EXT is vlaa to the cytotoxic action of antibodies anti-tumor activity of cytotoxic or cytostatic drugs. Examples of such conjugates are trastuzumab-DM1, an amplified version of trastuzumab (Herceptin) (WO201169074) and a series of conjugates with auristatin E (US20120003248).

An alternative direction was greater individual cytotoxicity of antibodies by enhancing the interaction with the receptor, causing cytotoxicity. Company Roche has developed antibody Abanotubani with enhanced binding with the receptor CD16. This effect is achieved by engineering the glycosylation of antibodies. Abanotubani has ten times stronger antibody-dependent cellular cytotoxicity (WO2005044859, EA).

Antibody-dependent cellular cytotoxicity is one of the main mechanisms of cytotoxic action of antibodies that bind to the antigen on the surface of target cells by variable domains, whereas the constant part associated with CD16 receptor on the cell surface killers. This intercellular contact leads to the secretion of the killers of perforin and Gerasimov. The first form pores in the cell membrane of target cells, and the latter activates caspase and other molecules of apoptosis. CD16 receptor is a member of a large family Fc-recetaron that communicates with the constant domain of antibodies and different localization, function and affinity constant domain.

The following is about the determining the terms which are used in the description of this invention.

"Alkyl" means an aliphatic hydrocarbon of linear or branched group with 1-12 carbon atoms in the chain. Branched means that the alkyl chain has one or more "lower alkyl" substituents. The alkyl may have one or more identical or different substituents ("alkyl substituents") including halogen, alkenylacyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, aroyl, cyano, hydroxy, alkoxy, carboxy, alkyloxy, Alcoxy, aryloxy, aryloxyalkyl, alkylthio, heteroaromatic, Uralkali, arylsulfonyl, alkylsulfonates, annelirovannymi heteroarylboronic, annelirovannymi heteroalicyclic, annelirovannymi heterooligomerization, annelirovannymi heteroalicyclic, annelirovannymi arylchloroalkanes, annelirovannymi aristically, annelirovannymi arylheteroacetic, annelirovannymi arylheteroacetic, alkoxycarbonyl, arelaxation, heteroarylboronic or RkaRk+1aN-, RkaRk+1aNC(=O)-, RkaRk+1aNC(=S)-, RkaRk+1aNSO2-, where Rkaand Rk+1aindependently from each other represent "amino substituents", the value of which is determined by the this section, for example, a hydrogen atom, alkyl, aryl, aralkyl, heteroalkyl, heterocyclyl or heteroaryl, or Rkaand Rk+1atogether with the N atom to which they are bound, form a through Rkaand Rk+1a4-7 - membered heterocyclyl or heterocyclyl. Preferred alkyl groups are methyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, ISO-propyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl, methoxyethyl, carboxymethyl, methoxycarbonylmethyl, ethoxycarbonylmethyl, benzyloxycarbonylation methoxy-carbonylmethyl and pyridinedicarboxylate. Preferred "alkyl substituents" are cycloalkyl, aryl, heteroaryl, heterocyclyl, hydroxy, alkoxy, alkoxycarbonyl, Alcoxy, aryloxy, alkylthio, heteroaromatic, Uralkali, alkylsulfonyl, arylsulfonyl, alkoxycarbonyl, arelaxation, heteroarylboronic or RkaRk+1aN-, RkaRk+1aNC(=0)-, annelirovannymi arylheteroacetic, annelirovannymi arylheteroacetic.

"Aminoalkyl" means CnH2n+1NH - or (CnH2n+1)(CnH2n+1)N - group in which alkyl is defined in this section. The preferred alkylamino groups are methylamino, ethylamino, n-propylamino, out-propylamino and n-butylamine. "The anti-Christ. ate" - protein (immunoglobulin)produced by b-lymphocytes in the body in response to the ingress of foreign matter and which has specific affinity for this substance. They are a major factor specific humoral immunity. Antibodies have two functions: antigennegative and effector (cause one or the other of the immune response).

"Autoantigens" - available molecules of substances or molecules in the structure of cells, organs and tissues that are recognized under certain conditions the immune system as foreign and therefore cause cellular or humoral immune response from your body. This is usually normal proteins or protein complexes (complexes of proteins with DNA or RNA)that are recognized by the immune system in patients with autoimmune diseases. Such antigens normally does not have to be recognized by the immune system, but, due to genetic factors or environmental conditions, immunological tolerance to these antigens may be lost.

Properties of autoantigens may have so-called natural autoantigens (sequestered). These include proteins, the synthesis of which begins after the maturation of the immune system (semen, milk); macromolecules bodies, separated from the immune system blood-tissue interfaces barrier; macromolecules, in adasia in the nuclei and cytoplasm of cells; macromolecules with new alien determinant groups due to the actions of endogenous (immune complexes, necrosis, inflammation) or exogenous (temperature, chemical substances, including drugs, microbes and their toxins, viruses and other) factors; embryonic proteins with renewable under certain conditions the synthesis (for example, when tumors). They can induce an immune response, leading to the formation of antibodies or sensitized T-lymphocytes and the development of autoimmune diseases. Starts the development of autoimmune reactions. They can lead to the development of a variety of autoimmune diseases. These include, in particular, include autoimmune polyendocrinopathy of the first type, autoimmune hemolytic anemia, idiopathic thrombocytopenia, hemolytic disease of the newborn, multiple sclerosis, autoimmune thyroiditis, etc.

Autoimmunity - process and related diseases caused by the acquisition of the immune system's ability to recognize native antigens (autoantigens) of the body and respond to the formation of autoantibodies or autoimmune T-lymphocytes. Autoimmune process and related diseases, tissue damage due to the effects of interaction of autoantibodies or AU is ommuni T-lymphocytes with autoantigens.

"Conjugate" is modified chemical compounds, protein, antigen or antibody. The formation of the conjugate is one of the important stages of immune-enzyme assay (ELISA). When forming the conjugate choose the best method of introducing chemical compounds to the component of the conjugate, antigen or antibody retained its biological activity - antigenicity and antigennegative activity, respectively. The ability of foreign compounds and metabolites to join in the conjugation reaction depends on the presence in their molecules of certain functional groups.

"The drug (the drug), a substance (or mixture of substances in the form of pharmaceutical compositions in the form of tablets, capsules, injections, ointments and other ready-made forms, designed to restore, correct or modify physiological functions in humans and animals, as well as for treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology and others. "Receptors" (from Latin recipere to receive, to learn) are biological macromolecules that are located on the plasma membrane of cells or intracellular able to interact specifically with a limited set of physiologically active substances (ligands) and transform the signal about this interaction to determine the certain cell response.

"Solvate" is the addition products of solvent to solute; a special case of the solvate is a hydrate (solvent is water). Typically, the solvate formed in solution, but often (when cooling solution, evaporation of the solvent, etc. can be obtained in the form of crystalline phases - crystallosolvate.

"Pharmaceutical composition" means a composition comprising the active ingredient (modified protein) and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, auxiliary, distributing and perceiving means, means of delivery, such as preservatives, stabilizers, fillers, shredders, moisturizers, emulsifiers, suspendresume agents, thickeners, sweeteners, flavors, fragrances, antibacterial agents, fungicides, lubricants, regulators prolonged delivery, the choice and the value of which depends on the nature and method purpose and dosage. Examples suspendida agents are ethoxylated isostearoyl alcohol, polyoxyethylene, sorbitol and sorbitol ester, microcrystalline cellulose, Metagalaxy aluminum, bentonite, agar-agar and-tragakant, as well as mixtures of these substances. Protection from the action of microorg the mechanisms can be provided using a variety of antibacterial and antifungal agents, for example, such as parabens, chlorobutanol, sorbic acid and similar compounds. The composition may also include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged action of the composition can be achieved with agents that slow the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles include water, ethanol, polyalcohol, and mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as etiloleat). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of shredders and distributes funds are starch, aginova acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulphate, talc, and polyethylene with high molecular weight. Pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, local or rectal injection of the active principle, one or in combination with other active early, can be introduced animals and people in the standard form of administration, mixed with conventional pharmaceutical carry the firs. Usable standard form of introduction include oral forms such as tablets, gelatin capsules, pills, powders, granules, chewing gum and oral solutions or suspensions, sublingual and transbukkalno forms of administration, aerosols, implants, local, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular forms of administration and rectal forms of administration. Pharmaceutical compositions typically obtained using standard procedures involving mixing the active compound with a liquid or finely powdered solid carrier.

"Pharmaceutically acceptable salt" refers to the relatively non-toxic organic and inorganic salts of acids and bases, as claimed in the present invention. These salts can be obtained in situ during the synthesis, separation, or purification of compounds or prepared. In particular, salts of bases can be obtained specifically on the basis of the purified free base of the claimed compounds and a suitable organic or inorganic acid. Examples of the thus obtained salts are hydrochloride, hydrobromide, sulphates, bisulfate, phosphates, nitrates, acetates, oxalates, valeriote, oleates, palmitate, stearates, laurate, borate, benzoate, lactates, tozilaty, citrates, maleate, fumarate is, succinate, tartratami, mesylates, malonate, salicylates, propionate, econsultancy, bansilalpet, sulfamate and the like (for a Detailed description of the properties of such salts are described in Berge S.M., et al., “Pharmaceutical Salts” J. Pharm. Sci. 1977, 66: 1-19). Salts of the stated acids can also be specially obtained by the reaction of purified acid with a suitable base, can be synthesized metal salts and amines. The metal include sodium, potassium, calcium, barium, zinc, magnesium, lithium and aluminum, the most desirable of which are sodium and potassium salts. Suitable inorganic bases which can be obtained metal salts are the hydroxide, carbonate, bicarbonate and sodium hydride, hydroxide and bicarbonate of potassium, potash, lithium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide. As organic bases, of which can be obtained salts of the stated acids, selected amines and amino acids with sufficient basicity to form a stable salt, and suitable for use in medical purposes (in particular, they should have a low toxicity). Such amines include ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, benzylamine, dibenzylamine, dicyclohexylamine, piperazine, ethylpiperidine, Tris(hydroxymethyl)aminomethan and under the ing them. In addition, for the salt formation can be used tetraalkylammonium hydroxide, such as choline, Tetramethylammonium, tetraethylammonium and the like. As amino acids can be used basic amino acids such as lysine, ornithine and arginine.

The purpose of the present invention is to provide new compounds and modified their proteins (conjugates)that are able to interact with the CD16a receptor used for the induction of antibody-dependent cellular cytotoxicity and removal thus from the body of a certain target group of cells, for example, cancer cells or autoimmune lymphocytes.

This goal is achieved detected by the authors of the new compounds with affinity to CD16a receptor containing the activated group capable of join the amino group of the protein, namely the new substituted 5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]thiazepine General formula 1 or 5,6,7,8,9,10-hexahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-a][1,4]benzodiazepines of General formula 2, or pharmaceutically acceptable salts or solvate,

in which R1 represents (CH3)2N-,

R2 represents

,

where R3 is as stake is avago Deputy represents the unsubstituted aminoalkyl,

-NH2,or,

a R4 represents H or C1-C3alkyl.

Preferred are compounds of General formula 1 in which R1 represents:

,

,

,

;

R2 is selected from the group including:

,

,

where R4=H or C1-C3alkyl.

Preferred are also compounds of General formula 2, in which

R1 represents (CH3)2N - or,

a R2 represents

.

More preferred compounds are:

2,5-dioxopiperidin-1 silt ether (3 chlorbenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carboxylic acid 1(1),

2,5-dioxopiperidin-1 silt ether (4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenoxy)-acetic acid 1(2),

2,5-dioxopiperidin-1 silt ether 4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenylcarbinol acid 1(3),

2.5 dioxo-pyrrolidin-1 silt ester of 3-[8-(3,4-dimethoxycinnamoyl)-5,5,11-trioxo-5,11-dihydro-is benzo[b,f][1,4]diazepin-10-ylmethyl]-benzoic acid 1(4),

2.5 dioxo-pyrrolidin-1 silt ether (4-{8-[3-(4-benzylpiperidine-1-yl)-propellerblades]-5,5,11-trioxo-5,11-dihydro-dibenzo[b,f][1,4]diazepin-10-ylmethyl}-phenyl)-acetic acid 1(5),

10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid (2-amino-ethyl)-amide 1(6),

10-{4-[(2-amino-ethylcarbazole)-methyl]-benzyl}-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide 1(7),

2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethyl ester of 10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f|[1,4]diazepin-8-carboxylic acid 1(8),

10-(4-{[2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethylcarboxyl]-methyl}-benzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide 1(9),

N-[2-({N-(methoxycarbonyl)-N-[(1,5-dimethoxy-1,5-dioxetane-2-yl)carbarnoyl]-β-alanyl}amino)ethyl]-10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydrobenzo[b,f][1,4]diazepin-7-carboxamide 1(10),

N5-(2-{[(4-{[7-{[[3-(4-benzylpiperidine-1-yl)propyl](phenyl)amino]-carbonyl}-5,5,11-trioxo-dibenzo[b,f][1,4]thiazepine-10(11H)-yl]methyl}phenyl)acetyl]-amino}ethyl)-N2-{[(1,3-dicarboxylate)amino]carbonyl}glutamine 1(11),

4-[4-(dimethylamino)phenyl]-N-(4-{[(2,5-dioxopiperidin-1-yl)oxy]carbonyl}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-α][1,4]diazepin-5(6H)-carboxamide 2(1),

4-[4-(dimethylamino)phenyl]-N-(4-{2[(2,5-dioxopiperidin-1-yl)oxy]-2-oksidoksi}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno [3,2-f]pyrrolo[1,2-α][1,4] diazepin-5(6H)-carboxamide 2(2),

2,5-dioxopiperidin-1-yl N-[4-(5-{[(3,4-acid)amino] -carbonyl}-5,6,7,8,9,10-hexahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-α][1,4]diazepin-4-yl)phenyl]-N-methylglycine 2(3).

The authors first discovered the modified protein (conjugate), active against CD16 receptor. Therefore, the subject of this invention is a modified protein (conjugate), active against CD16 receptor, obtained by the interaction of the protein with the modifying compound which has affinity to CD16 receptor.

More preferred is a modified protein (conjugate), active against CD16 receptor, obtained by the interaction of the protein with the modifying compound of General formula 1 or 2.

More preferred is a modified protein (conjugate derived from antibodies and compounds of General formula 1 or 2, wherein the antibody is a rituximab, trastuzumab or cetuximab.

More preferred is a modified protein (conjugate), which is a rituximab-modified compound of General formula 1 or 2.

More preferred is a modified protein (conjugate), which is a trastuzumab-modified compound of General formula 1 or 2.

More preferred is a modified protein (conjugate), not only at the store a cetuximab, modified compound of General formula 1 or 2.

More preferred is a modified protein (conjugate), obtained from autoantigen and compounds of General formula 1 or 2, in which the autoantigen is a interferon Alfa or major myelin protein, or a protein of the complement C1q.

More preferred is a modified protein (conjugate), representing interferon Alfa-modified compound of General formula 1 or 2.

More preferred is a modified protein (conjugate), which is the main protein of myelin, a modified compound of General formula 1 or 2.

More preferred is a modified protein (conjugate), a protein of the complement C1q-modified compound of General formula 1 or 2.

A study of the comparative efficacy of proteins and their conjugates with respect to CD16 receptor showed that conjugates 1-3 order more of their unmodified proteins.

The subject of this invention is also a method of obtaining modified protein (conjugate), according to which is subjected to the interaction of the protein with the compound of General formula 1 or 2, dissolved in a medium of an organic solvent, such as dimethylsulfoxide, in the range of molar ratios of 1:3 to 1:100 in the environment of phosphate saline buffer solution (pH 7.4) at room temperature with constant stirring.

The subject of this invention is also a pharmaceutical composition active against CD16 receptor containing the modified protein (conjugate) in a therapeutically effective amount and a pharmaceutically acceptable diluent, carrier or excipient.

Pharmaceutical compositions can include pharmaceutically acceptable excipients. Under the pharmaceutically acceptable excipients are meant to be applied in the field of pharmaceutical diluents, auxiliary agents and/or carriers. The pharmaceutical composition along with the modified protein (conjugate), obtained by the interaction of the protein with the modifying compound of General formula 1 or 2, or its pharmaceutically acceptable salt or MES, the present invention may include other active substances, including with anti-influenza activity, provided that they do not cause unwanted effects.

If you want to use the pharmaceutical compositions of the present invention in clinical practice it can be mixed with conventional pharmaceutical carriers.

The media used in the pharmaceutical compositions of the present invention, are media that are used in the pharmaceutical industry to obtain common forms, including the number of the e: oral, forms for injection, local forms.

The subject of this invention is also a drug that is active against CD16 receptor, in the form of tablets, capsules, or injections, placed in pharmaceutically acceptable packing intended for the treatment of diseases caused by pathological cells, incorporating new modified protein (conjugate) or a new pharmaceutical composition in a therapeutically effective amount.

Since the formation of the conjugate component, an antigen or an antibody that retain their biological activity - antigenicity and antigennegative activity, respectively, then the conjugates of the present invention can be used to treat the same diseases, which are unconjugated monoclonal antibodies as drugs, such as nizkozameshhennoj or follicular non-Hodgkin's lymphoma, breast cancer. It is also known that the activity of the antibodies against CD16 receptor is used for the treatment of autoimmune or cancer (R.L.Ferris, et al. J. Clinical Oncology, 2010, Oct 1, Vol.28, No 28: 4390-4399), including such as lymphoma (K.-.Heider, et al. Blood, 2011, 118: 4159-4168) or lymphatic leukemia (J.A.Bowels, et al. Blood 2006 108: 2648-2654).

The subject of this invention is a method of treating diseases caused by pathological cells, which can be treated by indirect effects on CD16 receptor, whereby the patient in need of treatment is administered a therapeutically effective amount of the modified protein (conjugate), or pharmaceutical composition or the medicinal product, active against CD16 receptor.

Preferred is a method of treating an autoimmune or cancer, as defined earlier in this section, including such as lymphoma, lymphatic leukemia or breast cancer.

Preferred is a method of treating autoimmune polyendocrinopathy of the first type.

Drugs can be administered orally or parenterally (e.g. intravenously, subcutaneously, intraperitoneally or topically). The clinical dosage of the modified protein (conjugate), or pharmaceutical composition or the medicinal product, active against CD16 receptor, patients may be adjusted depending on therapeutic efficacy and bioavailability of the active ingredients in the body, the speed of their metabolism and excretion from the body, and depending on age, gender and stage of disease of the patient, the daily dose in adults is usually 300~1200 mg, preferably 500~1000 mg in the case when the protein in the conjugate represents titulo, and 0.01~100 mg, preferably 0.1 to~10 mg in the case when the protein in the conjugate is an autoantigen. Therefore, during the preparation of pharmaceutical compositions of the present invention in the form of dosage units it is necessary to consider the above-mentioned effective dosage. In accordance with the instructions of the doctor or pharmacist these medications can be taken several times during a defined time period (preferably from one to six times).

The invention is illustrated by the following drawings:

Figure 1. Range of proton magnetic resonance (PMR spectrum) 2,5-dioxopiperidin-1 silt ether (3 chlorbenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f|[1,4]diazepin-7-carboxylic acid 1(1).

Figure 2. PMR-spectrum of 2,5-dioxopiperidin-1 silt ether (4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenoxy)-acetic acid 1(2).

Figure 3. PMR-spectrum of 2,5-dioxopiperidin-1 silt ether 4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f|[1,4]diazepin-7-carbonyl]-amino}-phenylcarbinol acid 1(3).

Figure 4. LCMS-spectrum of 2,5-dioxo-pyrrolidin-1 silt ester 3-[8-(3,4-dimethoxycinnamoyl)-5,5,11-trioxo-5,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-10-ylmethyl]-benzoic acid 1(4).

Fig 5. PMR-spectrum of 2,5-dioxo-pyrrolidin-1 silt ether (4-{8-[3-(4-benzylpiperidine-1-yl)-propellerblades]-5,5,TiOx-5,11-dihydro-dibenzo[b,f][1,4]diazepin-10-ylmethyl}-phenyl)-acetic acid 1(5).

6. The PMR spectrum of 10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid (2-amino-ethyl)-amide 1(6).

7. The PMR spectrum of 10-{4-[(2-amino-ethylcarbazole)-methyl]-benzyl}-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide 1(7).

Fig. The PMR spectrum of 2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethyl ester 10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5TH-dibenzo[b,f][1,4]diazepin-8-carboxylic acid 1(8).

Fig.9. PMR-spectrum 10-(4-{[2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethylcarboxyl]-methyl}-benzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide 1(9).

Figure 10. PMR spectrum of the compound N-[2-({N-(Methoxycarbonyl)-N-[(1,5-dimethoxy-1,5-dioxetane-2-yl)carbarnoyl]-β-alanyl}amino)ethyl]-10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydrobenzo [b,f][1,4]diazepin-7-carboxamide 1(10).

11. The PMR spectrum of the N5-(2-{[(4-{[7-{[[3-(4-Benzylpiperidine-1-yl)propyl](phenyl)amino]carbonyl}-5,5,11-trioxo-dibenzo[b,f][1,4]thiazepine-10(11H)-yl]methyl}phenyl)acetyl]amino}ethyl)-N2-{[(1,3-dicarboxylate)amino]-carbonyl}glutamine 1(11).

Fig 12. LCMS-spectrum of 4-[4-(dimethylamino)phenyl]-N-(4-{[(2,5-dioxopiperidin-1-yl)oxy]carbonyl}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-α][1,4]diazepin-5(6H)-carboxamide 2(1).

Fig PMR spectrum of 4-[4-(dimethylamino)phenyl]-N-(4-{2-[(2,5-dioxobis is lidin-1-yl)oxy]-2-oksidoksi}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno[3,2-/]pyrrolo[1,2-α][1,4]diazepin-5(6H)-carboxamide 2(2).

Fig. LCMS-spectrum of 2,5-dioxopiperidin-1-yl N-[4-(5-{[(3,4-acid)amino]carbonyl}-5,6,7,8,9,10-hexahydro-4H-[1]benzothieno[3,2-f)pyrrolo[1,2-α][1,4]diazepin-4-yl)phenyl]-N-methylglycine 2(3).

Fig. Chromatogram conjugate KR1(1), on a column of TSK GEL SUPER SW3000.

Fig 16. Immune-enzyme assay (ELISA) binding conjugates of rituximab with CD16a receptor. The dependence of the optical density at a wavelength of 450 nm on the concentration introduced in ELISA conjugate.

Fig 17. Immune-enzyme assay (ELISA) binding of interferon (s) And conjugate CI(1) with CD16 receptor. The dependence of the optical density at a wavelength of 450 nm on the concentration introduced in ELISA conjugate.

Fig. Comparison of the effectiveness of rituximab (R) and conjugate KR1(1) in the test antibody-dependent cytotoxicity.

Fig. Comparison of the effectiveness of rituximab (R) and conjugate KR1(2) in the test antibody-dependent cytotoxicity.

Fig 20. Immune-enzyme assay (ELISA) binding of rituximab (R), trastuzumab (T) and their conjugates KR1(7), KT1(7) in relation to the CD16a receptor.

The following examples illustrate, but not limit the invention.

Example 1. 2,5-Dioxopiperidin-1 silt ether (3 chlorbenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carboxylic acid 1(1) receive the following Scheme 1.

To a solution of 31.8 g of KOH in 170 ml in the water sprinkled portions 25 g of compound 4, after dissolution, add 30 g of compound 3 and stirred at 60°C for 15 hours and Then the reaction mixture is cooled, acidified with 10% hydrochloric acid to pH 3, filtered, washed with water and dried. Get the connection 5 with the release of 70%. Dissolve 50 g of compound 5 in 500 ml of aqueous ammonia dissolved and portions sprinkled 80 g dithionite, after which the reaction mixture is refluxed 1 h Then the reaction mixture is cooled, evaporated on a rotary evaporator ammonia, and the aqueous solution acidified with conc. hydrochloric acid to pH 1, stirred for 1 h, the precipitate is filtered, washed with water and dried. Get connection 6 with the release of 60%. Add portions 45 g of compound 6 to 200 ml of polyphosphoric acid at 50°C, then stirred for 10 h at 90°C. was Poured onto 500 ml of ice, filtered, washed with water and dried. Get the connection 7 with the release of 60%. Dissolve 30 g of compound 7 in 500 ml of acetic acid, add 70 ml of 33% hydrogen peroxide is stirred overnight at 70°C. Then cooled, acetic acid evaporated on a rotary evaporator and to the residue add 600 ml of water. The precipitation was filtered, washed with water and dried. Get connection 8 with the release of 70%. To a solution of 22 g of compound 8 in 300 ml of ethanol was added dropwise 8 ml of chloride tiomila at 10°C, after which the mixture is boiled for 5 hours. Cooled, evaporated methanol on a rotary evaporator, add 300 ml of water is. The precipitation was filtered, washed with water and dried. Get the connection 9 with the release of 90%. Dissolving 8.2 g of compound (9) in 80 ml of DMF, add 7 g of potash, and then 5.2 g m-chlorobenzylchloride. The mixture was stirred at 50°C overnight, the DMF evaporated on a rotary evaporator, the residue is added 200 ml of water. The precipitation was filtered, washed with water and dried. Get the connection 10 with the release of 90%. Dissolve 11 g of compound 10 in 150 ml of 50% aqueous ethanol, added 2.7 g of KOH and stirred overnight at room temperature. Then the ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. Get the connection 11 with the release of 80%. Dissolve 1.8 g of compound (11) in 50 ml of THF in an argon atmosphere with stirring 0.48 g of N-hydroxysuccinimide and 0.87 g dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get connection 1(1) output 50%. Range of proton magnetic resonance (PMR spectrum of compound 1(1) is presented in figure 1.

Example 2. 2,5-Dioxopiperidin-1 silt ether (4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenoxy)-acetic acid 1(2) receive the following Scheme 2.

To a suspension of 30 g of potash in 200 ml of acetonitrile is added 15 g of p-NITROPHENOL 12, stirred 1 h, then was added dropwise to 19.8 g of ethyl ether bromoxynil acid. Stirred overnight at 60°C, filtered, evaporated on a rotary evaporator. The resulting product 13 is used without further purification. Dissolve 14.3 g of compound 13 in 200 ml of 50% aqueous acetic acid, heated to 70°C., and then sprinkled with small portions of 10 g of iron so that the mixture is boiling. Then stirred for further 15 minutes, cool, add 500 ml of water. Extracted with 3x150 ml of ethyl acetate, the combined extracts washed with a concentrated solution of NaHCO3, dried, the solvent evaporated on a rotary evaporator. Get the product 14 with a yield of 75%. Mix in 50 ml of dioxane 1.9 g of compound (11) and 0.87 g of compound 14. Stirred for 1 h, then add 1.2 ml of triethylamine and 0.89 g of phosphorus oxychloride. Stirred for 3 h at 50°C, add 150 ml of water, precipitated precipitate is filtered, washed with water and dried. Get the product 15 with the release of 60%. Dissolve 2.7 g of compound 15 in 20 ml of 50% aqueous ethanol, added 0.93 g of LiOH and stirred overnight at room temperature. Then the ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. The product is distilled chronochromie in the system chloroform-methanol-triethylamine 10-1-1. Get product 16 with a yield of 11%. Dissolve 290 mg of compound 16 in 50 ml of THF in an argon atmosphere with stirring 58 g of N-hydroxysuccinimide and 104 mg dicyclohexylurea-diimide. The mixture is stirred at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get product 1(2) with the release of 50%. PMR spectrum of the compound 1(2) is presented in figure 2.

Example 3. 2,5-Dioxopiperidin-1 silt ether 4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5N-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenylcarbinol acid 1(3) receive the following Scheme 3.

Dissolve 1 equiv of n-aminobenzoic acid in tert-butyl alcohol and added 1.1 EQ of EDC. Boil the reaction mixture for 18 hours Cooled to 0°C, add water and extracted with diethyl ether. One stripped off the organic layer is allowed to the next stage without purification. Get product 17 with the release of 60%. Mix in 50 ml of dioxane 2.2 g of compound (11) and 0.85 g of tert-butyl methyl ether, t-aminobenzoic acid 17. Stirred for 1 h, after which add 1.4 ml of triethylamine and 1.0 g of phosphorus oxychloride. Stirred for 3 h at 50°C, add 150 ml of water, precipitated precipitate is filtered, washed with water and dried. Get the product 18 with the release of 60%. The connection 18 is dissolved in triperoxonane acid is the same and stirred overnight at 50°C. The reaction mass is evaporated to dryness. Get product 19 with the release of 88%, which is used in the next stage without purification. Dissolve 760 mg of compound 19 in 50 ml of THF in an argon atmosphere with stirring to 160 mg of N-hydroxysuccinimide and 287 mg of dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature, the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent-ethyl acetate). Get product 1(4) with the release of 50%. PMR spectrum of the compound 1(4) is presented in figure 3.

Example 4. 2.5 Dioxo-pyrrolidin-1 silt ester of 3-[8-(3,4-dimethoxycinnamoyl)-5,5,11-trioxo-5,11-dihydro-dibenzo[b,f][1,4]diazepin-10-ylmethyl]-benzoic acid 1(4) receive the following Scheme 4.

Boil 10 g of 2,4-dimethoxybenzaldehyde and 9.2 g of 3,4-dimethoxyaniline in 150 ml of toluene with a nozzle Dean-stark to stop shedding water. After that, the mixture is cooled, evaporated toluene on a rotary evaporator, the product 20 is used without further purification. Dissolve the connection 20 in 100 ml of methanol and stirring sprinkled 2.8 g of sodium borohydride, stirred at room temperature for another 1 h, evaporated methanol on a rotary evaporator, add 100 ml of water, alkalinized with 10% KOH solution to pH 9, extracted with chloride of methylene is, dried, evaporated. Get product 21 with the release of 80%. Mix in 50 ml of dioxane 2.2 g of compound (9) and 3.0 g of compound 21. Stirred for 1 h, then add 5.5 ml of triethylamine and 2.0 g of phosphorus oxychloride. Stirred for 3 h at 50°C, add 150 ml of water, precipitated precipitate is filtered, washed with water and dried. Get the product 22 with the release of 60%. Dissolve 3.0 g of compound 22 in 30 ml of DMF, added 1.7 g of potash, and 1.3 g of ethyl 3-bromoethylamine. The mixture was stirred at 50°C overnight, the DMF evaporated in vacuo, to the residue add 70 ml of water. The precipitation was filtered, washed with water and dried. Get product 23 with the release of 90%. Dissolve 3.0 g of compound 23 in 40 ml of methylene chloride, add 1.4 g triperoxonane acid and refluxed overnight. After that, the mixture was washed with conc. solution Panso3, dried, evaporated methylene chloride. Clean the product column chromatography, eluent chloroform / methanol 40/1. The output 24 is 30%. Dissolve 0.9 g of compound 24 in 20 ml of 50% aqueous ethanol, add 0.25 g of LiOH and stirred overnight at room temperature. Then the ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. The product was then purified column chromatography in the system chloroform-methanol-triethylamine 10-1-1. Get the product 25 from the output of 0%. Dissolve 140 mg of compound 25 in 20 ml of THF in an argon atmosphere with stirring 42 mg of N-hydroxysuccinimide and 76 mg dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get product 1(4) with the release of 50%. LCMS-spectrum of compound 1(4): (M+1=670) is presented in figure 4.

Example 5. 2.5 Dioxo-pyrrolidin-1 silt ether (4-{8-[3-(4-benzylpiperidine-1-yl)-propellerblades]-5,5,11-trioxo-5,11-dihydro-dibenzo[b,f][1,4]diazepin-10-ylmethyl}-phenyl)-acetic acid 1(5) receive the following Scheme 5.

Boil 3.8 g of 2,4-dimethoxybenzaldehyde and 5.3 g of compound 26 in 100 ml of toluene with a nozzle Dean-stark to stop shedding water. After that, the mixture is cooled, evaporated toluene on a rotary evaporator. The resulting product 27 is used without further purification. Dissolve the connection 27 in 50 ml of methanol and stirring sprinkled 1.3 g of sodium borohydride, stirred at room temperature for another 1 h, evaporated methanol on a rotary evaporator, add 50 ml of water, alkalinized with 10% KOH solution to pH 9, extracted with methylene chloride, dried, and evaporated. Get a connection 28 with the release of 80%. Dissolve 2.4 g of compound (9) in 100 ml of THF in an argon atmosphere with stirring 1.0 g of N-hydroc is succinimide and 1.1 g of dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature overnight, then add 3 g of compound 28 and stirred for 6 h at 60°C. the precipitation is filtered off, the mother liquor is evaporated on a rotary evaporator. The remainder of the cleaning column chromatography, eluent chloroform:methanol 19:1. Get the connection 29 with the release of 70%. A mixture of 10.0 g of p-tolyloxy acid, 13.0 g of N-bromosuccinimide and 0.1 g of 2,2'-azobisisobutyronitrile in 60 ml of carbon tetrachloride is refluxed 4 hours the Mixture is cooled to room temperature, poured onto 100 ml of water, precipitated precipitate is filtered off and dried. Dissolved in 50 ml of ethanol, added at 0°With 3.7 ml of thionyl chloride, is stirred overnight at room temperature, evaporated the solvent.

Get a connection 30 with the release of 50%. The resulting product is used without further purification. Dissolve 4.5 g of compound 29 in 50 ml of DMF, add 2.2 g of potash, and then 2.0 g of ethyl ester of p-bromomethylphenyl acid 30. The mixture was stirred at 50°C overnight, the DMF evaporated on a rotary evaporator and to the residue was added 100 ml of water. The precipitation was filtered, washed with water and dried. Get a connection 31 with the release of 80%. Dissolve 3.5 g of compound 31 in 40 ml of methylene chloride, add 2.0 g triperoxonane the acid and refluxed overnight. After that, the mixture is shaken out several times with conc. a solution of NaHCO3, dried, evaporated methylene chloride on a rotary evaporator. Clean the product column chromatography, eluent chloroform / methanol 40/1. Get a connection 32 with a yield of 55%. Dissolve 1.6 g of compound 32 in 20 ml of 50% aqueous ethanol, added 0.29 g of LiOH and stirred overnight at room temperature. Then the ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. The product was then purified column chromatography in the system chloroform-methanol-triethylamine 10-1-1. Get the connection 33 with the release of 15%. Dissolved 180 mg of compound 33 in 20 ml of THF in an argon atmosphere with stirring 47 mg of N-hydroxysuccinimide and 84 mg of dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get connection 1(5) with a yield of 50%. PMR spectrum of the compound 1(5) is shown in Figure 5.

Example 6. 10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid (2-amino-ethyl)-amide 1(6) receive the following Scheme 6.

Compound 11 was dissolved in chloroform, added 1.1 EQ CBI and PE is amerivault at room temperature for an hour, then add the side-Ethylenediamine and stirred overnight at room temperature. Washed with water and the organic layer evaporated. Clean on the column in eluent chloroform-methanol 20:1. Get the connection 34 with the release of 70%.

The connection 34 is stirred in dioxane saturated with HCl, at room temperature 1 h, the precipitate filtered off. Get connection 1(6) with a yield of 78%. PMR spectrum of the compound 1(6) is shown in Fig.6.

Example 7. 10-{4-[(2-Amino-ethylcarbazole)-methyl]-benzyl}-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide 1(7) receive the following Scheme 7.

Compound 35 was dissolved in chloroform, added 1.1 EQ CBI and stirred at room temperature for 1 h, then added the side-Ethylenediamine and stirred overnight at room temperature. Washed with water and the organic layer upriver. Chromatographic on silica gel with a mixture of chloroform with methanol (20:1). Get a connection 36 with the release of 70%. The connection 36 is stirred in dioxane saturated with Hcl at room temperature for 1 h, filter out sediment. Get connection 1(7) with the release of 78%. PMR spectrum of the compound 1(7) is shown in Fig.7.

Example 8. 2-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-ethyl ester of 10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic kislota(8) receive the following Scheme 8.

Connections 37 and 11 are dissolved in THF in a ratio of 1:1 and with stirring was added 1 EQ of dicyclohexylcarbodiimide and 1 EQ Et3N. Stirred overnight at 50°C. is Cooled, the precipitated residue was filtered and the mother liquor evaporated and purified HPLC. Get connection 1(8) with the release of 10%. PMR spectrum of the compound 1(8) presented on Fig.

Example 9. 10-(4-{[2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethylcarboxyl]-methyl}-benzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide 1(9) receive the following Scheme 9.

Connections 35 and 37 is dissolved in THF in a ratio of 1:1 and with stirring was added 1 EQ of dicyclohexylcarbodiimide and 1 EQ Et3N. Stirred overnight at 50°C. is Cooled, the precipitated residue was filtered and the mother liquor evaporated and purified HPLC. Get connection 1(9) with the release of 10%. PMR spectrum of the compound 1(9) is shown in Fig.9.

Example 10. N-[2-({N-(Methoxycarbonyl)-N-[(1,5-dimethoxy-1,5-dioxetane-2-yl)carbarnoyl]-β-alanyl}amino)ethyl]-10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydrobenzo[b,f][1,4]diazepin-7-carboxamide 1(10) receive the following Scheme 10.

To a suspension of triphosgene (4.2 g, 0014 mol) in CH2Cl2at 0°C. slowly added dropwise a solution, sustasis 11 g (0.038 mol) of the compound 38 and 12.6 g (0.098 mol) of DIPEA in CH 2Cl2. Stir the mixture for 30 minutes at 0°C, then add one portion a solution of 8.1 g (0.038 mol) of the compound 39 and 10.8 g (0.084 mol) of DIPEA in CH2Cl2. Then stirred for further 15 min, the organic layer was separated, washed with water and chromatographic on silica gel l3/Meon (98:2). Get a connection 40 to exit 53%. Dissolve 6 g of compound 40 in ethyl acetate, add 0.6 g Pd/VC (10%). The mixture hydronaut in an autoclave at 2 ATM 17 hours the Catalyst is filtered off, the filtrate was evaporated. Get 5 g of compound 41, which is used in the next stage without additional purification. To a solution of 4.8 g (0.013 mol) of the compound 41 in CH2Cl2at room temperature, covered 2.6 g CBI (0.016 mol). After 2 hours, add 2.2 g (0.013 mol) BOC-Ethylenediamine. The mixture is stirred for about 18 hours, diluted with water, the product extracted with CH2Cl2and chromatographic on silica gel with a mixture l3/Meon (99:1). Obtain 4.4 g (66%) of compound 42. To a solution of 4.4 g of compound 42 in dioxane add 5 EQ 3N Hcl in dioxane. The mixture is stirred overnight at room temperature, then evaporated to dryness. Get the connection 43 with the release of 90%. Mix 0.8 g (0.002 mol) of the compound 43, 0.85 g (0.002 mol) of compound (11), 0.4 g NEt3(0.004 mol) and 0.4 g (0.002 mol) of N-(dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride in THF. The reaction mass is stirred at 60°C for two days. Then to ablaut 0.5 g (0.012 mol) of LiOH·H 2O and 2 ml of water. After 24 hours add 0.72 g (0.012 mol) of acetic acid. The reaction mass is evaporated and purified HPLC. Get a connection 44 with the release of 7%. PMR spectrum of the compound 1(10) is shown in Figure 10. Hydrochloride 1(10)*Hcl is produced by adding to the compound 1(10) dissolution in ethyl acetate, saturated with Hcl. Obtained to precipitate the hydrochloride is filtered, dried, purified by recrystallization. Similarly receive hydrochloride compounds obtained in examples 1-9 and 11-14.

Example 11. N5-(2-{[(4-{[7-{[[3-(4-Benzylpiperidine-1-yl)propyl](phenyl)amino]-carbonyl}-5,5,11-trioxo-dibenzo[b,f][1,4]thiazepine-10(11H)-yl]methyl}phenyl)acetyl]-amino}ethyl)-N2-{[(1,3-dicarboxylate)amino]carbonyl}glutamine 1(11) receive the following Scheme 11.

Mix 1.6 g (0.0024 mol) of the acid 35, 1 g (0.0024 mol) of the ester 43, 0.5 g NEt3(0.0048 mol) and 0.45 g(0.0024 mol) of N-(dimethylaminopropyl)]-N of ethylcarbodiimide hydrochloride in THF. The reaction mass is stirred at 60°C for two days. Then, without releasing the product 45 add 0.0144 mol, 0.6 g of LiOH·H2O and 2 ml of water. After 24 h added 0.86 g (0.014 mol) of acetic acid. The reaction mass is evaporated and purified HPLC. Get connection 1(11) with the release of 10%. PMR spectrum of the compound 1(11) figure 11 presents.

Example 12. 4-[4-(Dimethylamino)phenyl]-N-(4-{[(2,5-dioxopiperidin-1-yl)oxy]carbonyl}phenyl)-7,8,9,10-tetrahydro-4H-[benzothieno[3,2-f]pyrrolo[1,2-α][1,4]diazepin-5(6H)-carboxamide 2(1) receive the following Scheme 12.

A mixture of 3.0 g of compound 46 and 1.7 g of 4-dimethylaminobenzaldehyde in 40 ml of ethanol is refluxed for 10 h, cooled to room temperature, filtered loose sediment. The residue is dissolved in 50 ml of water and alkalinized with 10% KOH to pH 10. The precipitation was filtered, washed with water and dried. Get a connection 47 with the release of 35%. Dissolve 0.6 g of compound 47 and 0.32 g of ethyl 4-isocyanatobenzene in 30 ml of dioxane and stirred overnight at room temperature. The precipitation is filtered. Get the connection 48 with 30%yield. Dissolve 140 mg of compound 48 in 5 ml of 50% aqueous ethanol, added 32 mg of LiOH and stirred overnight at room temperature. The ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. Get a connection 49 with the release of 60%. Dissolve 80 mg of compound 49 in 10 ml of THF in an argon atmosphere with stirring 22 mg of N-hydroxysuccinimide and 39 mg dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get connection 2(1) output 50%. LCMS-spectrum of compound 1(12) presented on Fig (M+1)=624.

Example 13. 4-[4-(Dimethylamino)phenyl]-N-(4-{2-[(2,5-di is cooperrider-1-yl)oxy]-2-oksidoksi}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-α][1,4]diazepin-5(6H)-carboxamide 2(2) receive the following Scheme 13.

Dissolved in 30 ml of dioxane 0.8 g of compound 50 and 0.5 g of compound 47 and stirred overnight at room temperature. The solvent is evaporated, the residue is cleaned column chromatography. Eluent chloroform - methanol 39:1. Get a connection 51 with the output 5%. Dissolve 6 mg of compound 51 in 5 ml of 50% aqueous ethanol, added 14 mg of LiOH and stirred overnight at room temperature. Then the ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. Get a connection 52 with the release of 60%. Dissolve 50 mg of compound 52 in 10 ml of THF in an argon atmosphere with stirring, add 10 mg of N-hydroxysuccinimide and 19 mg dicyclohexylcarbodiimide. The mixture is stirred at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get connection 1(13) with the release of 50%. PMR spectrum of the compound of compound 2(2) presented on Fig.

Example 14. 2,5-Dioxopiperidin-1-yl N-[4-(5-{[(3,4-acid)amino]-carbonyl}-5,6,7,8,9,10-hexahydro-4H-[1] benzothieno[3,2-f]pyrrolo[1,2-α][1,4]diazepin-4-yl)phenyl]-N-methylglycine 2(3) receive the following Scheme 14.

A mixture of 1.68 g of compound 46 is 1.45 g of compound 53 in 25 ml of ethanol is refluxed 10 h, cooled to room temperature, filtered, precipitated is dissolved in 50 ml of water and alkalinized with 10% KOH to pH 10. The precipitation was filtered, washed with water and dried. Get the connection 54 with the release of 19%. Dissolved in 10 ml of dioxane 0.54 g of compound 54 and 0.29 g dimethoxyaniline and stirred overnight at room temperature. The solvent is evaporated, the residue is cleaned column chromatography. Eluent - chloroform: methanol 39:1. Get a connection 55 with the release of 65%. Dissolve 500 mg of compound 55 in 5 ml of 50% aqueous ethanol, added 32 mg of LiOH and stirred overnight at room temperature. The ethanol is evaporated on a rotary evaporator, the aqueous solution is acidified with 10% hydrochloric acid to pH 3. The precipitation was filtered, washed with water and dried. Get a connection 56 with the release of 60%. Dissolve 360 mg of compound 56 in 10 ml of THF in an argon atmosphere with stirring 71 mg of N-hydroxysuccinimide and 127 mg of dicyclohexylcarbodiimide. The mixture is stirred overnight at room temperature overnight, then the precipitation is filtered, the mother liquor is evaporated, clean flash chromatography on silica gel (eluent ethyl acetate). Get connection 2(3) with the release of 50%. LCMS-spectrum of compound 2(3) presented on Fig (M+1)=684.

Example 15. A common method of obtaining modified proteins (conjugates). Protein modify the substance of General formula 1 or 2 in melanomatosis from 1:1 to 1:100. A portion of the substances of General formula 1 or 2 are dissolved in 50 µl of DMSO, and then slowly add phosphate saline buffer solution (FSB, pH 7.4) and add the protein solution to the FSB. The final concentration of protein in the reaction mixtures was 1 mg/ml Reaction is carried out at room temperature for 20 h with constant stirring. The reaction mass is centrifuged, the filtrate is applied to the gel Superdex 75 (GE, USA); column height of the carrier - 23 cm; mobile phase - FSB; flow rate - 57,3 cm/h; volume of injection is 2.2% of the volume of the media. The purity of the conjugate was determined using HPLC on affinity column (Bio-Monolith Protein A (Agilent, USA).

In this way obtained, in particular, conjugates:

KR1(1) - rituximab (R) connection 1(1), in the ratio of 1:10,

KR1(2) - rituximab (R) connection 1(2), in the ratio of 1:100,

KR1(3) - rituximab (R) connection 1(3), in the ratio of 1:100,

KR1(4) - rituximab (R) connection 1(4), in the ratio of 1:100,

KR1(5) - rituximab (R) with compound 1(5), in the ratio of 1:10,

CR(1) - rituximab (R) with compound 2(1), in the ratio of 1:100,

CC(1) - cetuximab (C) connection 1(1), in the ratio of 1:10,

CI(1) interferon - alpha (And) connection 1(1), in the ratio of 1:10,

KM1(1) - protein myelin (R) connection 1(1), in the ratio of 1:10,

KS1(1), a protein of the complement C1q (C) connection 1(1), in the ratio of 1:10.

On Fig listed on the I sample chromatogram conjugate KR1(1), indicating the basic substance content of more than 98%.

Example 16. The determination of the activity of conjugates with respect to CD 16A receptor. In the experiment using standard materials and equipment for immuno-enzymatic analysis. To the wells of the 96-hole tablet absorb CD16 from a solution of 3.3 mg/ml in phosphate-buffered saline in a volume of 75 μl/well. Sorption was conducted at 4°C for 16 hours. The solution CD16 removed from the tablet, the wells were filled with 2%solution of BSA in the FSB-P (150 µl/well). Tablets incubated at 37°C for 2 hours and washed three times with buffer FSB-P (each 300 μl/well). Then the wells were filled with solutions of the conjugates in the FSB-P at 50 μl/well, in dilutions from 0.25 to 256 µg/ml of Each dilution of one of the conjugate in the tablet corresponded to three wells. To control nonspecific sorption samples part of the hole was filled with 2%solution of BSA. The tablet is incubated for one hour at 37°C in a shaker for ELISA (speed 180 rpm). The tablet was washed five times with 300 µl/well of the FSB-P and was filled with a solution of a protein modified with horseradish peroxidase (75 μl/well, in a working breed that is specified in the description of immunoconjugate). The tablet is incubated for 30 minutes, washed five times with 300 µl/well of the FSB-P and was filled with a solution of TMB (100 μl/well). Visually estimated the depth of flow enzymatic R. the action and stopped the last addition of 50 µl/well 500 mm solution of sulfuric acid. Measure the optical density in the wells at room temperature at a wavelength of 450 nm. For processing of the experimental results using the program GraphPad Prism 5.0.

On Fig presents the dependence of the binding of some conjugates with respect to CD 16A receptor, which is designed (table 1) dissociation constants of the complexes of the conjugates with CD16 receptor (activity of conjugates with respect to CD16 receptor - Kd). As can be seen from the table, the activity of conjugates of rituximab in 10-100 times higher than the activity of unconjugated rituximab.

Table 1. Activity (Kd) of rituximab and its conjugates with respect to CD16 receptor.
PConjugates of rituximab
KR1(4)CR(1)KR1(3)KR1(5)KR1(1)KR1(2)
KdMof 1.5×10-6of 1.9×10-7of 7.4×10-86,5×10-88,0×10-8 of 1.3×10-81,0×10-8

On Fig presents the dependence of the binding of interferon alpha and interferon alpha conjugate connection 1(1) CD16 receptor. The dependence of the optical density at a wavelength of 450 nm on the concentration introduced in ELISA conjugate. As can be seen from table 2, the activity of a conjugate of interferon higher activity of unconjugated interferon alpha.

Table 2. Activity (Kd) interferon alpha (S), and its conjugate KI(1) in relation to CD16 receptor.
AndCI(1)
KdM2,3x10-76,0×10-8

Example 17. The efficiency of the conjugates in the test for antibody-dependent (rituximab-dependent cell cytotoxicity. The efficacy of conjugates of rituximab in comparison with unconjugated rituximab was evaluated in the test for antibody-dependent cellular toxicity. As target cells were used D20-positive cell line Daudi (b-cell lymphoma Bernita). As effector cells use polerowanie peripheral mononuclear blood cells from 5 healthy is anorov, selected according to the standard Protocol.

Cultivation of the studied conjugates and rituximab were prepared in RPMI medium 1640. 50 μl of solutions of the studied conjugates and rituximab were placed in wells of 96-well round-bottom of the tablet. Cell line Daudi resuspended in medium RPMI 1640 to a concentration of 8×105cells/ml 75 µl of the obtained cell suspension is added to the prepared solution of rituximab in the tablet. Tablet incubated in a thermostat at 37°C for 50 minutes. Add 75 ál of cell suspension RVMS (thawed immediately before the experiment) in the wells to Daudi cells in a ratio of 50:1 (RVMS: Daudi cells). As control solutions add medium RPMI 1640 (negative control, which contained only effector cells), as well as a 4% solution of Triton X-100 (positive control containing only target cells). Tablets centrifuged for 3 minutes at 500 rpm Incubate the plates in an incubator for 8 hours at 37°C.

After incubation, the samples were precipitated by centrifugation at 1200 rpm for 10 minutes. 50 μl of supernatant were transferred to a new tablet, where were mixed with 50 ál of reaction mixture (kit for the detection of lactate dehydrogenase, Promega (USA), catalog number G1780). The resulting mixture was incubated at room temperature for 25 minutes, the Le which the reaction was stopped. Measure the absorption at a spot spectrophotometer at a wavelength of 490 nm.

Cytotoxicity was calculated by the formula (all values in units of optical density):

cytotoxicity, %=100·(A-(SE-ST))/(HE-ST),

where a is the signal level in a mixture of effector cells and target cells;

SEthe signal level of effector cells;

STthe signal level of the target cells;

HEthe signal corresponding to the maximum lysis of target cells.

The result of measuring the effectiveness of the obtained conjugates shown in example conjugate KR1(1) (Fig) CON 4564. The resulting conjugate had enhanced cytotoxic effect on target cells (Daudi). In particular, ~35%lysis of the cells is achieved at a concentration of rituximab ~1 µg/ml, at the same time, a similar cytotoxic effect of the conjugate KR1(1) CON 4564 achieved at a concentration of ~0.001 microg/ml, a Similar dependence is observed in the case of conjugate KR1(2) (Fig). Thus, the activity of the conjugates of rituximab by 2-3 orders of magnitude higher than that of rituximab.

Example 18. Obtaining a conjugate of rituximab KR1(7) and trastuzumab KT1(7). To 1.5 g of the sugar part of the antibody in a solution of 100 mm sodium acetate, 200 mm sodium chloride, pH 5.5 add 2.0 g of periodate sodium, incubated for 30 min at room temperature in the dark under stirring. Dissolve 1200 mg with the unity 1(7) in 4 ml of DMSO. To a solution of antibody added to 8.4 g of sodium bicarbonate, then added dropwise 1230 μl dissolved in DMSO connection 1(7) and incubated for 2 hours.

Purification was performed on a SP-Sepharose (column HK GE) step gradient of NaCl (buffer 1: 25 mm sodium citrate, pH 4.5. Buffer 2: 25 mm sodium citrate, pH ~ 10,270 mm). Conjugate antibodies are concentrated in the cell through the membrane Amicon Millipore (NMWL 30000, cat. # PLTK07610) to a final concentration of conjugate (10 mg/ml After filtration conjugate add Polysorbate-80 to a final concentration of conjugate KR1(7) or KT1(7) of 0.01%.

Example 19. Comparative activity of rituximab (P) and trastuzumab (T) and their conjugates KR1(7), KT1(7) in relation to the CD16a receptor. Study of the binding of the obtained conjugates of trastuzumab and rituximab with receptor CD16 conducted identically to example 17.

Sravnenitelno efficiency and activity (Kd) with respect to CD16 receptor rituximab (R), trastuzumab (T) and their conjugates KR1(7), KT1(7) in the test antibody-dependent cytotoxicity presented on Fig.

As can be seen from the obtained data, the activity of the conjugates KR1(7), KT1(7) in relation to CD16 receptor by 1-2 orders of magnitude higher activity of unmodified rituximab (R), trastuzumab (T) (table 3).

Table 3. Activity (IQ) of rituximab (R), trastuzumab (T) and the conjugates KR1(7), KT1(7) in relation to CD16 receptor.
PKP1(7)TKT1(7)
KdM1,104×10-75,880×l0-92,341×l0-73,030×10-8

1. The compounds having affinity to the CD16a receptor, representing 5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin General formula 1 or 5,6,7,8,9,10-hexahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-a][1,4]diazepin General formula 2, or its pharmaceutically acceptable salt,

in which R1 is selected from the group represents (CH3)2N-,

R2 is selected from a group representing

where R3 as a terminal substituent represents-NH2,
,or
a R4 represents H or C1-C3alkyl.

2. The compound of General formula 1 according to claim 1, in which R1 is selected from the group including:

R2 is selected from the group including:

,
where R4=H or C1-C3alkyl.

3. The compound of General formula 2 according to claim 1, in which
R1 represents (CH3)2N - or;
R2 is selected from the group including:
.

4. The compound according to claim 1, selected from the group comprising 2,5-dioxopiperidin-1 silt ether (3 chlorbenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carboxylic acid,
2,5-dioxopiperidin-1 silt ether (4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenoxy)-acetic acid,
2,5-dioxopiperidin-1 silt ether 4-{[10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-7-carbonyl]-amino}-phenylcarbinol acid,
2.5 dioxo-pyrrolidin-1 silt ester of 3-[8-(3,4-dimethoxycinnamoyl)-5,5,11-trioxo-5,11-dihydro-dibenzo[b,f][1,4]diazepin-10-ylmethyl]-benzoic acid,
2.5 dioxo-pyrrolidin-1 silt ether (4-{8-[3-(4-benzylpiperidine-1-yl)-propellerblades]-5,5,11-trioxo-5,11-dihydro-dibenzo[b,f][1,4]diazepin-10-ylmethyl}-phenyl)-acetic acid,
10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f|[1,4]diazepin-8-carboxylic acid (2-amino-ethyl)-amide,
10-{4-[(2-amino-ethylcarbazole)-methyl]-benzyl}-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide,
2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethyl ester 0-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f] [1,4]diazepin-8-carboxylic acid,
10-(4-{[2-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-ethylcarboxyl]-methyl}-benzyl)-5,5,11-trioxo-10,11-dihydro-5H-dibenzo[b,f][1,4]diazepin-8-carboxylic acid [3-(4-benzyl-piperidine-1-yl)-propyl]-amide,
N-[2-({N-(methoxycarbonyl)-N-[(1,5-dimethoxy-1,5-dioxetane-2-yl)carbarnoyl]-β-alanyl}amino)ethyl]-10-(3-Chlorobenzyl)-5,5,11-trioxo-10,11-dihydrobenzo[b,f][1,4]diazepin-7-carboxamid,
N5-(2-{[(4-{[7-{[[3-(4-benzylpiperidine-1-yl)propyl](phenyl)amino]-carbonyl}-5,5,11-trioxo-dibenzo[b,f] [1,4]thiazepine-10(11H)-yl]methyl}phenyl)acetyl]-amino}ethyl)-N2-{[(1,3-dicarboxylate)amino]carbonyl}glutamine,
4-[4-(dimethylamino)phenyl]-N-(4-{[(2,5-dioxopiperidin-1-yl)oxy]carbonyl}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-a][1,4]diazepin-5(6H)-carboxamide,
4-[4-(dimethylamino)phenyl]-N-(4-{2-[(2,5-dioxopiperidin-1-yl)oxy]-2-oksidoksi}phenyl)-7,8,9,10-tetrahydro-4H-[1]benzothieno [3,2-f]pyrrolo[1,2-a][1,4]diazepin-5(6H)-carboxamide, and
2,5-dioxopiperidin-1-yl N-[4-(5-{[(3,4-acid)amino] -carbonyl}-5,6,7,8,9,10-hexahydro-4H-[1]benzothieno[3,2-f]pyrrolo[1,2-a][1,4]diazepin-4-yl)phenyl]-N-methylglycine.

5. The modified protein is active against CD16a receptor selected from antibodies or autoantigen conjugated modifying compound which has affinity to the CD16a receptor selected from the compounds of General formula 1 according to claim 1 or 2 in mass. the ratio of 1:1-1:100.

6. The modified protein according to claim 5, the tives such as those what antibody is a rituximab.

7. The modified protein according to claim 5, characterized in that the antibody is a trastuzumab.

8. The modified protein according to claim 5, characterized in that the antibody is a cetuximab.

9. The modified protein according to claim 5, wherein the autoantigen is an interferon alpha.

10. The modified protein according to claim 5, wherein the autoantigen is a major protein of myelin.

11. The modified protein according to claim 5, wherein the autoantigen is a protein of the complement C1q.

12. The method of obtaining modified protein according to claim 5, according to which is subjected to the interaction of the protein with the compound of General formula 1 or 2 according to claim 1 dissolved in a medium of an organic solvent, such as dimethylsulfoxide, in the range of molar ratios from 1:3 to 1:100 in the environment of phosphate saline buffer solution (pH 7.4).

13. The pharmaceutical composition active against CD16a receptor, in the form of tablets, capsules, or injections, placed in pharmaceutically acceptable packing containing the modified protein according to claim 5 in a therapeutically effective amount and a pharmaceutically acceptable diluent, carrier or excipient.

14. The drug is active against the CD16a receptor, in the form of tablets, cap the street or injection, placed in pharmaceutically acceptable packing intended for the treatment of diseases caused by pathological cells, incorporating the modified protein according to claim 5, or a pharmaceutical composition according to item 13 in therapeutically effective amounts.

15. A method of treating diseases caused by pathological cells, which can be treated by indirect effects on the CD16a receptor, whereby administered a therapeutically effective amount of the modified protein according to claim 5, or a pharmaceutical composition according to item 13, or the medicines at 14.

16. The method of treatment according to § 15 of autoimmune or cancer.

17. The method of treatment according to article 16, lymphoma, lymphocytic leukemia or breast cancer.

18. The method of treatment according to article 16, autoimmune polyendocrinopathy of the first type.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: chimeric monoclonal antibody is described, which specifically connects to human erythropoietin, characterised by the following criteria: a) Kd=2.4×10-9 M, isoelectric point in the range pH 7.5-8.0; b) sequence of the heavy chain SEQ ID NO:12; c) sequence of the light chain SEQ ID NO:14. A mouse hybridome strain is proposed, which is a producent of a monoclonal antibody to human erythropoietin, deposited in the Russian Academy of Agricultural Sciences, Specialised Collection of Cell Cultures of Farm and Game Animals under the No.84. Also a mouse anticlonal antibody is described, which specifically connects to human erythropoietin, produced by the specified hybridome and characterised by the following criteria: a) Kd=0.95×10-9 M, molecular weight = 160 kD, isopoint in the range pH 6.8-7.1; b) sequence of variable area of light chain SEQ ID NO:1; c) sequence of variable area of heavy chain SEQ ID NO:2; d) sequence of areas that define antibody complementarity: CDRH-1 - SEQ ID NO:5, CDRH-2 - SEQ ID NO:6, CDRH-3 - SEQ ID NO:7, CDRL-1 - SEQ ID NO:8, CDRL-2 - SEQ ID NO:9, CDRL-3 - SEQ ID NO:10.

EFFECT: invention makes it possible to expand arsenal of mouse antibodies against human erythropoietin.

3 cl, 3 dwg, 5 ex, 2 tbl

Antibodies to her // 2504553

FIELD: biotechnologies.

SUBSTANCE: invention describes versions of bispecific antibodies specifically bound to EGFR and HER3, which contain amino-acid sequences of variable regions of heavy and light chains respectively, SEQ ID NO: 30 and 29; or SEQ ID NO: 28 and 27; or SEQ ID NO: 28 and 29; or contain complementary regions CDR of heavy and light chains of the above sequences of variable regions. The invention describes nucleic acid coding a versions antibody, and a host cell containing the above nucleic acid and used for expression of the anitbody. Immunoconjugate containing antibody versions and cytotoxic agent used for treatment of cancer containing cells that express EGFR and HER3 are presented. A method for obtaining a bispecific antibody, which involves cultivation of a host cell so that an antibody is generated, is described. The invention describes a pharmaceutical composition for treatment of cancer containing EGFR- and HER3-expressing cells, which contains effective amount of bispecific antibody and pharmaceutically acceptable carrier. The invention proposes a treatment method of cancer containing EGFR- and HER3-expressing cells and an inhibition method of biological activity of EGFR and/or HER3 of a specimen, which involve introduction of effective amount of a bispecific antibody. Use of the above antibody in production of a remedy for treatment of cancer, the cells of which express EGFR and HER3, is described.

EFFECT: invention allows obtaining bispecific antibodies binding EGFR and HER3, which are not conjugates of two antibodies.

22 cl, 33 dwg, 4 tbl, 19 ex

FIELD: biotechnologies.

SUBSTANCE: two antibodies against IL-21 of a human being are presented. The first antibody includes a variable region of a heavy chain, which includes SEQ ID NO: 31, 33 and 35, and a variable region of a light chain, which includes SEQ ID NO: 39, 41 and 43. The second antibody includes a variable region of heavy chain, which includes SEQ ID NO: 47, 49 and 51, and variable region of light chain, which includes SEQ ID NO: 55, 57 and 59. Besides, the invention describes hybridomes producing the first and the second antibodies against IL-21 of a human being and deposited in the collection of cultures "American Type Culture Collection" and have numbers "ATCC Patent Deposit Designation PTA-8790" and "ATCC Patent Deposit Designation PTA-8786" respectively.

EFFECT: invention allows obtaining antibodies to IL-21 of a human being.

48 cl, 4 dwg, 16 tbl, 23 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes variable domains of heavy (VH) and light (VL) chains of murine antibody against tumour necrosis factor alpha (TNF-α) of a human being, as well as antigen-binding fragment Fab, which are selectively bound to TNF-α of the human being and neutralise it.

EFFECT: invention can be further used in development of medicines for therapy of TNF-α-mediated diseases and for diagnostics of such diseases.

3 cl, 5 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to a molecule of nucleic acid, which is a cyclic or a linear vector fit for expression, of at least one target polypeptide in cells of mammals, including (a) at least one expressing cassette (POI) for expression of the target polypeptide; (b) an expressing cassette (MSM), including a gene of a selective marker of mammals; (c) an expressing cassette (MASM), including an amplificated gene of a selective marker of mammals; besides, the expressing cassette (POI) is flanked in direction 5' by the expression cassette (MASM), the expression cassette (MSM) is localised in direction 3' from the expression cassette (POI) and in which the expression cassettes (MASM), (POI) and (MSM) are arranged in the same orientation from 5' to 3'. Also the method is disclosed to produce the specified molecule of nucleic acid of the vector, as well as a cell of a host mammal, containing the specified molecule of nucleic acid of the vector, the method to produce a host cell containing the specified molecule of nucleic acid of the vector, and also the method to produce the target polypeptide, using the specified host cell.

EFFECT: invention makes it possible to efficiently produce a target polypeptide in mammal cells.

24 cl, 2 dwg, 4 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. There are presented: a method for tumour cell growth inhibition in an individual and a method for immune response enhancement in an individual involving the introduction of a PD-1 monoclonal antibody and a CTLA-4 10D1 monoclonal antibody to the individual. The PD-1 monoclonal antibody has the following properties: it binds to human PD-1 having the value KD equal to 1×10-8 M or less; however it binds neither to human CD28, nor to CTLA-4, nor to ICOS; it is able to enhance the T-cell proliferation in the mixed lymphocyte reaction (MLR) analysis; it is able to enhance the gamma interferon production in the MLR analysis; it is able to enhance the interleukine-2 (IL-2) secretion in the MLR analysis.

EFFECT: invention provides a synergic effect when using the above antibodies in a combination.

4 cl, 54 dwg, 7 tbl, 25 ex

Novel antibodies // 2490277

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed is an anti-α5β1 antibody, which is described through amino acid sequences of six hypervariable regions and an antigen-binding moiety thereof. Described are conjugates of the disclosed antibodies with a medicinal agent or a label, a pharmaceutical composition, use of the disclosed antibodies to prepare a medicinal agent, methods and an industrial product for inhibiting angiogenesis and/or vascular permeability in a subject, and for treating cancer, an ophthalmic disease and an autoimmune disease in a subject. The invention describes an isolated nucleic acid, an expression vector, a cell and a method of producing an antibody or an antigen-binding moiety thereof, as well as a method of detecting α5β1 protein in a sample.

EFFECT: present invention can find further use in therapy and diagnosis of α5β1-mediated diseases.

52 cl, 11 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: disclosed are versions of human IL13 specific antibodies and a producing hybridoma cell line deposited in ATCC under number PTA-5657. Described are: versions of encoding polynucleotides; an antibody expression vector; a host cell for antibody expression, as well as versions of a method of producing an antibody using a vector, polynucleotide, hybridoma or host cell. The invention discloses a pharmaceutical composition for treating IL13-mediated diseases and methods of treating allergic, inflammatory and other diseases, which employ an anti-IL13 antibody.

EFFECT: providing antibodies which do not bind with target IL13 and neutralise activity of human IL13.

39 cl, 29 dwg, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to expression constructs, and may be used for immunoglobulin expression. An expression vector contains one open reading frame (sORF) insert which contains a first sequence of nucleic acid coding a first polypeptide; a first intermediate sequence of nucleic acid coding a first protein cleavage site containing an autoprocessing element with an intein segment providing proteolytic sORF polypeptide cleavage between the first polypeptide and the intein segment and the second polypeptide, but not ligation of said first polypeptide with said second polypeptide; and a second sequence of nucleic acid coding the second polypeptide. The expression vector is able to express a mammalian polypeptide coding sORF and cleaved in said first protein cleavage site in a host cell; consisting of the first polypeptide - an immunoglobulin heavy chain, and the second polypeptide - an immunoglobulin light chain able to be assembled into a multimer.

EFFECT: invention provides functional antibody production with 'correct' setup and assembly.

40 cl, 9 dwg, 57 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: what is produced is a recombinant protein created by gene shuffling possessing higher antiviral and antiproliferative activities as compared with existing human interferon-alpha-2b (HulFN-α2b). The prepared polypeptide is used in a composition as an antiviral, antiproliferative, anticancer or immunomodulatory agent.

EFFECT: invention enables using the prepared high-active interferon-like protein for treating a condition sensitive to interferon therapy, cancer or viral disease.

49 cl, 9 dwg, 8 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. A method of cleaning thrombin solution from infectious particles is provided. Macromolecules are added to the starting thrombin solution. The obtained solution is then passed through a nanofilter to obtain a thrombin solution free from infectious particles. Said macromolecule is not a nonionic surfactant, is different from thrombin and can be selected from a polymer containing at least 3 monomers of sugar, amino acids, glycols, alcohols, lipids or phospholipids. A thrombin-containing solution obtained using said method is also provided.

EFFECT: invention increases efficiency of removing infectious particles from thrombin solution; thrombin output reaches 93% using said method.

20 cl, 2 dwg, 13 tbl

FIELD: biotechnology.

SUBSTANCE: the invention relates to producing new peptides and may be used for treatment and prophylaxis of cytokine-sensitive disorders. Peptides, having a size of 5 to 40 amino acids and arising from cytokines, are used in a vaccine for treatment and prophylaxis of autoimmune diseases, disseminated sclerosis, rheumatoid polyarthritis, psoriasis, autoimmune diabeteses, lupus, allergy, asthma, cancer and AIDS.

EFFECT: allows effective immunization of patients against said diseases while minimizing side effects.

11 cl, 2 dwg, 17 tbl

FIELD: gene engineering, in particular method for treatment of viral infections.

SUBSTANCE: protein ZCYTO21 has amino acid sequence which is nearly similar to amino acid sequence of interferon-α. Protein and antibodies thereto have antiviral activity and are useful in treatment of hepatitis B and C as well as other diseases.

EFFECT: new protein with antiviral activity.

71 cl, 1 dwg, 6 tbl, 7 ex

FIELD: preparative biochemistry, medicine, pharmacology.

SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.

EFFECT: improved purifying method.

8 cl, 1 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel spirocyclic azaindole derivatives of formula I: , where: one of A stands for N, and the other ones stand for CR7-10; W stands for NR4; X stands for O, S; R1 and R2, independently on each other stand for H; C1-5-alkyl each time saturated, branched or unbranched, non-substituted or monosubstituted with -OC1-6alkyl; phenyl, thienyl, morpholinyl, benzothiophenyl or benzodioxilyl, each time non-substituted or monosubstituted with F, C1-6alkyl; or 5-membered heteroaryl, containing three nitrogen atoms as heteroatoms, substituted with C1-3alkyl; bound with C1-3-alkyl group phenyl non-substituted or monosubstituted with F or C1-6alkyl; R4 stands for H; R5 stands for H; R6 stands for H; R7, R8, R9 and R10 stand for H or CP3; in form of siastereomers, mixtures of diastereomers or separate diastereomer; bases and/or salts of physiologically compatible acids. Compounds are suitable for treatment of a number of diseases, for instance, pain, stress, depressions, etc. Described is method of their obtaining.

EFFECT: compounds are suitable for treatment of a number of diseases, for instance, pain, stress, depressions.

11 cl; 1 tbl; 34 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to new substituted tertahydro-4H-tieno-pyrrolo[3,2- c] pyridines of general formula 1, their geometric isomers or a mixture whereof and their pharmaceutically acceptable salts. In the general formula 1 Th represents an annulated tien cycle; W represents an single bond (in this case R3 is immediately bonded to the pyrrole cycle nitrogen atom), methylene, 1,2-ethane, 1,2-ethylene, 1,2-acetylene, 1,3-propane or 1,3-allyl bridge, non-obligatorily substituted by a hydroxy group; R1 and R2 represent hydrogen, C1-C4alkyl, a halogen or -CH2OH; R3 represents hydrogen, non-obligatorily substituted phenyl, non-obligatorily substituted by azaheteroaryl; R4 represents C1- C4alkyl, CO2C2H5 or CO2C(CH3)3; R5, R6, R7 (independent of each other) represent hydrogen or C1- C4alkyl or R5 and R6 together form an ethylene bridge while R7 represents hydrogen or R5 and R7 together form an ethylene bridge while R6 represents a hydrogen atom, they representing substituted tetrahydro-4H-tieno-pyrrolo [3,2-c] pyridines pf general formula 1, their geometric isomers, a mixture of such geometric isomers, their pharmaceutically acceptable salts as per any of Items 1-5.

EFFECT: obtainment of compounds representing ligands with receptor activity with regard to alpha-oadrenoreceptors, dopamine receptors, histamine receptors and serotonin receptors which compounds may be used during prevention and treatment of central nervous system diseases and for study of peculiarities of physiologically active substances possession biological activity with regard to the said receptors.

11 cl, 3 tbl, 21 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula I or pharmaceutically acceptable salts thereof, which have receptor tyrosine kinase type I inhibiting properties and can be used in treating hyperproliferative disorders in mammals. In general formula

,

A is O or S; G is N; B is a 6-member aryl or 5-6-member heteroaryl ring containing a sulphur atom as a heteroatom; E is

, , , , , X is N or CH; D1, D2 and D3 independently denote N or CR19; D4 and D5 independently denote N or CR19 and D6 is O, S or NR20, where at least one of D4 and D5 is CR19; D7, D8, D9 and D10 independently denote N or CR19, where at least one of D7, D8, D9 and D10 is N; R1 is H or C1-C6 alkyl; each R2 independently denotes halogen, cyano, nitro etc, trifluoromethyl, difluoromethyl, fluoromethyl, fluoromethoxy, difluoromethoxy, trifluoromethoxy, azido, -SR18, -OR15, -C(O)R15, -C(O)OR15, -NR14C(O)OR18, -OC(O)R15, -NR14SO2R18, -SO2NR15R14, -NR14C(O)R15, -C(O)NR15R14, -NR15C(O)NR15R14, -NR13C(NCN)NR15R14, -NR15R14, C1-C12alkyl, C2-C12 alkenyl, alkynyl, saturated or partially unsaturated C3-C10cycloalkyl, C3-C10cycloalkyl-C1-C12alkyl, -S(O)p(C1-C6alkyl), -S(O)p(CR13R14)q-phenyl, phenyl, phenyl-C1-3-alkyl, 5-6-member heteroaryl, 5-6-member heteroaryl-C1-C3-alkyl, saturated or partially unsaturated 3-8-member heterocyclyl, 5-6-member heterocyclyl-C1-C3-alkyl, -O(CR13R14)q-phenyl, NR15(CR13R14)q-phenyl, O(CR13R14)q-(5-6-member heteroaryl), NR13(CR13R14)q-(5-6-member heteroaryl, -O(CR13R14)q-(3-8-member heterocyclyl) or -NR15(CR13R14)q-3-8-member heterocyclyl), each R3 denotes Z, where Z is selected from and , W is O or S; W2 is O or S;V is CR8R9, R8b is H or C1-C6alkyl; each of R6, R8, R8a and R9 independently denotes hydrogen, trifluoromethyl, C1-C12alkyl etc.

EFFECT: improved properties and high efficiency of using the compounds.

25 cl, 13 dwg, 1 tbl, 36 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds, specifically to 4-substituted-3-(1-alkyl-2-chloro-1H-indol-3-yl)furan-2,5-diones of general formula I , where R1=H, C1-C6 alkyl; R2=H, C1-C6 alkyl, C1-C6 alkoxy; R3=phenyl, naphthyl, 2-phenyl-1-ethenyl, thienyl, furyl, pyrrolyl, benzothiophenyl, benzofuranyl, indolyl, synthesis method thereof and use as compounds capable of photochemical generation of stable fluorophores of formula II, which can be used, for instance in information storage systems, particularly as photosensitive components of material for three-dimensional recording and storage of information. The invention also relates to novel 4,5-substituted-6-alkyl-1H-furo[3,4-c]carbazole-1,3(6H)diones of general formula II , where R1=H, C1-C6 alkyl; R2=H, C1-C6 alkyl, C1-C6 alkoxy; R4=H, R5=phenyl, R4, R5=benzo, naphtho, thieno, furo, pyrrolo, benzothieno, benzofuro, indolo, method for synthesis of said compounds and use as fluorophores.

EFFECT: obtaining novel compounds and possibility of using said compounds as fluorophores.

14 cl, 2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention refers to novel compounds with affinity to MC4 receptors, of the general formula (I): , where A is -CH2- or -C(O)-; R1 is (C1-C8)alkyl; R2 is (C1-C8)alkyl; R3 is radical of the formula -(CH2)s-R'3; R'3 is 5-6-member heterocycloalkyl containing one or two nitrogen atoms and possibly one oxygen atom possibly substituted with (C1-C6)alkyl or benzyl; or radical of the formula -NW3W'3; W3 is hydrogen atom or (C1-C8)alkyl; W'3 is radical of the formula -(CH2)s'-Z3; Z3 is hydrogen atom, (C1-C8)alkyl; s and s' are independently an integer within 0 to 6; B is 5-6-member monocyclic unsaturated, aromatic or non-aromatic radical which can be condensed with 5-6-member unsaturated, aromatic or non-aromatic radical forming bicyclic condensed system, and B is possibly containing one or more equal or different heteroatoms selected out of O, S and N, and possibly substituted with one or more equal or different radicals selected out of halogen atom, nitro group, cyano group, oxy group, -XB-YB and phenyl possibly substituted with one substitute selected out of halogen atom and (C1-C6)alkyl; XB is a covalent bond, -O-, -S-, -C(O)-, -C(O)-O-; YB is (C1-C6)alkyl; or pharmaceutically acceptable salt of claimed compounds.

EFFECT: improved obtainment and application efficiency of compounds for production of drug for treatment of diseases related to MC4 receptor activation.

20 cl, 4 dwg, 2 tbl, 81 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I , where R1 is C1-C7-alkyl; R2 is C1-C7-alkyl, C1-C7-haloalkyl, C3-C8-cycloalkyl; R3 is -NRaRb; possibly substituted phenyl, thiophenyl, furanyl, where the substitutes are selected from a group consisting of halogen, C1-C7-alkoxy, C1-C7-alkylsulphonyl and -C(O)O-C1-C7-alkyl; R4 is hydrogen or C1-C7-alkyl; R5 is hydrogen, halogen, C1-C7-alkyl, phenyl; or R5 together with R4 can form a ring selected from a group consisting of C5-C7-cycloalkyl, tetrahydrofuranyl, piperidine, tetrahydropyran, phenyl or pyridinyl, which can possibly be substituted with -C(O)O-C1-C7-alkyl; Ra and Rb together with the nitrogen atom to which they are bonded form piperidine; and to pharmaceutically acceptable salts thereof. The invention also relates to a medicinal agent based on the said compounds which has GABA-B receptor allosteric enhancement effect.

EFFECT: obtaining novel compounds and a medicinal agent based on the said compounds, which can be used in medicine for treating central nervous system disorders.

13 cl, 42 ex

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