Conjugates, containing hydrophilic spacers of linkers

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to conjugates for the delivery of medications, which bind receptors on the cell surface, containing hydrophilic linker spacers.

EFFECT: claimed conjugates are intended for visualisation, diagnostics and treatment of painful conditions, caused by populations of pathogenic cells.

38 cl, 19 dwg, 49 ex

 

CROSS REFERENCES TO RELATED APPLICATIONS

For this application under 35 U.S.C. §119(e) priority is claimed based on provisional applications U.S. patent No. 60/946092 and 61/036186, filed on June 25, 2007 and March 13, 2008, respectively, the contents of which are incorporated into the present application by reference in its entirety.

The technical FIELD TO WHICH the INVENTION RELATES.

The present invention relates to compositions and methods intended for use in targeted drug delivery. More specifically, the invention is directed to conjugates that ensures the delivery of drugs by binding to receptors on the cell surface and containing linkers with hydrophilic spacers, which are intended for use in the treatment of painful conditions caused by populations of pathogenic cells, and to methods and pharmaceutical compositions, uses and includes such conjugates.

BACKGROUND of the INVENTION

The immune system of mammals includes tools for recognition and elimination of tumor cells, other pathogenic cells and invading the body external pathogens. Although usually, the immune system creates a strong line of defense of the organism, there is a significant number of cases where cancer cells, other pathogenic CL is TCA or infectious agents avoid the immune response of the host organism and proliferate or survive, accompanied pathogenic manifestations in relation to the host organism. Were developed chemotherapeutic and radiation therapy to eliminate, for example, can replicate tumors. However, many of the currently available chemotherapeutic agents and types of radiation therapy have unwanted side effects, because they destroy not only the pathogenic cells, but also kill normal cells of the host organism, such as cells of the haematopoietic system. Unwanted side effects mentioned anticancer funds emphasize the need to develop new therapies, selective in relation to the population of pathogenic cells, and reduced toxicity to normal cells of the host organism.

Researchers have developed therapeutic methods for destruction of pathogenic cells by targeting these cells cytotoxic compounds. Many of these techniques apply toxins conjugated with antibodies that bind to antigens that are characteristic only for pathogenic cells, or redundantly expressed by them, in an attempt to minimize the delivery of the toxin to normal cells. Using this approach were developed by some immunotoxins consisting of antibodies aimed at any specific the e antigens on the pathogenic cells, where antibodies were associated with toxins, such as ricin, Pseudomonas exotoxin, Diptheria toxin, and tumor necrosis factor. These immunotoxins targeted to pathogenic cells, such as tumor cells bearing specific antigens recognized by antibodies (Olsnes, S., Immunol. Today, 10, pp. 291-295, 1989; Melby, E.L., Cancer Res., 53(8), pp. 1755-1760, 1993; Better, M.D., PCT Publication Number WO 91/07418, published May 30, 1991).

Another approach to the targeting of a population of pathogenic cells, for example, cancer cells or external pathogens, the host organism is to strengthen its own immune response against pathogenic cells in order to avoid the necessity of introducing compounds that can demonstrate independent toxicity to the host. One of these strategies of immunotherapy is the binding of the antibody, for example, a multimeric antibodies obtained by genetic engineering, with the surface of tumor cells, so that the constant region of these antibodies were on the surface of cells, which causes the destruction of tumor cells by various processes, mediated by the immune system (De Vita, V.T.,Biologic Therapy of Cancer, 2d ed., Philadelphia, Lippincott, 1995; Soulillou, J.P., U.S. Patent 5672486). However, these approaches are complicated by problems identifying tumor-specific antigens. Accordingly, the required additional connections and ways to select the main target population of pathogenic cells.

The INVENTION

It was found that a therapeutic agent, diagnostic tools, and visualization tools can be conjugated with other compounds to regulate or modify their behavior, bearsdley, metabolism and/or clearance in vivo. In one of the illustrative embodiments of the present invention described conjugates of compounds that include a hydrophilic spacer linker. In one aspect of the described conjugates of compounds that include both hydrophilic spacer linker, and a targeting ligand. Illustrative examples of such conjugates are described in the application of the compounds of the following formula

B-L-A,

where B is a receptor-binding ligand that binds to a target-cell receptor, L is a linker that includes one or more hydrophilic spacer linker, and A represents a diagnostic or therapeutic agent, or the renderer, which shall be delivered to the cell.

In another embodiment, in the application described not bind to the receptors is not aimed compounds of the following formula:

L-A,

where L represents a linker that includes one or more hydrophilic spacer linker, and A is A diagnostic or therapeutic environments is the your, or renderer. In one embodiment, the linker L does not include the split fragment, in another embodiment, the linker L includes split fragment. In another embodiment, at least one of the hydrophilic spacer linker is formed by carbohydrates or includes at least one carbohydrate residue. In one embodiment, the carbohydrates form part of the chain of the linker connecting the fragments B and A. In another embodiment, the carbohydrate form part of the side chain attached to the chain linker linking the fragments B and A.

Have in mind that in each of the above embodiments to the linkers described in this application can be attached to more than one receptor-binding ligand B. Further, it is assumed that described in this application, the linkers can be attached to more than one remedy A. Such multiligand or multiplikatsionnye conjugates are also described in this application, where the linkers include hydrophilic fragments.

In another embodiment, the application describes compounds that reduce the uptake of the conjugates in the liver and reduce the possibility of passing through the liver. In one aspect, these compounds preferably are displayed in the activity of the kidneys, not the liver.

Means designated with the letter a include therapeutic drugs, di is the Gnostic means, renderer and any other compounds that are desirable or mainly delivered to the cells by targeting cell receptors. Illustrative examples of drugs include cytotoxic tools, anti-inflammatories, etc. Illustrative diagnostic tools and visualization tools include PET renderer, fluorescent renderer, radio, tools, forming complexes with radio and others.

In various embodiments, the implementation of the compounds, compositions and methods described in this application, the cells, which can make targeting therapeutic, diagnostic and/or visualization tools And include a wide range of cells, for example, not limited to the above, cancer cells, bacterial cells, tumor cells, monocytes, activated macrophages, precursor cells, such as endothelial precursor cells, other inflammatory cells, atherosclerotic plaques, and infectious agents, and others. The target cells is carried out by an appropriate choice of ligand B, binding of cellular receptors. Assume that selective or specific targeting of cells in vivo may be due to the choice of the receptor, which is the advantage of the but or redundantly expressed by the target cell. As an illustration, the target cell is predominantly or excessively expresses the receptor for the vitamin, for example, the folate receptor.

In another embodiment described in this application conjugates are included in the pharmaceutical composition in amounts effective for the treatment of diseases and painful conditions associated with pathogenic populations of cells.

In another embodiment described in this application conjugates containing pharmaceutical compositions are used in methods of treating diseases and painful conditions associated with pathogenic populations of cells.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the relative affinity for binding EC234, DPM for folic acid (●) and EC0234 (■).

Figure 2 shows the activity EC0258 against KB cells (2 h pulse/72 h chase) for EC258 (●) and EC258 + excess folic acid (○).

On figa shows the effect EC0234 and EC0246 on M109 tumors in mice, control animals that were not injected drugs (■), standard EC145 (TIW (three times per week) 3 µmol/kg, 3 weeks) (●), EC0234 (TIW 3 µmol/kg, 3 weeks) (▼) and EC0246 (TIW 3 µmol/kg, 3 weeks) (▲).

On FIGU shows the effect EC0234 and EC0246 on the change of body weight in percent of the control animals that were not injected drugs (■), standard EC145 (TIW 3 µmol/kg, 3 weeks) (●), EC0234 (TIW 3 µmol/kg, 3 weeks) (▼) and EC0246 (TIW 3 µmol/kg, 3 weeks) (▲); Dan is haunted show during the injection was not observed obvious toxicity.

On figa shows the effect on tumor volume KB in mice EC0396 (▼), EC145 (▲) and PBS control (■) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration).

On FIGU shows the impact on the change in body weight in percent EC0396 (▼), EC145 (▲) and PBS control (■) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration); the data show that during the introduction of drugs was not observed obvious toxicity.

On figa shows the effect on tumor volume KB EC0400 (●), EC145 (▲) and PBS control (■) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration).

On FIGU shows the impact on the change in body weight in percent EC0400 (●), EC145 (▲) and PBS control (■) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration); the data show that during the introduction of drugs was not observed obvious toxicity.

In Fig. 6A shows the effect on tumor volume EC0429 (and EC145 (▲) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration) compared with the control animals, which do not CC the Dili drugs (●), for tumors M in Balb/c mice.

In Fig. 6B shows the effect on the change of body weight in percent EC0429 (and EC145 (▲) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration) compared to the control animals that were not injected drugs (●); the data show that during the introduction of drugs was not observed obvious toxicity.

In Fig. 7A shows the effect on tumor volume EC0434 (∆) and EC145 (▲) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration) compared to the control animals that were not injected drugs (●), s.c. tumors M in Balb/c mice.

In Fig. 7B shows the effect on the change of body weight in percent EC0434 (∆) and EC145 (▲) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration) compared to the control animals that were not injected drugs (●); the data show that during the introduction of drugs was not observed obvious toxicity.

On figa shows the effect on tumor volume EC0305(●), EC0436(▼) and PBS control (■) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration) for s.c. the M109 tumors in mice Bulb/c.

On FIGU shows the impact on change is the weight percentage EC0305(●), EC0436(▼) and PBS control (■) at a dose of 2 μmol/kg TIW for two weeks (the vertical line indicates the last day of administration); the data show that during the introduction of drugs was not observed obvious toxicity.

Figure 9 shows the percentage change of body weight of Balb/c mice with s.c. the M109 tumor, which intravenously three times a week for one week were administered PBS (control animals not treated with medications) (●), EC0436 (TIW 2 µmol/kg) (▲), EC0436 (TIW 2.5 µmol/kg) (▼), EC0436 (TIW 3 µmol/kg) (■), EC0305 (TIW 2 µmol/kg) (∆), EC0305 (TIW 2.5 µmol/kg) (and EC0305 (TIW 3 µmol/kg) (□).

On figa shows the impact on s.c. (subcutaneous) tumors KB in micenu/nuEC0565 with the introduction of 3 µmol/kg (qdx5 for two weeks) (●), compared to control animals treated with PBS (■). From these data we can determine the value of Log Cell Kill (LCK), equal to 1.2 (values greater than approximately 0.7 indicate active anticancer compound).

On FIGU shows the impact on weight change in percent AS with the introduction of 3 µmol/kg (qdx5 for two weeks) (●), compared to control animals treated with PBS (■); data show that during the introduction of drugs was not observed obvious toxicity.

Figure 11 shows the total excretion DAVLBH with bile from various conjugates DAVLBH when abolution i.v. the introduction of 2 µmol/kg in the hepatobiliary excretion in the study of the bile ducts kanalirovannykh rats. The measured percentage of the total dose in the bile was for EC145=8,7% (●), EC409=7,9% (♦), EC0429=8,6% (Œ), EC0434=2,8% (▼). In addition, EC145 showed AUC=1092 (●); the last sampling time was 139 min; and EC0434 showed AUC=260 (œ); at all time points 120, 135 and 360 minutes the results were below limit of quantitative detection, ie <0.65 micron.

On Fig shows the effect of the spacers on the basis of the ribose on the excretion of bile, as well as the introduction of the linker derivatives of greater length. Numbers above the rectangles of the histogram correspond to the number of hydrophilic spacers in the linker.

On Fig shown that EC0565 causes a dose-dependent inhibition of RPS6 and p70S6K in KB cells (1 h pulse/4 h chase) when using the camera exposure 30 min, where C=control (untreated cells); FAC=control sample folic acid (100 μm).

On Fig shows the cytotoxicity of bortezomib compared with methylthioninium derivative of bortezomib (EC0501). IC50bortezomib, 20 nm (●), EC0501, 240 nm (œ).

On Fig shown that hydrophilic spacers of linkers make possible specific activity of conjugates of mono - and bis-thio-Velcade-folate against RAW264.7 cells. Cell survival after 5 h pulse and 72 h of chase (MTT); bortezomib (■), EC0501 (□), EC0522(▲), EC0522 plus excess folic acid ().

On Fig shows the survival rate of cells (5 h pulse/72 h chase) (Templ) after processing EC0595 (13 nm IC50) (▼), EC0595 plus excess folic acid (), bortezomib (■), EC0525 (46 nm, IC50) (●), EC0525 plus excess folic acid (○).

On Fig shows the survival rate of cells after 24 h incubation (Templ) with bortezomib (■), EC0587 (●), EC0587 plus excess folic acid (○).

On Fig shows inhibition of LPS-stimulated activity calpaine in cells RAW 264.7 (5 h pulse/24 h chase), LPS 100 ng/ml, the reaction time is 30 m 20S proteasome/substrate under the action of bortezomib (■), EC0522 (▼), EC0522 plus excess folic acid (), EC0525 (●), EC0525 plus excess folic acid (○), EC0595 (♦), EC0595 plus excess folic acid (□); IC50is approximately 30 nm for EC0595 and EC0525.

On Fig shown activity against RAW cells (5 h pulse/72 h chase) after treatment with α-amantina (■), EC0592 (IC503,7 nm) (●), EC0592 plus excess folic acid (○).

DETAILED description of the INVENTION

In the present application is described conjugates for drug delivery, comprising the receptor-binding ligand (B), the polyvalent linker (L), including the subsequent one or more hydrophilic spacers, as well as diagnostic or therapeutic agent, or renderer (A), which shall be delivered to the cell. The binding ligand (B) covalently attached to the polyvalent linker (L), and diagnostic or therapeutic agent, or a renderer (A) or its analogue or derivative is also covalently attached to the polyvalent linker (L). It should be understood that the diagnostic or therapeutic agent, or a renderer (A) includes its analogs or derivatives, which are attached to the linker (L). The polyvalent linker (L) comprises one or more spacer linker and/or cleaved fragments, and combinations thereof in any order. In one embodiment, the split fragments of the linker and optional spacer linker covalently linked to each other to form the linker. In another embodiment, a biodegradable linker is directly attached to the tool (A), its analogue or derivative. In another embodiment, a biodegradable linker is directly attached to the binding ligand. In another embodiment, or a ligand-binding, or agent (A), or an analogue or derivative, or both of these structural units attached to the cleaved fragment linker through one or more spacer linker. In another embodiment, and a ligand-binding, and the agent (A) or anal is g or a derivative thereof, attached to split the linkers, each of which can be directly associated with other biodegradable linker, or covalently connected to it through one or more spacer linker.

From the preceding description it should be understood that the location of the binding ligand and the means (A), its analogue or derivative, and various split and optional spacer linker may vary within wide limits. In one aspect of the binding ligand and the agent (A) or its analogue or derivative, and various split and optional spacers of linkers attached to each other via heteroatoms, such as nitrogen, oxygen, sulfur, phosphorus, silicon, etc. In various embodiments, the heteroatoms, with the exception of oxygen, can have different oxidation States, for example, N(OH), S(O), S(O)2, P(O)P(O)2, P(O)3etc. In other embodiments, the heteroatoms can be grouped with the formation of divalent radicals, such as, for example, in the remains of hydroxylamines, hydrazines, hydrazones, sulfonates, Phosphinates, phosphonates and the like, including radicals of the formulas -(other1Other2)-, -SO-, -SO2)- and-N(R3)O-, where each of the fragments of R1, R2and R3independently selected from hydrogen, alkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl, replaced the on heteroaryl and alkoxyalkyl. In another embodiment, to the polyvalent linker attached to more than one binding ligand. In another embodiment, to the polyvalent linker attached to more than one tool (A). In another embodiment, to the polyvalent linker attached to more than one binding ligand, and more than one tool (A).

In one embodiment, the implementation of the receptor-binding ligand is a ligand that binds the receptor for the vitamin, such as vitamin or analogue or derivative, capable of contacting the receptor of vitamin. In another embodiment, the binding ligand is a vitamin, or analog or derivative that is attached to split the linker that is attached to the drug via a linker formed by one or more spacer linker, and/or cleaved fragments of the linker, and/or hydrophilic spacer linker. In one embodiment, and drug, vitamin, or analog or derivative can be attached to the spacers of the linkers, where the above-mentioned spacers of linkers are connected to each other through one or more cleaved fragments of the linker. In addition, as a drug, or vitamin, or analog or derivative, can be attached to one or more split the linkers, where the degradable linkers connect the us with each other or through a spacer linker. Each of these radicals can be connected with others through existing or additional heteroatoms included in a ligand-binding agent or A biodegradable, hydrophilic spacer, or an additional spacer linker.

The binding site binding ligand (B) may include the receptors for any binding ligand (B), its derivative or analog, capable of specific contact with the receptor, where the receptor or other protein is expressed exclusively, redundantly expressed or is expressed predominantly by a population of pathogenic cells. Presented on the surface of a protein that is expressed exclusively, redundantly expressed or is expressed predominantly pathogenic cells, as a rule, is a receptor that is either not present in non-pathogenic cells or present in lower concentrations, allowing selective destruction, labeling or diagnosing pathogenic cells. Conjugates for drug delivery containing a ligand-binding, may have the ability with high affinity to bind to receptor sites on cancer cells or other types of pathogenic cells. Binding with high affinity may be inherent in the most binding ligand or affinity for binding can be improved is the label of chemically modified ligand (for example, analogue or derivative of vitamin).

Described in the present invention conjugates for drug delivery containing a ligand-binding, can be obtained, for example, from a wide range of vitamins or receptor-binding analogs/derivatives of vitamins, linkers and medicines. Described in the present invention conjugates for drug delivery containing a binding ligand capable of selective targeting a population of pathogenic cells in the body of an animal host due to predominant expression on the surface of pathogenic cells receptor binding ligand, for example, vitamin available for binding with the ligand. Illustrative vitamin fragments that can be used as the binding ligand (B), include carnitine, Inositol, lipoic acid, pyridoxal, ascorbic acid, nicotinic acid, Pantothenic acid, folic acid, Riboflavin, thiamine, Biotin, vitamin B12other water-soluble vitamins, B vitamins and fat-soluble vitamins A, D, E and K. These vitamins, and their receptor-binding analogs and derivatives are illustrative target fragments, which can be sewn with drug using bivalent linker (L) to obtain the described in this application to Nyugati for delivery of medicines containing a binding ligand (B). It is implied that the term "vitamin" includes analogs and/or derivatives of vitamins, unless otherwise noted. To illustrate, vitamins, analogues of vitamins and derivatives of vitamins are peroia acid, which is a derivative of folate, Biotin analogues such as biocytin, Biotin sulfoxide, Exibition and other compounds that bind to the receptor, Biotin, etc. Should be understood that the term "analogs or derivatives of vitamins" in the present description refers to vitamins, which include heteroatom through which the analogs or derivatives of vitamins are covalently bound to the bivalent linker (L).

Illustrative examples of vitamin fragments include folic acid, Biotin, Riboflavin, thiamine, vitamin B12and the receptor-binding analogs and derivatives of these vitamins molecules, as well as other related receptor-binding molecules of vitamins.

In another embodiment, the cellular receptor is the receptor for folate, and target B ligand is a ligand that binds to the folate receptor. In another embodiment, B is a folate, for example, folic acid, or an analogue or derivative of folic acid, which binds to the receptors of folic acid. It should be understood that in this C the turnout, the term "folate" is used in individual and General sense to refer to most of folic acid and/or analogs and derivatives of folic acid, able to contact the folate receptor. In another embodiment, B represents a connection that is capable of selectively or specific contact folate receptor, for example, an antibody.

Illustrative embodiments of analogues and/or derivatives of folate include folinievu acid, penopoliuretanovuju acid and pteridine linking folate receptor, for example, tetrahydrofurane, dihydrofolate, tetrahydrofolate and their deaza and dideaza counterparts. The terms "death" and "dideaza" analogs are known in the art to analogues in which one or two atoms of nitrogen in the natural structure of folic acid, its analogue or derivative, substituted on the carbon atoms. For example, deaza analogues include 1-deaza, 3-deaza, 5-deaza, 8-deaza and 10-deaza analogs of folate. Dideaza analogs include, for example, 1.5-dideaza, 5,10-dideaza, 8,10-dideaza and 5.8-dideaza analogs of folate. Other folate, applicable as complexing ligands include receptor-binding analogs of folate, aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-diaminophenoxyethanol, deaza analogues such as 1-deathmetal or 3-deathmetal and 3',5'-dichloro-4-amino-4-deoxy-N10-methylethanolamine acid (dichloromethotrexate). Mentioned analogues and/or derivatives of folic acid is usually referred to as the foul is Tami, that reflects their ability to bind to folate receptors, and these ligands in the case of formation of conjugates with exogenous molecules are effective in improving transmembrane transport, for example, by means described in the present application is mediated by folate endocytosis. Other suitable binding ligands capable of binding to the folate receptor for the initiation mediated by receptors endocytotic transport complex include antibodies to the folate receptor. Exogenous molecule in complex with antibody to folate receptor is used to run the transmembrane transport of the complex.

Additional analogs of folic acid, which bind to receptors of folic acid, as described in the published patent applications U.S. No. 2005/0227985 and 2004/0242582, the contents of which are incorporated into the present application by reference. Illustrative such analogs of folate have the following General formula:

where each of the substituents X and Y are independently selected from the group consisting of halogen, R2, OR2, SR3and NR4R5;

U, V and W represent a bivalent fragments, each of which is independently selected from the group consisting of -(R6a)C=, -N=, -(R6a)C(R7a)- and-N(R4a)-; fragment Q is selected from the group consisting of C and CH; FR is gment T selected from the group consisting of S, O, N and-C=C-;

each of the fragments of A1and A2independently selected from the group consisting of oxygen, sulfur, -C(Z), -C(Z)O-, -OC(Z)-, -N(R4b)-, -C(Z)N(R4b)-, -N(R4b)C(Z)-, -OC(Z)N(R4b)-, -N(R4b)C(Z)O-, -N(R4b)C(Z)N(R5b)-, -S(O)-, -S(O)2-, -N(R4a)S(O)2-, -C(R6b)(R7b)-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C12alkylene and C1-C12alkylene, where Z represents oxygen or sulfur;

Deputy R1selected from the group consisting of hydrogen, halogen, C1-C12the alkyl and C1-C12alkoxy; each of the substituents R2,R3,R4,R4a,R4b,R5,R5b,R6band R7bindependently selected from the group consisting of hydrogen, halogen, C1-C12of alkyl, C1-C12alkoxy, C1-C12alkanoyl, C1-C12alkenyl, C1-C12the quinil, (C1-C12alkoxy)carbonyl, and (C1-C12alkylamino)carbonyl;

each of the substituents R6and R7independently selected from the group consisting of hydrogen, halogen, C1-C12the alkyl and C1-C12alkoxy; or R6and R7together form a carbonyl group; each of the substituents R6aand R7aindependently selected from the group consisting of hydrogen, halogen, C1-C12the alkyl and C1-C1 alkoxy; or R6aand R7atogether form a carbonyl group;

L represents a divalent group that is described in this application; and

each of the coefficients n, p, r, s and t independently is 0 or 1.

It should be understood that in the present description, the term "folate" refers directly to folic acid, used in obtaining the conjugate, or, alternatively, analogs or derivatives of folate, which are able to bind with the receptors of folate or folic acid.

In one aspect, these analogs of folate, if s is 1, t is a 0, and if s is 0, t is 1. In another aspect, in the above analogs of folate both coefficients n and r equal to 1, and the linker Larepresents a natural amino acid that covalently associated with A2and its alpha-amino group through an amide bond. Illustrative examples of amino acids include aspartic acid, glutamic acid, lysine, cysteine, etc.

Vitamin C may consist of folate, which includes nitrogen, and in this embodiment, a spacer linker can be alkilenkarbonatov, cycloalkylcarbonyl, carbonylchloride, 1-alkylresorcinol-3-yl, 1-(carbonylethyl)succinimide-3-yl, where each of the spacer linker optionally substituted Deputy X1, and the spacer linker is points the user to the nitrogen atom of folate with the formation of imide or alkylamide. In this embodiment, the Deputy X1can be an alkyl, hydroxyalkyl, amino, aminoalkyl, acylaminoalkyl, dialkylaminoalkyl, sulfgidrilny, alkylthiol, aryl, substituted aryl, arylalkyl, substituted arylalkyl, carboxy, carboxylic, guanidinate, R4is carbonyl, R5-carbonylethyl, R6-acylamino and R7-acylaminoalkyl, where each of the substituents R4and R5independently selected from amino acids, derivatives of amino acids and peptides, and where each of the substituents R6and R7independently selected from amino acids, derivatives of amino acids and peptides.

Illustrative embodiments of analogues and/or derivatives of vitamins also include analogs and derivatives of Biotin, for example, biocytin, bioconcentrated, Exibition, as well as other compounds that bind to the receptor, Biotin, etc. it is Assumed that analogues and derivatives other vitamins that are described in this application, are also addressed in this invention. In one embodiment, the implementation of vitamins, which can be used as the binding ligand (B) conjugates for drug delivery according to the present invention include ligands that bind to receptors vitamins that are specific expressed on activated macrophages, for example, the folate receptors, which bind folate or their analogues, or derivatives described in this application.

In addition to the described in the application of vitamins, it is assumed that other binding ligands can be sewn with drugs and linkers described and discussed in the present invention, with the formation of the conjugates bind to the ligand-linker-drug that can facilitate the delivery of drugs to the desired goal. These other binding ligands, in addition to the above vitamins and their analogues and derivatives, may be used for formation of conjugates for drug delivery capable contact with the target cells. In General, any binding ligand (B) receptor on the cell surface can be successfully applied as a targeting ligand to which can be attached conjugate linker-drug.

Illustrative examples of other ligands described in this application include peptide ligands identified by screening libraries, peptides, demonstrating the specificity against tumor cells, aptamers exhibiting specificity against tumor cells, carbohydrates, showing specificity against tumor cells, monoclonal or polyclonal antibodies exhibiting specificity against the AI of tumor cells, antibody fragments Fab or scFv (i.e. single-chain variable region), for example, Fab fragments of antibodies aimed at EphA2 or other proteins that have been specifically expressed or are only on metastatic cancer cells, small organic molecules derived from combinatorial libraries, growth factors such as EGF, FGF, insulin and insulin-like growth factors and homologous polypeptides, somatostatin and its analogs, transferrin, lipoprotein complexes, salts of bile acids, selectins, steroid hormones, Arg-Gly-Asp containing peptides, retinoids, various galectin, the ligands of the δ-opioid receptor the ligands of the receptor cholecystokin A, ligands specific for receptors of angiotensin AT1 or AT2, the ligands of peroxisome proliferator-activated receptor λ, β laktamovogo antibiotics, such as penicillin, small organic molecules including antimicrobial agents, as well as other molecules that have been specifically bind to receptors predominantly expressed on the surface of tumor cells or infectious microorganisms, antimicrobial and other means, the structure of which is designed to match the binding pocket of a specific receptor based on the crystal structure of the receptor or other cell surface proteins that bind is their ligands tumor antigens or other molecules preferentially expressed on the surface of tumor cells, or fragments of any of these molecules.

Examples of tumor-specific antigens that could serve as binding sites for a ligand-binding conjugate-drug, include extracellular epitopes of the members of the protein family Aminov, such as EphA2. Expression of EphA2 in normal cells is limited to the places of connection of the cells to each other, but in metastatic tumor cells EphA2 distributed throughout the cell surface. Thus, EphA2 on metastatic cells could be available for binding, for example, with the Fab fragment of the antibody conjugated with the drug, while this protein would not be available to bind to the Fab fragment in normal cells, leading to specific binding of the conjugate binds the ligand-drug with metastatic cancer cells.

The linker L comprises one or more hydrophilic spacer linker. In addition, the linker L may include other optional spacer linker and/or biodegradable linkers. It is implied that the linker can include additional spacer linker, if to separate the binding ligand B and medicines A required group of predetermined length. It is also assumed that in some configurations of the linker can be included splitting the linkers. For example, one described in the application options for the implementation of targeted conjugates of ligands can be used to deliver drugs for the treatment of cancer or other diseases involving pathogenic cells. In such scenarios, the implementation mean that after delivery is desirable for the release of drug from the conjugate. For example, in a configuration where the target ligand is folate or its analogue or derivative, conjugate may contact the folate receptor. After binding conjugate is often subjected to the process of endocytosis and enters into the interior of the cell. Cellular mechanisms can biologically destroy conjugate with the release of "payload", i.e. drugs, and folate.

In an alternative configuration aimed conjugate can be used in immunotherapy. In this configuration, the composition of the conjugate, as a rule, is not biodegradable linker. For example, conjugates of folate or other compounds that bind receptors vitamins, and immunogenic, after delivery will contact the appropriate receptor and mark the appropriate box antigenic fragment. In another alternative configuration aimed conjugate can be used in diagnosis. In this configuration, the split linker may be included is not included in the structure of the conjugate. For example, conjugates that include visualization tools, can be delivered to the target cell using a suitable ligand that binds a cellular receptor, such as folate or other compounds that bind the receptor for the vitamin. In one aspect of this conjugate may remain on the cell surface to obtain an image. In another aspect of this conjugate may be subjected to endocytosis in the internal volume of the cell. In the latter situation, the structure of the conjugate can be introduced biodegradable linker.

Accordingly, in other aspects described in this application conjugates B-L-A also include structures corresponding to the following General formula:

B-LS-LH-A

B-LH-LS-A

B-LS-LH-LS-A

B-LR-LH-A

B-LH-LR-A

B-LR-LH-LR-A

B-LS-LR-LH-A

B-LR-LH-LS-A

B-LR-LS-LH-LR-A

B-LH-LS-LH-LR-A

where B, L and A correspond to the data in the application definitions and LRis a split piece of structural linker, LSrepresents a structural fragment (spacer) linker and LHis a hydrophilic structural fragment linker L. it Should be understood that the above formulas are t is like illustrative and that the present invention should include other mutual arrangement of the hydrophilic linker fragments, cleaved fragments of the linker and structural fragments of the linker. In addition, it should be understood that the invention considers the additional conjugates that include a large number of hydrophilic spacer linker, and/or a large number of cleaved fragments of the linker and/or a large number of spacers linker.

Similarly, in other aspects described in the invention, the conjugates of L-A include compounds corresponding to the following General formula:

LS-LH-A

LH-LS-A

LS-LH-LS-A

LR-LH-A

LH-LR-A

LR-LH-LR-A

LS-LR-LH-A

LR-LH-LS-A

LR-LS-LH-LR-A

LH-LS-LH-LR-A

where L and A correspond to the data in the application definitions and LRis a split piece of structural linker, LSrepresents a structural fragment linker and LHis a hydrophilic structural fragment linker L. it Should be understood that the formulas above are only illustrative and that the present invention should include other mutual arrangement of the hydrophilic linker fragments, cleaved fragments of the linker and structural fragments of the linker. In addition, it is necessary to understand what, in the invention considers the additional conjugates that include a large number of hydrophilic spacer linker, and/or a large number of cleaved fragments of the linker and/or a large number of spacers linker.

Assume that the location and/or orientation of various hydrophilic linkers may be linear or branched type, or both types. For example, the hydrophilic structural fragments can form the skeleton of a linker, forming a conjugate of folate and medicines renderer or diagnostic tools. Alternatively, the hydrophilic part of the linker can be hung from or attached to the main chain of atoms connecting the binding ligand B with tool A. In this last structure of the hydrophilic part may be directly next to the main chain of atoms or removed from it.

In another embodiment, the linker is more or less linear and hydrophilic groups are mostly sequentially, forming the conjugate linker, similar to the chain. In this case, the hydrophilic group may form part of or the entire skeleton of a linker in this linear version of the implementation.

In another embodiment, the hydrophilic groups are branching linker. This branched hydrophilic variant of gr is PPI can be associated with the basic skeleton or be away from him. In each of these cases, the linker has a more spherical or cylindrical shape.

In one embodiment, the linker has the form of a brush for washing bottles. In one aspect of the skeleton of a linker formed by the linear sequence of amides and the hydrophilic portion of the linker is formed in parallel spaced razvetvlenii side chains obtained, for example, by the addition of monosaccharides, sulfonates and the like, and their derivatives and analogues.

Assume that the linkers can be neutral or can insulates in certain conditions, for example, physiological conditions, which can occur in vivo. In the case of an ionisable of linkers in the selected conditions, the linkers can deprotonates with the formation of a negative ion, or, alternatively, to protonemata with the formation of a positive ion. Have in mind that may be more than one case of deprotonation or protonation. In addition, it is assumed that the same linker can deprotonate and protonemata with the formation of internal salts or zwitterionic connections.

In another embodiment, the hydrophilic structural fragments of linkers are neutral, i.e. under physiological conditions, these linkers are not exposed to significant protonation or deprotonation. In another embodiment is sushestvennee hydrophilic fragments of linkers can protonemata, acquiring one or more positive charges. It is assumed that the ability to protonation depends on the conditions. In one aspect of these conditions are physiological conditions and the linker is protonated in vivo. In another embodiment, the structural portions include as neutral areas, and areas that can protonemata, acquiring one or more positive charges. In another embodiment, the structural fragments include both areas that can deprotonate, acquiring one or more negative charges, and areas that can protonemata, acquiring one or more negative charges. It is understood that in the latter embodiment can be formed zwitterionic or internal salts.

In one aspect of the linker region, which can deprotonate, acquiring a negative charge include carboxylic acids, for example aspartic acid, glutamic acid and the group of carboxylic acids with longer chains, as well as esters of sulfuric acid, for example, alkalemia esters of sulfuric acid. In another aspect of the field of linkers that can protonemata, acquiring a positive charge include amino groups, for example, polyamidoamine, including Ethylenediamine, Propylenediamine, b is etilendiamina etc. and/or heterocycles, including pyrrolidine, piperidine, piperazines and other amino groups, each of which optionally is substituted. In another embodiment, the linker region, which are neutral, include polyhydroxylated groups, such as sugar, carbohydrates, sugars, inosite, etc. and/or a polyester group, for example, polyoxyalkylene groups, including polyoxyethylene, polyoxypropylene etc.

In one of the embodiments described in this application hydrophilic spacers of linkers consist primarily of carbon, hydrogen and oxygen and have a ratio of carbon/oxygen about 3:1 or less, or about 2:1 or less. In one aspect described in the present application is hydrophilic fragments of linkers include a large number of functional groups of ester. In another aspect described in the application hydrophilic linkers include a large number of hydroxyl functional groups. Illustrative examples of fragments that can be used for the formation of such linkers include polyhydroxylated compounds, such as carbohydrates, polyester compounds, for example, parts of a polyethylene glycol and an acid group such as carboxylic group and alkylene acids. In one embodiment, the structure of the linker may also be included oligoamine and similar spacers.

Illustrative examples of carbohydrate spacers include sharoitida described in this application, which combine features of both peptides, and sugars; glucuronidase, which can be introduced into the structure of the linker via [2+3] Huisgen cyclization, also known as "Click chemistry"; β-Alkylglucoside, for example, 2-diethoxyacetophenone (2-deoxyglucose, 2-deoxyglucosone etc) and β-alkylaminocarbonyl. Illustrative examples of PEG-groups include groups with a length in a specific range, from about 4 to about 20 groups of PEG. Illustrative esters alkylene acid can also be entered directly into the circuit by using "click chemistry". Illustrative oligoamine fragments include fragments of EDTA and DTPA, β-amino acids, etc.

In another embodiment described in the present application is a fragment of the linker includes polyesters, for example, linkers of the following structure:

where m is an integer, in each case independently selected from integers from 1 to about 8; p is an integer selected from the integers from 1 to about 10; and n is an integer, in each case independently selected from the integers from 1 to about 3. In one aspect of m in each case independently denotes an integer from 1 to about 3. In another aspect, n is the distance between the m case is 1. In another aspect of p in each case independently represents an integer from about 4 to about 6. Illustrative, in this application discusses the polyesters of polypropylene, based on the previous description, and they can be included in the conjugates as a hydrophilic spacer linker. In addition to this, we mean that mixed polyesters of polyethylene and polypropylene can be included in the conjugates as hydrophilic spacers of linkers. In addition, the present invention considers the cyclic variations of the above-mentioned polyester compounds, for example those that include tetrahydrofuranyl, 1,3-dioxans, 1,4-dioxans, etc.

In another illustrative embodiment, the hydrophilic spacers of the linkers described in this application, include a significant number of hydroxyl functional groups, for example, are considered linkers that include monosaccharides, oligosaccharides, polysaccharides, etc. Should be understood that polyhydroxyvalerate the spacers of linkers include a large number of groups(CROH)-, in which R represents hydrogen or alkyl.

In another embodiment, the spacers of linkers include one or more of the following fragments:

where R represents H, alkyl, cycloalkyl or arylalkyl; m not only is em an integer from 1 to about 3; n is an integer from 1 to about 5; p is an integer from 1 to about 5; and r is an integer selected from the integers from 1 to about 3. In one aspect of the integer n is 3 or 4. In another aspect, the integer p is 3 or 4. In another aspect, the integer r is equal to 1.

In another embodiment, the spacer linker includes one or more of the following cyclic polyhydroxylated groups:

where n denotes an integer from 2 to about 5, p is an integer from 1 to about 5, and r is an integer from 1 to about 4. In one aspect of the integer n is 3 or 4. In another aspect, the integer p is 3 or 4. In another aspect, the integer r is equal to 2 or 3. Assumes that all stereochemical forms of the depicted fragments of linkers are included in the scope of the present invention. For example, in the above structural formulas fragment can be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, and it retains the spatial location of the lateral hydroxyl and alkyl groups present in these molecules. In addition, it should be understood that the above formulas cover a variety of dezoxidanti. As an illustration, covered by compounds of the following formulas:

where n is less than Il is equal to r, for example, if r is 2 or 3, n is 1 or 2, or 1, 2 or 3, respectively.

In another embodiment, the spacer linker include polyhydroxylated compounds of the following formula:

where each of the coefficients n and r is selected from integers from 1 to about 3. In one aspect, the spacer linker comprises one or more polyhydroxylated compounds of the following formulas:

Assumes that all stereochemical forms of such fragments linkers are included in the scope of the present invention. For example, in the above structural formulas fragment can be derived from ribose, xylose, glucose, mannose, galactose, or other sugars, and it retains the spatial location of the lateral hydroxyl and alkyl groups present in these molecules.

In another configuration described in this application hydrophilic linkers L include polyhydroxylated groups that are at a certain distance from the main chain of the linker. In one embodiment, the implementation of such carbohydrate group or polyhydroxylated group connected with the main circuit triazole group, forming a cross-linked triazole hydrophilic spacer linker. As an illustration of such linkers include fragments of the following formulas:

where the coefficients n, m and r are integers and each of them is in each case independently selected from integers from 1 to about 5. In one illustrative aspects of m in each case independently equal to 2 or 3. In another aspect, r in each case is 1. In another aspect, n in each case is 1. In one embodiment, the group linking polyhydroxyphenols group with the main chain of the linker, is another heteroaryl group, including, but not limited to, pyrrole, pyrazole, 1,2,4-triazole, furan, oxazole, isoxazol, thienyl, thiazole, isothiazol, oxadiazol etc. Similarly, in the invention are considered divalent 6-membered cyclic heteroaryl group. Other options described above are illustrative examples of the hydrophilic structural fragments of linkers include oxyalkylene group, for example, the following formulas:

where the coefficients n and r are integers and each of them is in each case selected from the integers from 1 to about 5; and p represents an integer from 1 to about 4.

In another embodiment, the above-mentioned carbohydrate or polyhydroxylated group linked to the main chain amide groups, forming a hydrophilic spacer linker with amide bonds. As an illustration of such linkers include fragments of the following forms is l:

where n is an integer selected from the integers from 1 to about 3, and m represents an integer selected from the integers from 1 to about 22. In one illustrative aspect, n is 1 or 2. In another illustrative aspect, the coefficient m is selected from integers from about 6 to about 10, for example, equal to 8. In one embodiment, a group that connects polyhydroxyphenols group with the main chain of the linker, is another functional group, including, but not limited to, the group of esters, ureas, carbamates, acylhydrazones etc. Similarly, the invention encompasses cyclic variations. Other options mentioned above illustrative hydrophilic spacer linker include oxyalkylene group, for example, having the following formula:

where the coefficients n and r are integers, and each of them is in each case independently selected from the integers from 1 to about 5; and p represents an integer selected from the integers from 1 to about 4.

In another embodiment, the spacer linker include one or more of the following fragments:

where R represents H, alkyl, cycloalkyl or arylalkyl; the coefficient m is an integer independently selected from integers from 1 to when is Erno 3; n is an integer from 1 to about 6, p is an integer from 1 to about 5, and r is an integer selected from the integers from 1 to about 3. In one embodiment, n is an integer equal to 3 or 4. In another embodiment, p is an integer equal to 3 or 4. In another embodiment, r is an integer equal to 1.

In another embodiment, the structural fragments of linkers include one or more of the following fragments:

where the coefficient m is an integer independently selected from integers from 1 to about 3; n is an integer from 1 to about 6, p is an integer from 1 to about 5; and r is an integer selected from the integers from 1 to about 3. In one embodiment, the integer n is 3 or 4. In another embodiment, the integer p is 3 or 4. In another embodiment, the integer r is equal to 1.

In another embodiment, the structural fragments of linkers include one or more of the following fragments:

where the coefficient m is an integer independently selected from integers from 1 to about 3; n is an integer from 1 to about 6, p is an integer from 1 to about 5, and r is an integer selected from the integers from 1 to about 3. In one embodiment, the integer n is 3 or 4. In another embodiment, celecia p is 3 or 4. In another embodiment, the integer r is equal to 1.

In another embodiment, the hydrophilic spacer linker is a combination of structural fragments of the main chain and razvitsya side of the group presented, for example, by the following formulas:

where the factor n is an integer, in each case independently selected from integers from 0 to 3. Have in mind that the formulas above show the cyclic sugar with the number of atoms in the cycle 4, 5, 6, and even more. In addition, it should be understood that the above formula can be modified to show detoxifer, in which one or more hydroxyl groups present in the formula is replaced by hydrogen, alkyl or amino group. In addition, it should be understood that the above formulas covered by the corresponding carbonyl compounds in which one or more hydroxyl groups are oxidized to the corresponding carbonyl. In addition, in this illustrative embodiment, the pyranose include both carboxyl and amino groups and (a) may be included in the main chain, and (b) can provide synthetic possibilities for branching of the side chains in the varieties of this variant implementation. Any of the available hydroxyl groups can be used for p is soedineniya other chemical fragments, including additional sugars to obtain the corresponding oligosaccharides. In addition, the invention includes other varieties of this variant implementation, including the inclusion of pyranose or other sugars in the main chain via one carbon atom, i.e. with the formation of spirotrichous, a couple genialnyh carbon atoms, and similar structures. For example, one or two end of the linker, or the agent A, or a ligand-binding B can be connected with a fragment of sugar, which is included in the main chain through provisions 1,1; 1,2; 1,3; 1,4; 2,3 or other provisions.

In another embodiment described in this application hydrophilic spacers of linkers include and primarily consist of carbon, hydrogen and nitrogen and have a ratio of carbon/nitrogen, equal to about 3:1 or less, or about 2:1 or less. In one aspect described in the present application is hydrophilic linkers include a significant number amidofunctional groups.

In another embodiment, the spacer linker include one or more amino groups of the following formulas:

where n is an integer, in each case independently selected from the integers from 1 to about 3. In one aspect of the integer n in each case independently represents 1 or 2. In another aspect of the integer n in su is s cases is 1.

In another embodiment, the hydrophilic spacer linker is an ester of sulfuric acid, for example, Elgiloy ether sulfuric acid. As an illustration, the spacer linker is shown by the following formulas:

where n is an integer, in each case independently selected from the integers from 1 to about 3. As an illustration, n in each case independently is 1 or 2.

It is understood that in such linkers that contain a large number of hydroxyl groups, amino groups, carboxyl groups, fragments of sulfuric acid and the like, which comprise free hydrogen atoms associated with heteroatoms, one or more of these free hydrogen atoms can be protected by a protective group suitable, respectively, for hydroxyl, amino and/ or acid groups or, alternatively, can be blocked with the formation of the corresponding prodrugs, the latter is chosen for a particular application, such as prodrugs that release of the original medicinal product in General or specific physiological conditions.

In each of the above illustrative examples of linkers L in some cases also includes an additional spacer linker LSand/or more degradable linkers L . The above-mentioned spacer linker and the split fragments of the linker may also include asymmetric carbon atoms. Next, you should understand that shown in the present description stereochemical configuration is only illustrative and other stereochemical configuration is also included in the scope of the present invention. For example, in one embodiment described in this application conjugates may include corresponding unnatural configuration amino acids, such as

where n denotes an integer from 2 to about 5, p is an integer from 1 to about 5 and r is an integer from 1 to about 4, as described above.

Further, it should be understood that in the above-described embodiments, the implementation of Unallocated provisions, such as the atoms marked with (*)are the places of joining binding ligand (B) or agent (A), the delivery of which is expected to be implemented. In addition, it should be understood that such accession of any of the fragments B and A, or both, can be performed directly or via an intermediate linker. Intermediate linkers include other spacer linker and/or cleaved fragments of the linker. Illustrative examples of additional spacer linker, and a biodegradable linker fragments, which are the conjugates described in n the standing of the application described in the patent application U.S. serial No. 10/765335, the contents of which are incorporated into this description by reference.

In one embodiment, the implementation of the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers. In another embodiment, the hydrophilic spacer linker comprises at least three carbohydrate-containing or polyhydroxystearic linker. In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers, as well as one or more fragments of aspartic acid. In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers and one or more pieces of glutamic acid. In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers, one or more fragments of glutamic acid, one or more fragments of aspartic acid and one or more residues of beta-aminoadenine. In some variants, in each of the above embodiments the hydrophilic spacer linker comprises one or more cysteine residues. The other is the option, in each of the above embodiments the hydrophilic spacer linker includes at least one arginine residue.

In another embodiment, the hydrophilic spacer linker comprises one or more divalent residues of 1,4-piperazine, which are included in the chain of atoms connecting the at least one binding ligand with at least one of the agents (A). In one embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers. In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers and one or more pieces of aspartic acid. In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers and one or more pieces of glutamic acid. In some variants, in each of the above embodiments the hydrophilic spacer linker comprises one or more cysteine residues. In another series of choices, each of the above embodiments the hydrophilic spacer linker includes at least one arginine residue.

In another embodiment, the hydrophilic spacer linker includes one or more Oli is aaminah hydrophilic spacers, for example, but not limited to, AMINOETHYLPIPERAZINE.

In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers connected through a fragment of a triazole. In another embodiment, the hydrophilic spacer linker comprises one or more carbohydrate-containing or polyhydroxystearic of linkers connected via amide fragment. In yet another embodiment, the hydrophilic spacer linker comprises one or more PEG groups, and one or more cysteine residues. In another embodiment, the hydrophilic spacer linker comprises one or more residues derived EDTE.

In another embodiment, an additional spacer linker can be a 1-alkylresorcinol-3-yl, optionally substituted Deputy X1defined below, and the split fragments of the linker can be a methylene, 1-alkoxyalkyl, 1-alkoxyalkyl, 1-alkoxycarbonyl, 1-alkoxycarbonyl, where each of the cleaved fragments linker optionally substituted Deputy X2defined below, and where as a spacer linker, and split the fragment linker associated with another spacer linker to form succinimide-1-ilal the Il acetal or Catala.

Additional spacer linker can be a carbonyl, tonekaboni, alkylen, cycloalkyl, alkylbenzoates, alkilenkarbonatov, cycloalkylcarbonyl, carbonylchloride, 1-alkylresorcinol-3-yl, 1-(carbonylethyl)succinimide-3-yl, alkylsulfonyl, sulfonylated, alkyloxyalkyl, alkylsulfonates, carboniteservice-2H-pyranyl, carbonitridation, 1-(carboniteservice-2H-pyranyl)succinimide-3-yl and 1-(carbonitridation)succinimide-3-yl, where each of these spacers linker optionally substituted Deputy X1that is defined below. In this embodiment, the spacer linker may include additional nitrogen atoms, and the spacer linker can be alkilenkarbonatov, cycloalkylcarbonyl, carbonylchloride, 1-(carbonylethyl)succinimide-3-yl, where each of these spacers linker optionally substituted Deputy X1defined below, and the spacer linker is linked to the nitrogen atom with the formation of the amide. Alternatively, the spacer linker may further include a sulfur atom, and the spacer linker can be alkylen and cycloalkyl, where each of the spacer linker optionally substituted carboxy, and the spacer linker is linked to grey with the formation of thiols. In another embodiment, peiser linker may include sulfur, moreover, the spacer linker can be a 1-alkylresorcinol-3-yl and 1-(carbonylethyl)succinimide-3-yl, and the spacer linker is associated with sulfur to form succinimide-3-itiola.

In embodiments, implementation of the alternative described above, an additional spacer linker may include nitrogen, and the split fragment linker can be a divalent radical, including alkylenediamine-1-yl, carbonylchloride-1-yl, sulfosalicylate-1-yl or sulphonylchloride-1-yl, where each of fissile linkers optionally substituted Deputy X2that is defined below. In this alternative embodiment, the spacer linker can be a carbonyl, thionocarbonate, allencaron.com, cycloalkylcarbonyl, carbonylchloride, 1-(carbonylethyl)succinimide-3-yl, where each of these spacers linker optionally substituted Deputy X1defined below, and where the spacer linker is associated with a biodegradable linker with the formation of the amide of aziridine.

The substituents X1can represent alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, acylaminoalkyl, dialkylaminoalkyl, halogen, halogenated, sulfgidrilny, alkylthiol, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted hetero is the Rila, carboxy, carboxylic, alkylcarboxylic, alkylamino, guanidinate, R4is carbonyl, R5-carbonylethyl, R6-acylamino and R7-acylaminoalkyl, where each of the substituents R4and R5independently selected from amino acids, derivatives of amino acids and peptides, and where each of the substituents R6and R7independently selected from amino acids, derivatives of amino acids and peptides. In this embodiment, the spacer linker may include nitrogen, and the Deputy X1, and the spacer linker, to which they are attached, form a heterocycle.

In another embodiment, biodegradable linker can be a divalent radical, including alkylenediamine-1-yl, alkilenkarbonatov-1-yl, carbonylchloride-1-yl, alkylsulfonamides-1-yl, sulfosalicylate-1-yl, sulphonylchloride-1-yl or alkylsulfonamides-1-yl, where each of fissile linkers optionally substituted Deputy X2that is defined below.

Additional illustrative examples of fissionable linkers include methylene, 1-alkoxyalkyl, 1-alkoxyalkyl, 1-alkoxycarbonyl, 1-alkoxycarbonyl, carbonylations, carbonyl(carboxyethyl)carbonyl, carbonyl(bicarboxylic)carbonyl, gelegenheitsarbeit, alkylen(dealkiller), kilen(alkylaryl), alkylen(diallelic), (dealkiller)aryl, (alkylaryl)aryl, (diallelic)aryl, oxycarbonate, oxycarboxylic, sulfonyloxy, oxysulphate, aminoalkylsilanes, carbonylcontaining, aminocyclohexanol, carbonylcontaining, alkylthio, alkalinity, carbonrally, where each of fissile linkers optionally substituted Deputy X2that is defined below.

In the previous embodiment, degradable linker may include oxygen, and the split linkers can be a methylene, 1-alkoxyalkyl, 1-alkoxyalkyl, 1-alkoxycarbonyl and 1-alkoxycarbonyl, where each of fissile linkers optionally substituted Deputy X2defined below, and the split linker is associated with oxygen, with the formation of the acetal or Catala. Alternatively, split the linker may include oxygen and split the linker can be a methylene, where the methylene is substituted by an optionally substituted aryl, and split the linker is associated with oxygen with the formation of the acetal or Catala. Next, split the linker may include oxygen and can be sulfonylated, where the biodegradable linker is associated with oxygen with the formation of alkylsulfonate.

In another embodiment, implementation is tvline the above-mentioned variant of degradable linker, split the linker may include nitrogen, and split the linker can be aminoalkylsilanes, carbonylcontaining, aminocyclohexanol and carbonylchloride.info, where each of these fissile linkers optionally substituted Deputy X2defined below, and split the linker is linked to the nitrogen with the formation of the hydrazone. In an alternative configuration, the hydrazone can be allerban derivative of carboxylic acid, a derivative of orthoformiate or carbamoyl derived with the formation of various acylhydrazones fissile linkers.

Alternatively, split the linker may include oxygen, and biodegradable linkers can be alkylen(dealkiller), alkylene(alkylaryl), alkylene(diallelic), (dealkiller)aryl, (alkylaryl)aryl and (diallelic)aryl, where each of fissile linkers optionally substituted Deputy X2defined below, and the split linker is associated with oxygen with the formation of silanol.

In the above-described embodiment, degradable linker drug may include a nitrogen atom, and split the linker may include nitrogen, and split the linker can be carbonylation, carbonyl(carboxyethyl)carbonyl, carbonyl(Biscarrosse the Rila)carbonyl and split the linker can be linked to a heteroatom of nitrogen with the formation of amide and in addition, linked to the nitrogen atom of the medicinal product with the formation of the amide.

In the above-described embodiment, biodegradable linker, the drug may include an oxygen atom, a biodegradable linker may include nitrogen, and the split linkers can be carbonylation, carbonyl(carboxyethyl)carbonyl, carbonyl(bicarboxylic)carbonyl and split the linker can form amide and, in addition, contact with the oxygen atom of the medicinal product with the formation of ester.

Deputy X2can represent alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, acylaminoalkyl, dialkylaminoalkyl, halogen, halogenated, sulfgidrilny, alkylthiol, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, carboxy, carboxylic, alkylcarboxylic, alkylamino, guanidinate, R4is carbonyl, R5-carbonylethyl, R6-acylamino and R7-acylaminoalkyl, where each of the substituents R4and R5independently selected from amino acids, derivatives of amino acids and peptides, and where each of the substituents R6and R7independently selected from amino acids, derivatives of amino acids and peptides. In this embodiment, degradable linker can vkluchaiut, and Deputy X2and split the linker can form a heterocycle.

These heterocycles can be pyrrolidinone, piperidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolinone, pyrrolidinone, piperidine, oxazolidinone, isoxazolidinone, thiazolidinone, isothiazolinone and suktinimida.

The tool may include a nitrogen atom, and split the linker can be gelegenheitsarbeit, optionally substituted Deputy X2and split the linker is linked to the nitrogen atom of the medicinal product with the formation of the amide.

The tool A may include an oxygen atom and split the linker can be gelegenheitsarbeit, optionally substituted Deputy X2and split the linker is linked to the oxygen atom of the medicinal product with the formation of ester.

The tool may include A nitrogen atom linked by a double bond, and in this embodiment, degradable linkers can represent alkalinemanganese and 1-(alkalinemanganese)succinimide-3-yl, and split the linker can be linked to the nitrogen atom of the medicinal product with the formation of the hydrazone.

The tool A may include a sulfur atom, and in this embodiment, degradable linkers can not only is th alkylthio, carbonrally, and split the linker can be linked to a sulfur atom of a molecule drugs with the formation of disulfide.

The tool A can be a mitomycin derivative of mitomycin or similar mitomycin and in this embodiment, degradable linkers can represent carbonrally, carboniteservice-2H-pyranyl, carbonitridation, 1-(carboniteservice-2H-pyranyl)succinimide-3-yl and 1-(carbonitridation)succinimide-3-yl, where each of fissile linkers optionally substituted Deputy X2and where aziridinyl fragment of mitomycin associated with a biodegradable linker, forming acylanilides.

A ligand-binding B may consist of folate, which includes the nitrogen atom, and in this embodiment, fragments of linkers can be alkilenkarbonatov, cycloalkylcarbonyl, carbonylchloride, 1-alkylresorcinol-3-yl, 1-(carbonylethyl)succinimide-3-yl, where each of these spacers linker optionally substituted Deputy X1, and the spacer linker is linked to the nitrogen atom of folate with the formation of imide or alkylamide. In this embodiment, the Deputy X1can be an alkyl, hydroxyalkyl, amino, aminoalkyl, acylaminoalkyl, dialkylaminoalkyl, sulfgidrilny, alkylthiol, aryl, substituted the reel, arylalkyl, substituted arylalkyl, carboxy, carboxylic, guanidinate, R4is carbonyl, R5-carbonylethyl, R6-acylamino and R7-acylaminoalkyl, where each of the substituents R4and R5independently selected from amino acids, derivatives of amino acids and peptides, and where each of the substituents R6and R7independently selected from amino acids, derivatives of amino acids and peptides.

The term "cycloalkyl" in the present description refers to a divalent chain consisting of carbon atoms, part of which forms a cycle, as, for example, cycloprop-1,1-Dyilo, cycloprop-1,2-Dyilo, cyclohex-1,4-Dyilo, 3-ethylcyclohexane-1,2-Dyilo, 1-metalenclosed-4-DRS, etc.

The term "heterocycle" in the present description refers to a monovalent chain of carbon atoms and heteroatoms, where the heteroatoms selected from nitrogen, oxygen and sulfur, some of which, including at least one heteroatom, forms a cycle, such as, aziridine, pyrrolidine, oxazolidine, 3-ethoxypyrrolidine, 3-methylpiperazin etc.

The term "aryl" in the present description refers to an aromatic mono - or polycycle consisting of carbon atoms, for example, phenyl, naphthyl, etc. in Addition, the aryl may also include heteroaryl.

The term "heteroaryl" in the present description refers to an aromatic mono - or polycycle consisting of and the Ohm carbon and at least one heteroatom, selected from nitrogen, oxygen and sulfur, for example, pyridinyl, pyrimidinyl, indolyl, benzoxazolyl etc.

The term "optionally substituted" in the present description refers to replacing one or more hydrogen atoms, usually carbon atoms, corresponding to the number of substituents, such as halogen, hydroxy, amino, alkyl - or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro, etc. in Addition, two hydrogen atoms from the same carbon atom, the neighboring carbon atoms or neighboring carbon atoms can be substituted divalent Deputy with the formation of the corresponding cyclic structure.

The term "aminoalkylsilanes" in the present description refers to a bivalent radical containing alkylene corresponding to the above definition, and the nitrogen atom, where the terminal carbon atom of alkylene connected by a double bond to the nitrogen atom, which corresponds to the formula -(CH)=N-, -(CH2)2(CH)=N-, -CH2C(Me)=N -, etc.

The term "amino acid" in the present application refers mainly to aminoalkylsilanes, in which the alkyl radical is optionally substituted, for example, alkyl, hydroxyalkyl, sulfgidrillnaya, aminoalkyl, carboxylation, etc. that include groups corresponding to the natural amino acids, for example, serine, cysteine, methionine, aspartic acid, glut mirovoi acid, etc. It should be understood that such amino acids may be one of the stereoisomers or mixtures of stereoisomers, including racemic mixtures. In addition, the term "amino acid" refers to beta-, gamma - or more amino acids long, for example, the amino acids of the formula:

-N(R)-(CR'R”)q-C(O)-,

where R represents hydrogen, alkyl, acyl or a group suitable for the protection of the nitrogen atom, R' and R” represent hydrogen or Deputy, each of which is in each case selected independently, and q is an integer, e.g. 1, 2, 3, 4 or 5. As an illustration, R' and R” independently are not limited to, hydrogen or the side chain found in natural amino acids, for example, methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxymethyl, guanidinopropionic, etc. and their derivatives and protected derivatives. The above formula includes all stereoisomeric options. For example, amino acids can be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, etc. In another illustrative aspect of the intermediate conjugate for delivery of drugs that bind the receptor vitamin that is described in this application, the drug or its analog or derivative in the cancel alkylthiophenes nucleophile.

It should be understood that the above terms can be combined, forming from them the names of corresponding chemical groups, such as, for example, the term "alkoxyalkyl"related methoxymethyl, ethoxyethyl etc., "halogenoacetyl"related triftormetilfosfinov, 1,2-debtor-2-floret-1-aloxiprin etc., "arylalkyl"related to benzyl, fenetre, α-methylbenzyl etc., and other groups.

The term "derived amino acid" in the present application generally refers to optionally substituted by aminoalkylsilanes, where the amino group and/or carboxylate group optionally substituted, for example, alkyl, carboxylation, alkylamino and the like, or optionally protected. In addition, optionally substituted intermediate divalent alkyl fragment may include additional groups, for example, protective groups etc.

The term "peptide" in the present description refers mainly to the sequences of amino acids and/or analogs and derivatives of amino acids, covalently linked to each other amide bonds.

The term "biodegradable linker" in the present description refers to a linker that includes at least one bond which can be split under physiological conditions (for example, under certain pH, acid, oxidation or under the action of enzymes). Should the imat, these physiological conditions, leading to the breakdown of relationships include standard chemical hydrolysis, which are, for example, at physiological pH values, or as a result of contact with the cell organelle, for example, endosome with lower pH compared to pH of the cytoplasm.

Split link or connection may be present within the circuit split linker and/or with one or two ends of degradable linker. Assume that the ease of destruction split links can be adjusted by the introduction of polyvalent linker L functional groups or fragments, which are able to facilitate the disconnection or to facilitate it, which is also referred to as anchiornis assistance. In addition, it is assumed that the polyvalent linker L may include additional functional groups or fragments, which are able to promote or facilitate additional fragmentation of conjugates of the receptor-binding ligand-agent after the destruction of the communication cleaved the linker. Ease of fracture split links may be adjusted, for example, substitutions at or near the split connection, for example, the introduction of the alpha-chain branching in the neighboring position to the cleaved disulfide bonds, increasing the hydrophobicity of the substituents at silicon, FR is gente, includes a link to a silicon-oxygen, which may be subjected to hydrolysis, the use of homologues of substituents in alkoxygroup, which form an integral part of ketala or acetal, which can be subjected to hydrolysis, etc.

Assume that the split link can connect two adjacent split the atom in the linker and/or connect other linkers or fragments V and/or D, which are described in this application, with either or both ends of the cleaved linker. When split link connects two adjacent split the atom in the linker, after the destruction of the connection split the linker splits into two or more fragments. On the contrary, if the split link is split between the linker and the other fragment, for example, additional heteroatom, an additional spacer linker, other biodegradable linker tool And its analog or derivative or binding ligand B, its analogue or derivative, after the destruction of communication, biodegradable linker is separated from the other fragments.

It is assumed that each of the additional spacer linker and the split fragments of the linker is a divalent. Next, you need to understand that education is the relationship between each of a variety of additional spacers and split by the linker, and is between different additional spacers and fissionable linkers and A and/or B, defined in the present description may be implemented through any atom present in a variety of additional spacers or split the linkers.

In one aspect described in the present application is conjugate for drug delivery, binding the receptor, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment 3-disuccinimidyl-1 jalkiliitemerkki, where methyl optionally substituted alkyl or substituted aryl.

In another aspect, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment 3-disuccinimidyl-1-ilgilerinin, where the carbonyl forms allsidedly fragment with A tool, its analogue or derivative.

In another aspect, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment 1 alkoxyalkanols.

In another aspect, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment alkylaminocarbonyl(decarboxylation)carboxylate.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together with the way the t fragment detailcontroller, where the hydrazide forms an hydrazone with A tool, its analogue or derivative.

In another aspect, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment 3-disuccinimidyl-1-illconsidered, where the hydrazide forms an hydrazone with A tool, its analogue or derivative.

In another aspect, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment 3-dialkylaminoalkyl (disubstituted silyl)hydroxy, where disubstituted silyl substituted alkyl or optionally substituted aryl.

In another aspect, the polyvalent linker includes a large number of additional spacer linker selected from the group consisting of natural amino acids and their stereoisomers.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form the fragment 3-detailsilluminator, where the carbonyl forms a carbonate with A tool, its analogue or derivative.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form the fragment 3-dithioerythritol, where the carbonyl forms a carbonate with A tool, it is a tax or a derivative, and aryl optionally is substituted.

In another aspect, the polyvalent linker includes an additional spacer linker, and a biodegradable linker, which together form the fragment 3-disuccinimidyl-1 iliskilendirilebilen where alkylidene forms an hydrazone with A tool, its analogue or derivative, each alkyl independently selected, and a fragment of oxyalkylene independently substituted by alkyl or optionally substituted aryl.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form the fragment 3-detailgenauigkeit.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form the fragment 3-citibankonline, where the amino forms winelog amide with A tool, its analogue or derivative.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form the fragment 3-citibankonline, where the amino forms winelog amide with A tool, its analogue or derivative, and alkyl represents ethyl.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker and races is epsey linker, which together form a 3-detailsilluminator, where the carbonyl forms a carbamate with the tool And its analogue or derivative.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form a 3-detailsilluminator, where the carbonyl forms a carbamate with the tool And its analogue or derivative, and alkyl represents ethyl.

In another aspect, the polyvalent linker includes a biodegradable linker, an additional spacer linker, and a biodegradable linker, which together form a 3-dithioerythritol, where the carbonyl forms a carbamate or carbamoylation tool And its analogue or derivative.

In another embodiment, the polyvalent linker (L) comprises a disulfide biodegradable linker. In another embodiment, the polyvalent linker (L) comprises at least one degradable linker that is not a disulfide biodegradable linker.

In one aspect of the cleaved fragment and the spacer linker can be located so that after the breakdown of communication in the polyvalent linker released functional groups contributed to the rupture or splitting of additional bonds, which is also called anchiornis assistance splitting or tearing of the. Illustrative embodiments of such a polyvalent linker or its fragment include compounds having the formula:

where X is a heteroatom, such as nitrogen, oxygen, or sulfur, or carbonyl group; n indicates an integer selected from the integers from 0 to 4; figure 2; R is hydrogen or a Deputy, including the Deputy, capable of stabilizing a positive charge inductively or by resonance with aryl cycle, for example, alkoxy and the like, including methoxy; and the symbol (*) indicates the point of connection of additional structural fragment of heteroatom or fissile linkers forming the polyvalent linker, or, on the other hand, the connecting points of the medicinal product or its analog or derivative, or vitamin, or analog or derivative. In one embodiment, the implementation of n=2 and R represents methoxy. It is assumed that other substituents may be present on the aryl cycle, benzyl carbon atom, the fragment alanovoy acid or methylene bridge, including, but not limited to, hydroxy, alkyl, alkoxy, alkylthio, halogen, etc. Splitting with anchiornis assistance may include mechanisms that participate benzylamine intermediate compounds, gasoline temporarily the passed connection cyclization of lactones, oxonium intermediate compounds, beta-elimination, and so Forth meant that in addition to fragmentation, following the disconnection split the linker, the original destruction of communication cleaved the linker may be facilitated by a mechanism animaroo assistance.

Illustrative examples of intermediate compounds useful for obtaining these linkers include

where Xarepresents an electrophilic group, for example, maleimide, vinylsulfonic, activated carboxylic acid derivative and the like, Xbrepresents NH, O or S; and each of the coefficients m and n are independently selected from integers in the range 0-4. In one embodiment, each of the coefficients m and n are independently selected from integers in the range 0-2. Such intermediate compounds can be associated with drugs that bind ligands or other linkers due to the nucleophilic attack on the electrophilic group Xaand/or due to the formation of ethers or derivatives of carboxylic acids. In one embodiment, the implementation of the benzyl hydroxyl group is transformed into the corresponding activated benzyloxycarbonyl compound by the action of phosgene or a synthetic equivalent of phosgene. This version of the linker may be svazas drugs binding ligands or other linkers due to the nucleophilic attack on the activated carbonyl group.

Illustrative mechanisms of splitting the bivalent linkers described in this application include the following mechanisms 1,4 - and 1,6-fragmentation.

where X is an exogenous or endogenous nucleophile, glutathione or biological reducing agent and the like, and either Z or Z' is a vitamin, its analogue or derivative, or a drug, its analogue or derivative, or a vitamin or a drug in combination with other fragments of polyvalent linker. It should be understood that although the above mechanisms of fragmentation depicted in the form of synchronous mechanisms for the final fragmentation of polyvalent linker prior to the final products can be any number of discrete steps. For example, mean that the splitting may also occur under the action of acid catalyzed elimination of a fragment of the carbamate, which may have anhimidae assistance stabilization provided or aryl group at the beta sulfur atom or by a disulfide fragment, as shown in the above examples. In these varieties of this case for splitting the second linker is a fragment of a carbamate. Alternatively, fragmentation can be initiated by nucleophilic attack by a disulfide group, which causes cleavage with the formation of thiolate. This tilt can intramolecular mechanism to replace a fragment or a carboxylic carbamino acid with formation of the corresponding Tallapoosa. In the case of polyvalent linker containing benzyl fragment, after illustrative cleavage of the disulfide bond formed penalties can be further segmented with segment selection carboxylic acid or carbamino acid, by forming a resonance-stabilized intermediate. In any of these cases, the degradability described in the present application is illustrative of polyvalent linkers can be implemented by any mechanism that can match existing chemical, metabolic, physiological or biological conditions.

Other illustrative mechanisms of splitting ties split the linker include splitting with intermediate formation of hydronium ion according to the following mechanism:

where Z is the vitamin, or analog or derivative, or a drug, its analogue or derivative, or fragment, consisting of a vitamin or cartonnage means, in combination with other fragments of polyvalent linker, for example, a fragment of a drug or vitamin that includes one or more spacer linker and/or other fissile linkers. Not limited to any theory, in this embodiment, the acid catalysts which are present for example, in endosomes, can initiate the splitting due to the protonation of urethane groups. In addition, acid-catalyzed elimination of the carbamate leads to the production of CO2and nitrogen-containing fragment attached to Z, and the formation of benzyl cation, which can be occupied by water or any other Lewis base.

Other illustrative linkers include compounds of the formulas:

where X represents NH, CH2or O; R represents hydrogen or a Deputy, including the Deputy, capable of stabilizing a positive charge, inductively or by resonance in the aryl cycle, for example, alkoxy and the like, including methoxy; and the symbol (*) indicates the point of connection of additional fragments, heteroatoms or fissile linkers forming the polyvalent linker, or, in the alternative, the attachment point of the medicinal product, its analog or derivative, or vitamin a, its equivalent or proizvodnog is.

Illustrative splitting mechanisms such polyvalent linkers described in this application include the following mechanisms 1,4 - or 1,6-fragmentation, followed by facilitated anchiornis assistance acylated cleavage fragment Z' in the cyclization with hydrazine powered attack group.

where X is an exogenous or endogenous nucleophile, glutathione or biological reducing agent and the like, and any of the fragments of the Z or Z' is a vitamin, its analogue or derivative, or drug analogue or derivative, or the rest of the vitamin or drug in combination with another part of polyvalent linker. It should be understood that although the above mechanisms of fragmentation depicted in the form of synchronous mechanisms for the implementation of the final fragmentation of polyvalent linker prior to the final products, can be any number of discrete steps. For example, it is assumed that the splitting may also occur by acid catalyzed elimination of a fragment of the carbamate, which can be facilitated anchiornis assistance due to the stabilization provided or aryl group at the beta sulfur atom or by a disulfide fragment, as shown in the you the e examples. In these varieties of this variant implementation of the biodegradable linker is a fragment of a carbamate. Alternatively, fragmentation can be initiated by nucleophilic attack by a disulfide group, which causes cleavage with the formation of thiolate. This tilt can intramolecular replace the fragment carboxylic acid or carbamino acid to form the corresponding talkloop. In the case of polyvalent linker containing benzyl fragment, after illustrative cleavage of the disulfide bond formed penalties can be further segmented with segment selection carboxylic acid or carbamino acid, by forming a resonance-stabilized intermediate. In any of these cases, the degradability described in the present application is illustrative of polyvalent linkers can be implemented by any mechanism that can match existing chemical, metabolic, physiological or biological conditions. Not limited to any theory, in this embodiment, the acid catalyst present, for example, endosomes, can also initiate the splitting due to the protonation of urethane groups. In addition, acid-catalyzed eliminated the e carbamate leads to the production of CO 2and nitrogen-containing fragment attached to Z, and the formation of benzyl cation, which can be occupied by water or other Lewis base, in the same way as has been described above in this application.

In one of the embodiments described in this application polyvalent linkers are compounds having the following formulas:

where n is an integer selected from the integers from 1 to about 4; each of the fragments of Raand Rbindependently selected from the group consisting of hydrogen and alkyl, including lower alkyl, for example, C1-C4of alkyl, which optionally is branched; or Raand Rbtogether with the carbon atom to which they are attached, form carbocycle; R represents an optionally substituted alkyl group, optionally substituted acyl group or a group suitable for protecting a nitrogen atom; and the symbol (*) denotes the attachment point of the medicinal product, vitamin, visualization tools, diagnostic tools, other polyvalent linkers or other parts of the conjugate.

In another embodiment, the polyvalent linkers described in this application include compounds of the following formulas

the de m is an integer, selected from the integers from 1 to about 4; R represents an optionally substituted alkyl group, optionally substituted acyl group, or a suitable protective group for the nitrogen atom; and the symbol (*) denotes the attachment point of the medicinal product, vitamin, visualization tools, diagnostic tools, other polyvalent linkers or other parts of the conjugate.

In another embodiment described in this application polyvalent linkers include compounds of the following formulas

where m is an integer selected from the integers from 1 to about 4; R represents an optionally substituted alkyl group, optionally substituted acyl group, or a suitable protective group for the nitrogen atom; and the symbol (*) denotes the attachment point of the medicinal product, vitamin, visualization tools, diagnostic tools, other polyvalent linkers or other parts of the conjugate.

Another illustrative mechanism includes such an arrangement cleaved fragment linker and spacer linker that after the breakdown of communication in the polyvalent linker released functional group chemically promotes rupture or splitting of additional bonds, which is also called anchiornis the assistance splitting or tearing. Illustrative version of the exercise of such polyvalent linker or part includes compounds having the formula:

where X is a heteroatom, for example nitrogen, oxygen or sulfur, n means an integer selected from 0, 1, 2, and 3, R is a hydrogen or a Deputy, including the Deputy, capable of stabilizing a positive charge inductively or by resonance involving aryl cycle, for example, alkoxy and the like, and any of the symbols Z or Z' means the vitamin, its analogue or derivative, or a drug, its analogue or derivative, or the rest of the vitamin or medication combined with the other parts of the polyvalent linker. Implies that other substituents may be present at the aryl cycle, benzyl carbon atom, the nitrogen atom of the carbamate, fragment alanovoy acid or methylene bridge, including but not limited to the above, hydroxy, alkyl, alkoxy, alkylthio, halogen, etc. promoting the cleavage may include mechanisms are involved in benzylamine intermediate compounds, petrol intermediate compounds, cyclization of lactones, oxonium intermediate compounds, beta-elimination, and so Forth, it is understood that in addition to fragmentation, following the destruction with the ides split in the linker, the original breakdown of the bond split in the linker can be facilitated mechanism comprising anhimidae assistance.

In this embodiment, is capable of cyclization hydroxyalkanoate acid facilitates the splitting of the methylene bridge due to the formation of, for example, oxonium ion, and facilitates the splitting of the other links or subsequent fragmentation after disconnection split the linker. On the other hand, acid catalyzed and is carried out using ion hydronium cleavage of the methylene bridge can cause a number of consecutive slices mentioned illustrative of polyvalent linker or its fragment. Alternatively, acid-catalyzed hydrolysis of the carbamate can facilitate beta-elimination hydroxyalkanoate acid, which is capable of cyclization, and facilitates the splitting of the methylene bridge, for example, the formation of oxonium ion. Implies that other chemical mechanisms of rupture or splitting of ties in metabolic, physiological or cellular conditions described in this application can initiate such a series of slices.

Another illustrative variant implementation of the linkers described in this application, includes biodegradable linkers, which are destroyed in these in savtuslly, through chemical mechanism, including beta-elimination. In one aspect of such degradable linkers include beta-thio, beta-hydroxy and beta-amino substituted carboxylic acids and their derivatives, e.g. esters, amides, carbonates, carbamates and urea. In another aspect of such degradable linkers include 2 - and 4-tierraviva esters, carbamates and carbonates.

In another embodiment, the polyvalent linker includes additional spacers of linkers and biodegradable linkers, which are connected with the formation of polyvalent 3-disuccinimidyl-1-jalkiliitemerkki shown by the following formula:

where n is an integer from 1 to 6; an alkyl group optionally is substituted, and methyl optionally replaced by alkyl or optionally substituted aryl group, each of these substituents represented independently selected the group R. the Symbol (*) indicates the point of attachment of the fragment of polyvalent linker to other parts of the conjugate described in this application.

In another embodiment, the polyvalent linker includes an additional spacer linker, and a degradable linkers, United in such a way that they form a polyvalent 3-disuccinimidyl-1-iliskiler niloy group, shown by the following formula:

where n is an integer from 1 to 6, and the alkyl group optionally is substituted. The symbols (*) indicate the point of attachment of the fragment of polyvalent linker to other parts of the conjugate described in this application. In another embodiment, the polyvalent linker includes structural fragments and biodegradable linkers, which are connected so as to form a polyvalent 3-dialkylaminoalkyl(disubstituted silyl)oxygraph where disubstituted silyl substituted by alkyl and/or optionally substituted aryl group.

In another embodiment, the polyvalent linker includes an additional spacer linker, and a degradable linkers, United so as to form a polyvalent group detailcontroller or polyvalent group 3-disuccinimidyl-1-illconsidered, which are displayed by the following formulas:

where n means an integer from 1 to 6, the alkyl group is optionally substituted and the hydrazide forms an hydrazone with (B), (D) or other part of the polyvalent linker (L). The symbols (*) indicate the point of attachment of the fragment of polyvalent linker to other parts of the conjugate described in the present is her request.

In another embodiment, the polyvalent linker includes an additional spacer linker, and a degradable linkers, United so as to form a polyvalent group 3-disuccinimidyl-1-iliskilendirilmelidir, represented by the following formula:

where the factor of n in each case independently selected from integers from 1 to 6, each alkyl group is independently selected and optionally substituted, e.g. by alkyl and optionally substituted aryl, and where alkylidene forms an hydrazone with (B), (D) or other part of the polyvalent linker (L). The symbols (*) indicate the point of attachment of the fragment of polyvalent linker to other parts of the conjugate described in this application.

Other illustrative additional spacer linker include alkylene--amino--alkilenkarbonatov, alkylene--tio--carbonylcontaining-3-yl and the like, which further shown by the following formulas:

where integers x and y is equal to 1, 2, 3, 4 or 5.

In another illustrative embodiment, the linker includes one or more amino acids. In one embodiment, the linker comprises one amino acid residue. In another embodiment, the linker includes a peptide containing from 2 to about 50, from about 2 to about 30, or from 2 to about 20 amino acid residues is so In another embodiment, the linker includes a peptide containing from about 4 to about 8 amino acids. These amino acids as an illustration, choose from natural amino acids and their stereoisomers. The mentioned amino acids may also be any other amino acid, for example, any amino acid having the following General formula:

-N(R)-(CR'R”)q-C(O)-,

where R represents hydrogen, alkyl, acyl, or a suitable protective group for the nitrogen atom, R' and R” represent hydrogen or Deputy, each of which is in each case selected independently, and the coefficient q is an integer such as 1, 2, 3, 4 or 5. Illustrative of R' and R” independently are not limited to, hydrogen or the group, which is the side chains of natural amino acids, such as methyl, benzyl, hydroxymethyl, thiomethyl, carboxyl, carboxymethyl, guanidinopropionic, etc. and their derivatives and protected derivatives. The above formula includes all stereoisomeric options. For example, the amino acid may be selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, threonine, etc. In one embodiment, biodegradable linker comprises at least 2 amino acids selected from asparagine, aspartic acid, cysteine, glutamic the acid, lysine, glutamine, arginine, serine, ornithine, and threonine. In another embodiment, a biodegradable linker comprises from 2 to about 5 amino acids selected from asparagine, aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine, serine, ornithine, and threonine. In another embodiment, a biodegradable linker includes the Tripeptide, tetrapeptide, Pentapeptide or Hexapeptide consisting of amino acid residues selected from aspartic acid, cysteine, glutamic acid, lysine, arginine, and ornithine, as well as their combinations.

In another illustrative aspect, the intermediate conjugate for drug delivery, linking the receptor for the vitamin, described in the present invention, the drug, its analogue or derivative includes alkylthiophenes nucleophile.

Additional linkers are described in the following tables, where the atoms marked with this symbol (*)is the point of connection of additional spacer linker or a biodegradable linker, medicines and/or ligand binding.

Describes the following illustrative structural fragments of linkers.

Describes the following illustrative biodegradable linkers

On razumeetsa, such hydrophilic linkers can change the stability, metabolism and distribution conjugates in tissues, particularly in comparison with other forms conjugates, such as, for example, forms-based peptides described in application for U.S. patent No. 10/765336. For example, it is assumed that in some situations the interaction of carbohydrate-protein weaker than the interaction of the peptide-protein. Thus, it is assumed that in various embodiments, the implementation described in this invention, the conjugates may lead to lower serum protein binding. These and other physico-chemical differences between the conjugates described in this application, and other, previously described, may include improved targeting cellular targets and improved, i.e. more selective or differentially selective, profiles bearsdley. Increased cytotoxicity may be a natural consequence of the reduced protein binding serum or better or differentiated bearsdley (i.e. fewer medicines aimlessly spent in non-target tissues). This is especially true in the case of the use of hydrophilic, but neutral spacers. Not limited to any theory, I also believe that the hydrophilic spacer linker, described in this application, could the t to reduce toxicity, which could arise, at least partly, due to interactions based on nonspecific binding.

In an alternative embodiment, the drug is associated with the hydrophilic spacer linker directly or through an intermediate fragments to achieve such goals as reducing excretion through the liver. In the present invention it was found that attaching hydrophilic groups, regardless of otsepleniya or not, and more specifically, neutral hydrophilic groups, increases the intake of medicines it is in the kidneys.

It was found that hepatic clearance of conjugates of folate-drug, including disulfide linkers and peptide fragments, residual and sometimes significant adverse toxicity profile. The introduction of hydrophilic spacers described in this application leads to the appearance trends of receipt of the conjugates in the kidneys. Therefore, it is understood that the inclusion of such linkers in drug conjugates directed action can reduce the overall absorption of drugs by the liver and, therefore, reduce the overall toxicity. Not limited to any theory, I believe that the toxicity expressed as MTD, such as conjugates of Vinca alkaloids, can bydivision nonspecific hepatic clearance, leading to the metabolism, release of free drug, for example DAVLBH in the bile and then into the intestine. After that could be local toxicity and systemic toxicity (due to reabsorption). It is believed that by introducing a hydrophilic linkers in aims and anything conjugates described in this invention can mainly be clearance through the kidneys, which helps to reduce and/or avoid concomitant metabolism in the liver, which is the basis of toxicity. Accordingly, the measurement of the total excretion of bile medicinal component, for example DAVLBH from a number of conjugates of a drug-folate, can be used to forecast what the agent would be the least toxic.

As described above, is described in this application, the conjugates can be used for delivery of the target funds And the cells are selective or specific way. In one aspect of such delivery can also avoid unwanted clearance mechanisms. It was found that described in the present invention, the hydrophilic spacer linker in their application to obtain conjugates receptor-binding ligands B and means And can reduce the amount of drugs produced from the liver. It was further discovered that these hydrophilic spacers link the RA tend to facilitate clearance through urinary tract, for example, the kidneys. It was further found that the described invention conjugates exhibit lower toxicity than the original tools And themselves when introducing them in the same way. Not limited to any theory, I believe that the reason for this lower toxicity is observed decrease of the role of the mechanism of excretion via the liver and increasing the role of the mechanisms of renal excretion.

In another embodiment, the present invention describes compounds that are characterized by a reduced uptake by the liver and less likely to be output by the liver. In one aspect of such compounds preferably are derived renal processes compared with hepatic processes. Accordingly, in another embodiment, the invention described is not intended compound of the following formula:

L-A,

where L is a hydrophilic spacer, and A represents a diagnostic or therapeutic agent, or a renderer. It is assumed that these are not targeted compounds, although they do not have directional caused by the use of the receptor-binding ligand B, however, exhibit reduced toxicity compared to the original tool And when delivering them the same way. These are not targeted compounds and targeted conjugates described above is about the text, include hydrophilic spacer L. Hence, the agent does not reach the cell which is desired to be treated, will be put through the normal metabolic and biological mechanisms. However, it is assumed that the presence of hydrophilic spacer L will guide clearance through the renal route, and not by the hepatic route.

In another embodiment, the present invention is described multiplikatsionnye conjugates. The invention describes several illustrative configurations such multirecording conjugates, and they include compounds and compositions described in international PCT publication no WO 2007/022494, the contents of which are incorporated into the present application by reference. As an illustration, polyvalent linkers can connect the receptor-binding ligand B with two or more tools in A number of structural configurations, including the following illustrative of the General formula, but not limited to:

where B is a receptor-binding ligand, each L1), (L2) and (L3) is a polyvalent linker described in this application, including hydrophilic spacer linker, and optionally containing one or more fissile linkers and/or additional spacer linker, and each of (A1 ), (A2) and (A3) is a tool And its analogue or derivative. In addition, the invention encompasses other variations, including the presence of additional funds And their analogues, or derivatives, additional linkers, additional configurations of the location of each of (B), (L) and (A).

In one embodiment described in this application conjugates for drug delivery is more than one receptor-binding ligand B, including the following illustrative of the General formula, but not limited to:

where each fragment B is a receptor-binding ligand, each L1), (L2) and (L3) is a polyvalent linker described in this application, including hydrophilic spacer linker, and optionally containing one or more fissile linkers and/or additional spacer linker, and each of (A1), (A2) and (A3) is a tool And its analogue or derivative. In addition, the invention encompasses other variations, including the presence of additional funds And their analogues, or derivatives, additional linkers, additional configurations of the location of each of (B), (L) and (A). In one embodiment, the receptor-binding ligands B are ligands for the same Rotz the Torah, in another embodiment, the receptor-binding ligands B are ligands for different receptors.

In another illustrative embodiment, choose the medicines on the basis of activity against one or more populations of pathogenic cells with a specific mechanism of action. Illustrative mechanisms of action include the action of alkylating agents, inhibitors of microtubules, including those that stabilize and/or destabilize the formation of microtubules, including tools that affect beta-tubulin, inhibitors of cyclin-dependent kinases (CDK), topoisomerase inhibitors, inhibitors of protein synthesis, inhibitors of protein kinases, including Ras, Raf, PKC, PI3K and similar inhibitors, inhibitors of transcription, antifolates, blockers of protein thermal shock, etc.

Illustrative alkylating agents include, but are not limited to, mitomycin CBI etc. Illustrative inhibitors of cyclin-dependent kinase (CDK) include, but are not limited to the above, CYC202, seliciclib, R-roscovitine, AGM-1470, etc. Illustrative topoisomerase inhibitors include, but are not limited to, doxorubicin and other anthracyclines, etc. Illustrative inhibitors of protein synthesis include, but are not limited to, bracianti etc. Illustrative inhibitors of protein kinases, including Ras, Raf, PKC, PI3K and similar inhib the Torah, include, but are not limited to, L-779450, R115777, etc. Illustrative inhibitors of transcription include, but are not limited to the above, α-amanitin, actinomycin, etc. Illustrative antifolates include, but are not limited to, methotrexate, etc. Illustrative blockers temperature shock proteins include, but are not limited to, geldanamycin etc.

Illustrative inhibitors of microtubules, including those that stabilize and/or destabilize the formation of microtubules, include means acting on β-tubulin, poisons that destroy microtubules, etc. Illustrative poisons that destroy microtubules that are associated with the selected receptors include, but are not limited to, inhibitors, binding to the binding site of Vinca alkaloids, such as arenastaden, dolastatin, halichondrin B, maytansine, phomopsin A, rhizoxin, octyloxy, vinblastine, vincristine and the like, stabilizers, binding to the binding site of Taxol, such as discodermolide, epothilone, Taxol, paclitaxel, etc., inhibitors, binding to the binding site colchicine, such as colchicine, combretastatin, curacin a, podophyllotoxin, steganacin, etc. as well as other communicating with an unidentified sites, such as cryptophycin, tubulysin etc.

In one of the embodiments tubulysin is with the battle of natural tubulysin. In another embodiment, tubulysin is a synthetic or semisynthetic tubulysins. Additional varieties tubulysin that can be included in the conjugates described in this application are disclosed in international PCT application no PCT/US2008/056824, the contents of which are incorporated into the present application by reference.

In one of the embodiments of the conjugate for drug delivery described in the present invention, at least one of the drugs is a microtubule inhibitor, an analogue or derivative. In another embodiment, at least one of the drugs is DNA-alkylating agent. In another embodiment, at least one of the drugs is DNA-alkylating agent, and at least one other drug is an inhibitor of microtubules.

In another embodiment described in this application conjugates for drug delivery of at least one drug is an inhibitor of P-glycoprotein (PGP). In another embodiment, at least one of the drugs included in the conjugate described in this application, an inhibitor of PGP, and at least one of the other drugs included in to jugat, is a substrate of PGP. As an illustration, in this latter embodiment, the substrate of PGP is a DNA alkylating agent. In relation to this option, the implementation assumes that the use of the PGP inhibitor in combination with a substrate of PGP, for example, DNA alkylating agent, including, but not limited to, any of mitomycin, for example, mitomycin C, mitomycin A, etc. can improve the total efficiency of the medicinal product, which otherwise would be a PGP substrate. In splitting the conjugates described in this application, the drug, which is the inhibitor of PGP, and the drug, which is a substrate of PGP, jointly released into the cell after endocytosis. In this way the PGP inhibitor can improve the overall efficiency and/or due to the action of PGP substrate. In addition, the PGP inhibitor can decrease the expression of PGP, which in turn will reduce the excretion of pathogenic cells of one or more drugs included in multilocational conjugate described in this application. Assume that mitomycin, their analogues or derivatives, for example, mitomycin C, can act as inhibitors of PGP or step-down regulators PGP. Further assume that the Vinca alkaloid, or an analog or derivative, for example, analogs and derivatives vinyl is Stina, can be PGP substrates, which are protected from elimination of the pathogenic cells by the action of the inhibitor or a step-down regulator PGP.

In another embodiment, described in the invention conjugates for drug delivery of at least one drug is a Vinca alkaloid, an analogue or derivative. Vinca alkaloids described in the application, include all family members indole-dihydroindole of Vinca alkaloids, such as, but not limited to, vindesine, vinblastine, vincristine, catharanthine, ventolin, Laursen, vinorelbine, imidocarb, sibutramine, toltrazuril, vinblastine acid, etc. and their analogues and derivatives.

In another embodiment, the invention describes methods for treating diseases caused or manifested itself in the activity of populations of pathogenic cells. Conjugates for drug delivery, comprising a ligand-binding, can be used to treat a painful condition characterized by the presence of a population of pathogenic cells in the host organism, where the members of a population of pathogenic cells have an accessible binding site binding ligand (B), its analogue or derivative, where the specified binding site is expressed exclusively, redundantly expressed or preferably xpresroute pathogenic cells. Selective destruction of pathogenic cells is mediated by binding fragment binding ligand (B), which conjugate for delivery of drugs to the receptor or Transporter ligand or other protein on the cell surface, which are specific binds a ligand-binding (B), its analogue or derivative and which is expressed exclusively, redundantly expressed or preferentially expressed by the pathogenic cells. Present at the cell surface protein that is expressed exclusively, redundantly expressed or preferentially expressed by the pathogenic cells, is a receptor that is not present or is present in smaller concentrations in non-pathogenic cells, which provides a means for the selective destruction of pathogenic cells.

For example, expressed on the surface receptors of vitamins, for example Vysocany the folate receptor, over expressed on cancer cells. It was reported that all epithelial cancers of the ovary, breast, colon, lung, nose, larynx and brain Express elevated levels of folate receptors. In fact, it is known that more than 90% of all ovarian tumors person Express significant amounts of this receptor. Accordingly, described in the present is avce conjugate for delivery of medicines containing a binding ligand (B), can be used to influence several types of tumor cells, as well as other types of pathogenic cells, such as infectious agents, which predominantly Express the receptors for such ligands, as receptors of vitamins, and, thus, have on the surface available for binding with ligands sites, such as vitamins, or their analogues, or derivatives. In one aspect, the invention describes methods of targeting conjugates binding to the ligand-linker-drug in order to achieve maximum selectivity against pathogenic cells to destroy them.

Further, the invention considers the use of combinations of conjugates of binding the ligand-linker-drug in order to achieve maximum selectivity against pathogenic cells to destroy them.

Described in the invention conjugates for drug delivery, comprising a binding ligand (B), can be used for clinical treatment of humans and in veterinary medicine. So, for example, pet-owner, in the body which has a population of pathogenic cells and undergoes the treatment conjugates for drug delivery, comprising a ligand-binding (for example, vitamin)may be a person or, in the case of application in which VETERINARII, can be a laboratory, agricultural, domestic or wild animals. Described in the present invention methods can be applied to an animal host, including, but not limited to, humans, laboratory animals such as rodents (e.g. mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats and rabbits, farm animals such as cows, horses, pigs, sheep, goats, and wild animals in captivity, for example, bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and the whales.

The described methods are applicable to the populations of pathogenic cells that cause a number of abnormalities listed animal hosts. The term "pathogenic cells" includes, for example, cancer cells, infectious agents such as bacteria and viruses, cells infected with bacteria or viruses, activated macrophages, can cause painful conditions, as well as any other types of pathogenic cells that exclusively Express predominantly Express or redundantly Express the receptor binding ligand, for example, receptors vitamins or receptors that bind analogs or derivatives of vitamins. Pathogenic cells can also include any cells that cause the disease that needed treatment described in this invention conjugates for drug delivery, containing a ligand-binding, leads to the reduction of disease symptoms. For example, pathogenic cells can be cells of a host organism, which becomes pathogenic under certain circumstances, as, for example, cells of the immune system that are responsible for diseases such graft-versus-host, but are not pathogenic in other situations.

For example, the population of pathogenic cells may represent a population of cancer cells that give rise to neoplasms, including benign tumors and malignant tumors, or this population may not be carcinogenic. The population of cancer cells may occur spontaneously or as a result of this process, as the mutation is present in the germ line of an animal host, or somatic mutation, or its appearance may be caused by chemical, viral or radiation exposure. Described in the invention methods can be used to treat such cancers, such as carcinoma, sarcoma, lymphoma, Hodgkin's disease, melanoma, mesothelioma, Burkitt's lymphoma, nasopharyngeal carcinoma, leukemia, and myeloma. The population of cancer cells may include, without limitation, cells of cancer of the oral cavity, thyroid, endocrine system, skin, stomach, esophagus, larynx, pagelogo the Noah gland, colon, bladder, bone, ovary, cervix, uterus, breast, testicular, prostate, rectum, kidneys, liver and lungs.

In those versions of the implementation, where the population of pathogenic cells is a population of cancer cells, the effect of the introduction of the conjugate is a therapeutic response, as measured by the decrease or destruction of the tumor mass or inhibition of proliferation of tumor cells. In the case of tumor destruction can be the destruction of the cells of the primary tumor or cells that have metastasized, or are in the process of separation from the primary tumor. In the invention described preventive treatment conjugate for delivery of drugs, including binding ligand (B) (as the binding ligand is applied, for example, vitamin), to prevent tumor recurrence after removal by a therapeutic approach that includes surgical removal of the tumor, radiation therapy, chemotherapy, or biological therapy. Prophylactic treatment may be primary treatment with conjugate for delivery of drugs, including binding ligand (B), for example, by introducing in the mode of multiple doses during the day, and/or may be an additional treatment or series of remedial measures is an interval of a few days or months, after the initial treatment. Accordingly, the destruction of any of the populations of pathogenic cells, which were treated with the use described in the invention methods include reducing the number of pathogenic cells, inhibiting proliferation of pathogenic cells, preventive treatment, which prevents the re-emergence of pathogenic cells, or treatment of pathogenic cells, which leads to reduction of symptoms.

In cases where it is necessary to destroy cancer cells, the methods of the present invention can be used in combination with surgical removal of the tumor, radiation therapy, chemotherapy or biological therapies, for example, other forms of immunotherapy, including, but not limited to, treatment with monoclonal antibodies, treatment with immunomodulatory agents, adoptive transfer of immune effector cells, treatment with hematopoietic growth factors, cytokines and vaccines.

Methods of treatment according to the present invention is applicable also to the populations of pathogenic cells that cause a number of infectious diseases. For example, these methods are applicable to such populations of pathogenic cells, such as bacteria, fungi, including yeasts, viruses, cells infected by viruses, Mycoplasma, and parasites. Infectious microorganisms that cause Zabol the tion, which can be treated as described in the invention conjugates for drug delivery, comprising a binding ligand (B)represent any known in the field of engineering microorganisms that cause pathogenesis in animals, including microorganisms, both gram-negative bacteria or gram-positive cocci or bacilli. For example, using the steps described in the invention conjugates for drug delivery, comprising a ligand-binding, can be subjected to treatment of the disease caused in the host body by bacteria of the genus Proteus, Klebsiella, the genus Providencia, the genus Yersinia genus Erwinia, the genus Enterobacter, the genus Salmonella, genus Serratia, the genus Aerobacter, the genus Escherichia, the genus Pseudomonas, the genus Shigella, genus Vibrio, the genus Aeromonas, of the genus Campylobacter, genus Streptococcus, genus Staphylococcus, genus Lactobacillus, the genus Micrococcus, the genus Moraxella, genus Bacillus, genus Clostridium, the genus Corynebacterium, the genus Eberthella, kind Micrococcus, the genus Mycobacterium, the genus Neisseria, of the genus Haemophilus, of the genus Bacteroides, of the genus Listeria, genus Erysipelothrix, the genus Acinetobacter, the genus Brucella, of the genus Pasteurella, genus Vibrio, the genus Flavobacterium, of the genus Fusobacterium, genus Streptobacillus, genus Calymmatobacterium, of the genus Legionella, of the genus Treponema, of the genus Borrelia, of the genus Leptospira, of the genus Actinomyces, genus Nocardia, genus Rickettsia and any other kinds of bacteria.

Of particular interest are bacteria that are resistant to antibiotics, for example, resistant to antibiotics, the species of Streptococcus and Staphylococcus, or bacteria that osprey the Chiva to antibiotics, but when antibiotics cause recurrent infection, because over time develop resistant to antibiotic microorganisms. Bacteria that are susceptible to antibiotics, but cause recurrent infection with antibiotic treatment, because eventually develop drug-resistant microorganisms can be treated with the use described in this invention conjugates for drug delivery, comprising a binding ligand (B), in the absence of antibiotics, or in combination with lower doses of antibiotics compared with conventionally administered to patients to avoid the development of these antibiotic-resistant strains of bacteria.

Viruses, for example, DNA and RNA viruses, can also be treated by the described methods. These viruses include, but are not limited to the above, DNA viruses such as papilloma viruses, parvoviruses, adenoviruses, herpes viruses, and viruses ospowiki and RNA viruses, such as arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial viruses, influenza viruses, picornaviruses, paramyxoviruses, reoviruses, retroviruses, lentiviruses and rhabdovirus.

The methods of the present invention is also applicable to the treatment of pathologies caused by any fungi, including yeasts, various species of mycoplasmas, parasites or other infects the district microorganisms, which cause disease in animals. Examples of fungi that may be treated by the methods and compositions of the present invention, include fungi that grow in the form of mold or yeast are, including, for example, fungi that cause diseases such as ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, motorbikes, hromoblastomikoza, dermatophytes, prototokos, Fusarium, pytilias, mycetoma, paracoccidioidomycosis, phaeohyphomycosis, pseudoallescheria, sporotrichosis, trichosporon, Pneumocystis infection and candidiasis.

The methods of the present invention can also be used to treat parasitic infections including, but not limited to, infections, tapeworms, for example, related to the genera Taenia, Hymenolepsis, Diphyllothrium and Echinococcus, trematodes, such as, childbirth, Fasciolopsis, Heterophyes, Metagonimus, Clonorchis, Fasciola, Paragonimus and Schitosoma, round worms, for example the genera Enterobius, Trichuris, Ascaris, Ancylostoma, Necator, Strongyloides, Trichinella, Wuchereria, Brugia, Loa and Onchocerca Dracunculus, amoebae, for example, the genera Naegleria and Acanthamoeba spp., and protozoa, for example, delivery Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Giardia, Isospora, Cryptosporidium, Enterocytozoon.

Pathogenic cells targeted described in the present invention conjugates for drug delivery, including concerns about yuushi ligand, can also be cells, which contain endogenous pathogens, for example, cells infected with viruses, mycoplasmas, parasites or bacteria, if these cells predominantly Express the receptor ligand, for example, receptors of vitamin.

In one embodiment, the implementation of the conjugates for drug delivery, including binding ligands can be internalizable target pathogenic cell by binding fragment binding of ligand to the receptor, Transporter or other protein present on the surface, which is associated with specific ligand and which is predominantly expressed on pathogenic cells. This internalization may be, for example, by receptor-mediated endocytosis. If the conjugate for drug delivery, comprising a binding ligand (B), contains a biodegradable linker, a fragment of the binding ligand and the drug may be separated within the cell and the drug can act on its intracellular target.

In an alternative embodiment, the fragment binding ligand conjugate for delivery of medicines can communicate with the pathogenic cell, and the drug will be in close proximity with the surface of pathogenic cells. C is the fact this drug can be released by rupture of degradable linker, for example, the drug may be released under the action of the protein disulfide isomerase, if you split the linker is a disulfide group. Then the drug can be captured pathogenic cell, joined conjugate for delivery of drugs, including binding ligand (B), or the drug can be captured by other pathogenic cells that are in close proximity to the first. Alternatively, the drug could be released under the action of the protein disulfide isomerase inside the cell, if the biodegradable linker is a disulfide group. In addition, the drug could be released due to the hydrolytic mechanism, for example, acid-catalyzed hydrolysis, which is described above for some of the mechanisms of beta-elimination, or by cleavage with anchiornis assistance mechanism with the formation of oxonium or lactonovogo ion. The choice of degradable linker or linkers will determine the mechanism by which the drug will be chipped off from the conjugate. Assume that this choice can be predetermined conditions, which will apply the conjugate of the drug. Alternatively, the conjugates for taking the drug and can be internalizable in target cells upon binding, and linking the ligand and the drug can remain in a bound state inside the cell, and the drug exerts its effect separation of a fragment of the vitamin.

In another embodiment, in which the binding ligand is a vitamin, conjugate vitamin-drug may act by a mechanism that is not dependent on cell receptors vitamins. For example, the conjugates for drug delivery can be contacted with the soluble receptors of the vitamins present in the serum or serum protein, such as albumin, leading to increased circulation time conjugates relative to the unconjugated drug and to increase the activity of the conjugates in relation to the population of pathogenic cells, compared with unconjugated drugs.

In another embodiment, in which the linker does not contain fissile fragment, a fragment of the vitamin, which is conjugate for delivery of medicines can communicate with the pathogenic cell, placing the medicinal product on the surface of pathogenic cells in order to make it a target for attack other molecules capable of contact with the medicinal product. Alternatively, in this embodiment, con the gat for delivering medicines can be internality target cells after binding, and fragments of vitamin and drugs can stay bound within the cell, and the drug exerts its action without separation of a fragment of the vitamin.

In another embodiment, the present invention developed conjugate for drug delivery, cell receptor binding General formula B-L-A, where a fragment of L corresponds to the one given in the application description, and A is a drug, for example, the immunogen. The immunogen may be a hapten, for example, fluorescein, dinitrophenyl, etc. In this embodiment, the conjugate for delivery of drugs that bind the receptor vitamin, binds to the surface of pathogenic cells and "marks" cells immunogen, thereby triggering an immune response aimed at labeled population of pathogenic cells. Antibodies are introduced into the host organism in passive immunization, or antibodies that exist in the immune system of the host in the framework of innate or acquired immunity, are associated with the immunogen and start the endogenous immune response. The binding of the antibody with cells labeled conjugate vitamin immunogen, leading to complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, opsonization with the participation of antibodies is phagocytosis, clustering of receptors, induced antibodies, giving the signal for cell death or transition to a state of rest, or to any other humoral or cellular immune response stimulated by the binding of an antibody with an associated cell conjugate the ligand-immunogen. In cases where the immunogen can be directly recognized by immune cells without prior opsonization by antibodies may occur direct destruction of pathogenic cells. This version of the implementation are described in more detail in the application for U.S. patent No. 09/822379 included in the present application by reference. It is understood that in some varieties of this variant implementation, in which the drug is an immunogen, the polyvalent linker can be cleaved fragments, described above, for example, the conjugate for delivery of drugs that bind the receptor for the vitamin of General formula B-L-A, where L is a linker that includes one or more hydrophilic spacer linker and a biodegradable linker.

Described in the present invention conjugates for drug delivery include the binding ligand (B), the polyvalent linker (L), the drug and optional linkers based heteroatoms, which connect Swaziland and drug with polyvalent linker (L). The polyvalent linker (L) may include a spacer linker, split (i.e. destroy) the fragments of the linker, and linker fragments on the basis of the heteroatoms, or a combination of the above fragments.

The drug can be any molecule capable of modulating or any other way to modify the activity of cells, including pharmaceutically active compounds. Suitable molecules can include, but is not limited to the above, peptides, oligopeptides, retroverse oligopeptides, proteins, analogs of proteins in which at least one ones relationship replaces the peptide bond, apoprotein, glycoproteins, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, receptors and other membrane proteins; antigens and antibodies against them; haptens and antibodies against them; hormones, lipids, phospholipids, liposomes; toxins; antibiotics; analgesics; bronchodilating agents; beta-blockers; anti-microbial agents; protivogipertonicheskoe means; cardiovascular drugs, including anti-arrhythmic means, cardiac glycosides, antianginal tools and vasodilator; means for effects on the Central nervous system, including stimulants, psychotropic drugs, protivoradikulitnye means and depressants; antivirus the e means; antihistamines; anti-cancer tools, including chemotherapeutic agents; tranquilizers; antidepressants; antagonists H-2; anti-convulsants; protivotoshnotnoe means; prostaglandins and analogues of prostaglandins; muscle relaxants; anti-inflammatory agents; stimulants; decongestants; anti-emetics; diuretics; antispasmodic; anti-asthma remedies; anti-Parkinson's disease; expectorants; a means of suppressing the cough; mucolytics, mineral and dietary supplements.

In addition, the drug can be any drug known in the field of technology, which is cytotoxic, improves the permeability of tumors, inhibits proliferation of tumor cells, promotes apoptosis, reduces antiapoptotic activity in the target cells for the treatment of diseases caused by infectious agents, enhances endogenous immune response directed to pathogenic cells or used for the treatment of painful conditions caused by any type of pathogenic cells. Medicines which are suitable for use according to the present invention, include adrenocorticoid and corticosteroids, alkylating agents, antiandrogens, anties Raheny, androgens, allicin and derivatives glamuzina, estrogens, antimetabolites such as cytosine arabinoside, purine analogues, and pyrimidine analogues, as well as methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, Taxol, paclitaxel, derivatives of paclitaxel, Taxotere®, cyclophosphamide, daunomycin, daunorubicin, doxorubicin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycin, discodermolide inhibitors microtubules, epothilone, tubulysin, cyclopropenes[e]indole, seco-cyclopropanes[e]indole, O-Ac-seco-cyclopropanes[e]indole, bleomycin, and any other antibiotics, nitrogen mustards, nitrosamine, vincristine, vinblastine, its analogs or derivatives, for example, diacetylhydrazine monohydrate and other Vinca alkaloids, including the ones described in international PCT publication no WO 2007/022493, the contents of which are incorporated into the present application by reference, colchicine, colchicine derivatives, alocalized, thiocolchicine, trailsystem, halichondrin B, dolastatin, for example, dolastatin 10, amanitine, for example, α-amanitin, camptothecin, irinotecan and other derivatives camptothecin, maytansine, geldanamycin and derivatives geldanamycin, estramustin, nocodazole, MAP4 colcemid, inflammatory and proinflammatory and the coefficients, peptides and peptidomimetics, which are inhibitors of signal transmission, as well as any other known in the technical field of drugs or toxins. Other medicines that may be used in accordance with the present invention include penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and any other known in the technical field of antimicrobial compounds.

In another embodiment, the agent (A) is a drug selected from Vinca alkaloids, for example, DAVLBH, cryptophycin, bortezomib, Tiburtina, tubulysin, aminopterin, rapamycin, paclitaxel, docetaxel, doxorubicin, daunorubicin, everolimus, α-amanitin, verrucarin, didemnin B, geldanamycin, purvalanol A, everolimus, ispinesib, budesonide, dasatinib, epothilone, maytansine and tyrosine kinase inhibitors, including analogues and derivatives of listed medicines. In another embodiment, the conjugate includes at least two tools (A)selected from Vinca alkaloids, for example, DAVLBH, cryptophycin, bortezomib, Tiburtina, tubulysin, aminopterin, rapamycin, paclitax the ate, docetaxel, doxorubicin, daunorubicin, everolimus, α-amanitin, verrucarin, didemnin B, geldanamycin, purvalanol A, everolimus, ispinesib, budesonide, dasatinib, epothilone, maytansine and tyrosine kinase inhibitors, including analogues and derivatives of listed medicines. In one embodiment, the means (A) are the same. In another embodiment, means (A) are different.

In one embodiment, the implementation of a medicinal product intended for use in the methods described in this application, remain stable in serum for at least 4 hours. In another embodiment, the medicines are set IC50in the nanomolar range, and, in another embodiment, these drugs are water-soluble. If the drug is not water soluble, polyvalent linker (L) can be introduced additional functional groups to improve the solubility in water. The term "drug" means any of the analogs or derivatives of the drugs indicated above the application. It should be understood that in accordance with the present invention, the phrase "similar or derived medicinal product" means a drug, which includes heteroatom, through which an analogue or derivative drug covalently linked to a polyvalent linker (L).

Conjugate for drug delivery means may include a binding ligand (B), the bivalent linker (L), the drug and optional linkers based heteroatoms, for connecting the receptor-binding fragment of the binding ligand (B) and drugs with the bivalent linker (L). In one illustrative embodiment, it should be understood that the analogue or derivative of vitamin may mean vitamin, which includes heteroatom through which the analogue or derivative of vitamin covalently linked to the bivalent linker (L). For example, in this illustrative embodiment, the vitamin can be covalently bound to the bivalent linker (L) through a linker that includes a heteroatom, or an analogue or derivative of vitamin (i.e. vitamin that includes a heteroatom) can be directly linked to the bivalent linker (L). In a similar illustrative embodiments implement similar or derived medicinal product is a medicinal product, and an analogue or derivative drugs can mean a medicinal product, which includes heteroatom through which the analogue or derivative drugs Cova is into linked to the bivalent linker (L). For example, in these illustrative aspects of the drug can be covalently linked to the bivalent linker (L) through a linker based on the heteroatom, or an analogue or derivative drugs (i.e. including heteroatom) can be directly linked to the bivalent linker (L). The bivalent linker (L) may include a structural linker fragments, split (i.e. destroy) the fragment linker and linker-based heteroatom, which serves to link fragments of the linker with fissionable linker in conjugates containing both of these types of linkers.

As a rule, can be applied to any method of forming a conjugate between the bivalent linker (L) and the binding ligand (B) or its analogue or derivative, between the bivalent linker (L) and the medicinal product or its analogue or derivative, including the introduction of any intermediate linkers based on heteroatoms. In addition, to obtain the bivalent linker (L) may be any known in the art method of forming a conjugate between a structural fragment linker that is cleaved fragment linker and linker-based heteroatom. The conjugate can be formed by direct connection of any of these molecules, for example, by complexing or through hydrogen bonds, ionic or covalent St is zi. Covalent binding can be carried out, for example, through the formation of amide, ester, disulfide, or aminokwasy between carboxyl, aldehyde, hydroxyl, amino, sulfhydryl or hydratherapie.

In another embodiment, the bivalent linker (L) comprises a chain of atoms selected from C, N, O, S, Si and P, which are connected by covalent bonds binding ligand (B), hydrophilic fragments of the linker and/or tool (S). The length of the linker may vary within a wide range, for example, from about 2 to about 100 atoms. The atoms used in the formation of the linker can connect with each other with all matching from a chemical point of view ways, for example, chains of carbon atoms can constitute alkylene, alkenylamine and alkenylamine group and the like; chain of carbon atoms and oxygen can be a simple esters, polyoxyalkylene group or, in combination with carbonyl groups to represent esters, carbonates and the like; chain of carbon atoms and nitrogen can represent amines, imine, polyamine, hydrazines, hydrazones, or in combination with carbonyl groups to represent amides, urea, the semicarbazides, carbazide and the like; chain of atoms of carbon, nitrogen and oxygen can be alkoxyamine, alkoxyamine or in Combi is then with carbonyl groups, can be a urethane, amino acids, alloxylon, hydroxamic acids, etc. and much more. In addition, it should be understood that the atoms forming the chain in each of the previous illustrative embodiments, may be saturated or unsaturated, such as, for example, radicals which are included in the linker may be the remains of alkanes, alkenes, alkynes, Iminov, etc. in Addition, it should be understood that the atoms forming the linker, can also form loops with each other, with the formation of divalent cyclic structures, which are part of the linker, including cycloalkanes, cyclic ethers, cyclic amines, arylene, heteroaryl etc. in the linker.

In another embodiment, the invention describes a pharmaceutical composition comprising the conjugate for delivery of a medicinal product containing a binding ligand (B), in an amount effective to kill the population of pathogenic cells in the body of an animal host, with the introduction of one or more doses of the composition. Conjugates for drug delivery, comprising a ligand-binding, is preferably introduced into the body of an animal host parenteral, e.g., intradermally, subcutaneously, intramuscularly, administered intraperitoneally, intravenously or intrathecally. Alternatively, the conjugate for delivery of the medication is tion means, including a ligand-binding, you can enter into the body of an animal host other used in medicine ways, for example, orally, and can be any effective dose and any suitable dosage form, including dosage forms with prolonged release.

Examples of parenteral dosage forms include aqueous solutions of the active agent in isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carriers, such as liquid alcohols, glycols, esters and amides. Parenteral dosage forms can be obtained in the form of recoverable liofilizatow, including the dose of the conjugate for delivery of medicines. In one aspect of the present invention, the patient can enter any of the many known in the field of technology of medicinal forms of slow release, such as biorstwami carbohydrate matrix, described in U.S. patent No. 4713249; 5266333 and 5417982, the contents of which are incorporated into the present application by reference, or, alternatively, can be applied pumps delayed action (e.g., osmotic pumps).

In one illustrative aspect, at least one additional composition comprising terap ticheskoe active principle, may be injected into a host organism in combination or as an aid to the described above methods of the present invention, to improve the destruction of a population of pathogenic cells conjugate for delivery of a medicinal product containing a ligand-binding, or you can enter more than one additional therapeutic factor. This therapeutic factor can be selected from chemotherapeutic agents or other therapeutic agents that can Supplement the effectiveness of the injected conjugate containing a ligand-binding.

In one illustrative aspects can be used therapeutically effective combination of the mentioned factors. For example, in one of the embodiments together with the conjugate for delivery of a medicinal product containing a ligand-binding, can be used therapeutically effective amount of another therapeutic factor, e.g. in the range from about 0.1 MIU/m2/dose/day to about 15 MIU/m2/dose/day in injection mode, multiple doses per day, or, for example, amounts in the range from about 0.1 MIU/m2/dose/day to about 7.5 MIU/m2/dose/day in injection mode, multiple doses per day, to destroy, reduce or neutralize pathogenic glue is OK in the body of an animal host, bearing these cells (MIU=million international units; m2=approximate body surface area of an average human).

In another embodiment, a chemotherapeutic drug, which, for example, are cytotoxic by themselves or can improve the permeability of the tumor, also suitable for use in the described methods in combination with conjugates for delivery of a medicinal product containing a ligand-binding. Such chemotherapeutic agents include adrenocorticoid and corticosteroids, alkylating agents, antiandrogens, antiestrogens, androgens, allicin and derivatives glamuzina, estrogens, antimetabolites such as cytosine arabinoside, purine analogues, and pyrimidine analogues, as well as methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, Taxol, paclitaxel, derivatives of paclitaxel, Taxotere®, cyclophosphamide, daunomycin, daunorubicin, doxorubicin, rhizoxin, T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide, mitomycin, discodermolide inhibitors microtubule, epothilone, tubulysin, cyclopropenes[e]indole,seco-cyclopropanes[e]indole, O-Ac-seco-cyclopropanes[e]indole, bleomycin, and any other antibiotics, nitrogen mustards, nitrosamine, vincristin, vinblastine, its analogs or derivatives, for example diacetylhydrazine monohydrate, colchicine, colchicine derivatives, alocalized, thiocolchicine, trailsystem, halichondrin B, dolastatin, such as dolastatin 10, amanitine, for example, α-amanitin, camptothecin, irinotecan and other derivatives camptothecin, geldanamycin and derivatives geldanamycin, estramustin, nocodazole, MAP4, colcemid, inflammatory and proinflammatory agents, peptides and peptidomimetics, which are inhibitors of signal transmission, as well as any other known in the technical field of drugs and toxins. Other medicines that may find application include penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, maytansine, their analogues and derivatives, gemcitabine, and any other known in the technical field of antimicrobial compounds.

therapeutic factor can be administered into the body of an animal host before, after or during administration of the conjugates for the delivery of drugs containing a ligand-binding, and specified therapeutic factor can be administered as integral parts of the same composition, which includes to jugat for delivery of medicines containing a ligand-binding, or as a component of another song. Can be used any of such therapeutic compositions containing a therapeutic factor in therapeutically effective dosage.

In addition, can be used more than one type of conjugate for delivery of a medicinal product containing a ligand-binding. As an illustration, pet-owner can, for example, be treated with conjugates, including various vitamins, but the same drug, the method comprising co-administration. In other embodiments, the implementation of the animal owner may be subjected to treatment conjugates comprising the same binding ligand, coupled with different drugs or different binding ligands, combined with various drugs. In another illustrative embodiment could be applied conjugates for drug delivery comprising the same or different vitamins and the same or different drugs, including several vitamins and some medications as fragments of the same conjugate for drug delivery.

The total daily dosage of the conjugate for delivery of a medicinal product containing the binding is a first ligand, can vary significantly depending on the state of the host body, subjected to the treatment of painful conditions, the molecular weight of the conjugate, the way of its introduction and distribution in the tissues, as well as the possibility of joint use of other treatments, such as radiation therapy. An effective amount, which you must enter the patient depends on the surface area of the body weight of the patient and physician evaluation of the patient. In illustrative embodiments, the effective dosage may be in the range of, for example, from about 1 ng/kg to about 1 mg/kg, from about 1 μg/kg to about 500 μg/kg, from about 1 μg/kg to about 100 μg/kg, from about 1 μg/kg to about 50 μg/kg, from about 1 μg/kg to about 10 mg/kg

In another illustrative aspect can be applied to any effective mode of administration of the conjugates for the delivery of medicines, including a ligand-binding. For example, the conjugate for delivery of medicines, including a ligand-binding, may be injected in a single dose, or the dose can be divided and implement the introduction of mode multiple doses per day. In other embodiments, the implementation may apply intermittent modes of administration, for example, from one to three times in which the components, alternatively, the daily introduction, and it is believed that such intermittent or periodic mode is equivalent to the daily introduction and is within the limits of the methods described in the present invention. In one embodiment, the patient is subjected to treatment with multiple injections of the conjugate for drug delivery containing a ligand-binding, for the destruction of a population of pathogenic cells. In another embodiment, the patient performed a large number of injections (preferably from about 2 to about 50 injections) conjugate for drug delivery containing a ligand-binding, for example, at intervals of 12 to 72 hours or intervals of 48-72 hours. In another embodiment, the patient spend additional injections of the conjugate for drug delivery containing a ligand-binding, for example, in a few days or months after the first injection (injection), with additional injections prevent the recurrence of painful conditions caused by pathogenic cells.

In one embodiment, the implementation of vitamins, their analogues or derivatives, which can be used in the conjugates for drug delivery containing a ligand-binding, include those vitamins that are associated with receptors that specifically Express iroute on activated macrophages, for example, folate receptors, which are associated with folate, their analogs or derivatives. Conjugates, including associated folate, can for example be used to destroy or inhibit the activity of activated macrophages, which cause painful conditions in the host organism. Such conjugates targeted to macrophages when administered to a patient suffering from a disease state mediated by activated macrophages, concentrate and bind conjugated drug with a population of activated macrophages, destroying activated macrophages or suppressing their activities. Elimination, reduction or deactivation of a population of activated macrophages allows you to stop or decrease the pathogenesis mediated by activated macrophages, is characteristic of the disease condition being treated. Typical of the disease, which are known to be mediated by activated macrophages include rheumatoid arthritis, ulcerative colitis, Crohn's disease, psoriasis, osteomyelitis, multiple sclerosis, atherosclerosis, pulmonary fibrosis, sarcoidosis, systemic sclerosis, rejection of transplanted organ (GVHD) and chronic inflammation. The introduction of conjugate for drug delivery, as a rule, continue to until the symptoms boleznennogo the state is weakened or is not removed.

As an illustration, the conjugates for drug delivery containing a ligand-binding, which is administered for the destruction of activated macrophages or to suppress the activity of activated macrophages, you can enter the animal or patient suffering from painful conditions parenterally, for example, intradermally, subcutaneously, intramuscularly, administered intraperitoneally or intravenously, in combination with a pharmaceutically acceptable carrier. In another embodiment, the conjugates for drug delivery containing a ligand-binding, you can enter the animal or patient with others used in medicine, and the effective dose can be entered using a standard dosage forms or forms with slow release. In another aspect, the described method of treatment can be used by itself or in combination with other treatment modalities, which are known for the treatment of disease conditions mediated by activated macrophages.

Conjugates for drug delivery described in the present invention, it is possible to get known in the technical field of methods of synthesis. The synthesis methods are chosen depending on the choice of optional additional heteroatoms or heteroatoms, which are already present in the spacer linker, RAS is allaamah fragments linker, medicinal product and/or binding ligand. Reactions suitable for the formation of relationships, mainly described in Richard C. Larock, “Comprehensive Organic Transformations, a guide to functional group preparations”, VCH Publishers, Inc. New-York (1989), and Theodora E. Green & Peter G.M. Wuts, “Protective Group in Organic Synthesis”, 2ndedition, John Wiley & Sons, Inc. New York (1991), the contents of which are incorporated into the present application by reference. Additional details of the production of functional groups in the structure of the linker, including split fragments, including amides and esters, ketals and acetals, suktinimida, srilakshmi, hydrazones, acylhydrazone, semicarbazone, disulfides, carbonates, sulfonates, etc. described in published application US patent # US 2005/0002942 A1, incorporated into the present application in its entirety by reference.

The General methodology of the folate-peptides. Folate-containing pipidinny fragment Pte-Glu-(AA)n-NH(CHR2)CO2H (3) receive sequential synthesis on a polymer substrate, using standard techniques, for example Fmoc-strategy on acid-sensitive resin Fmoc-AA-Wang (1), as shown in figure 1.

(a) 20% piperidine/DMF; (b) Fmoc-AA-OH, PyBop, DIPEA, DMF; (c) Fmoc-Glu(O-t-Bu)-OH, PyBop, DIPEA, DMF; (d) 1.N10-(TFA)-Pte-OH; PyBop, DIPEA, DMSO; (e) TFAA, (CH2SH)2,i-Pr3SiH; (f) NH4OH, the pH of 10.3.

In this illustrative options the ante method, described in the present invention, R1represents Fmoc, R2is a suitably protected side chain of the desired amino acids and DIPEA means diisopropylethylamine. Apply standard methods of cross-linking are described in the present invention, or known in the technical field, for example, using PyBOP and others in which a crosslinking agent illustrative used as the activating reagent to ensure effective linkage. The protective Fmoc group is removed after each stage staple in standard conditions, for example, by treatment with piperidine, tetrabutylammonium fluoride (TBAF), etc. Use blocks of amino acids with the appropriate protection, such as, for example, Fmoc-Glu-OtBu,N10-TFA-Pte-OH and the like, as shown in figure 1 and presented on the stage (b) of the compound Fmoc-AA-OH. For example, AA refers to any original amino acid, which has adequate protection. It should be understood that the term "amino acid" in the present description is intended for the names of any agent, including both amino and carboxyl group separated by one or more carbon atoms, and the term encompasses natural alpha - and beta-amino acids and derivatives and analogues of these amino acids. In particular, described herein is the synthesis of folate-peptide can also be applied amino acids, it is the matter of side chains, which are secured, as, for example, protected serine, threonine, cysteine, aspartate and so Forth among the starting compounds for the synthesis of folate-peptides described in this application may include gamma, Delta or longer homologues of amino acids. In addition, analogs of amino acids, including homologous side chains or structure with a different type of branching, as for example, norleucine, isovaline, β-methylthionine, β-methylcysteine, β,β-dimetiltsistein, etc. can also be included among the starting compounds for the synthesis of folate-peptides described in this application.

The sequence of reactions stitching (stages (a) and (b)), which is used Fmoc-AA-OH, do “n” times, receiving peptide 2 on a solid substrate, where n means an integer, which can be from 0 to about 100. After the last stage of stitching the remaining Fmoc group is removed (stage (a)), and peptide consistently sew with glutamate derivative (stage (c)), remove protection and sew with paloemeu acid, TFA protected (stage (d)). Then the peptide otscheplaut from polymeric substrate processing triperoxonane acid, atadditional and triisopropanolamine (stage (e)). Conducting the reaction under these conditions leads to the simultaneous removal of the protective groups t-Bu, t-Boc and Trt, which may be fragments of the side chains of the amino acids with the corresponding protection of the Oh. The protective group is removed with TFA treatment with base (stage (f)), receiving folate-containing pipidinny fragment 3.

In addition, to obtain the compounds described in this application, you can apply the following illustrative method, where n is an integer from 1 to about 10:

It should be understood that although the above method of synthesis is depicted on the example of selected compounds, namely concrete shown sharovatov, using the same or similar method can be obtained, and other similar connections, simply by selecting the starting compounds and the standard optimization of reaction conditions.

Compounds described in this application can be obtained using standard techniques of synthetic organic chemistry. In addition, to obtain the compounds described can be applied to the following illustrative method, where n is an integer from 1 to about 10:

It should be understood that although the above method of synthesis is depicted on the example of selected compounds, namely concrete shown shareoption, using the same or similar method can be obtained, and other similar connections, simply by selecting the starting compounds and the standard optimization conditions for the second reaction.

In addition, to obtain the compounds described in this invention can be applied to the following illustrative method.

It should be understood that although the above method of synthesis is depicted on the example of selected compounds using the same or similar method can be obtained, and other similar connections, simply by selecting the starting compounds and the standard optimization of reaction conditions.

Each of the methods described above for the synthesis of intermediate compounds can be sewn with any additional hydrophilic spacer linker, other spacer linker, fissionable linkers or by means of A. In embodiments, each of the methods described above between the binding ligand B and shown hydrophilic spacers of linkers can be placed additional hydrophilic spacers of linkers, other spacers of linkers or split fragments of the linkers. In addition, it should be understood that the placement of divalent hydrophilic spacer linker from left to right is not limiting and, therefore, A tool, a ligand-binding B, additional hydrophilic spacer linker, other spacer linker and/or biodegradable linkers can be attached to any end of the hydrophilic spacer linker, described in the present is awke.

EXAMPLES of METHODS

Study on the relative affinity for binding. The affinity of compounds to the folate receptor (FR) relative affinity of folate was determined in accordance with the previously described method (Westerhof, G.R., J.H. Schornagel, et al. (1995) Mol. Pharm. 48: 459-471) with slight modifications. Briefly, FR-positive KB cells abundantly sown in 24-hole plates to cell cultures and allowed to attach to walls for 18 hours. Waste incubation environment in the planned wells was replaced with RPMI solution not containing folate (FFRPMI), with the addition of 100 nm3H-folic acid in the absence and presence of test compounds or folic acid in increasing concentrations. Cells were incubated for 60 minutes at 37°C and then three times washed with PBS, pH 7,4. Added five hundred microlitres 1% SDS in PBS, pH 7,4. Then collected cell lysates and added to separate vials containing 5 ml scintillation mixture, and then counting the radioactivity. Negative control samples contained only3H-folic acid in FFRPMI (no competing connections). Positive control samples contained folic acid at a final concentration of 1 mm, and the CPM values measured for these samples (reflecting non-specific binding of the label), subtracted from the data obtained for sexobrazil. It should be noted that the values of the relative affinity was determined as the reciprocal of the molar ratio of compound required to displace 50%3H-folic acid related receptors FR on KB cells, and the relative affinity of folic acid to FR took over 1.

Inhibition of cellular DNA synthesis. Compounds described in this invention were evaluated using analysis of cytotoxicityin vitrothat predicts the ability of drugs to inhibit the growth of folate-receptor positive KB cells. Compounds included folate associated with the appropriate chemotherapeutic drug, and they were received by the techniques described in this application. The KB cells were exposed conjugates of folate-drug at the indicated concentrations over time up to 7 hours at 37°C in the absence or in the presence of at least 100-fold excess of folic acid. Then the cells were washed once with fresh culture medium and incubated in fresh culture medium for 72 hours at 37°C. cell Survival was evaluated using analysis based on the inclusion of3H-thymidine.

Dependent on the concentration of the cytotoxic activity in vitro.Cells abundantly sown in 24-hole tablets Falcon and gave them to form p is almost confluently monolayers during the night. Thirty minutes before adding the test compounds emaciated medium was aspirated from all wells and replaced with fresh RPMI solution not containing folate (FFRPMI). It should be noted that in the planned wells have introduced a medium containing 100 μm folic acid; and the cells in these wells were used to determine the specificity of targeting, since the cytotoxic activity developed in the presence of excess folic acid (generating competition for binding to FR), could indicate a part of the total activity that is not associated with the FR-specific delivery of the conjugate. After one wash with 1 ml fresh FFRPMI containing 10% fetal calf serum inactivated by heating, to each well was added 1 ml of medium containing the test compounds at increasing concentrations (4 wells per sample) in the presence or absence of 100 μm of free folic acid (a substance that competes for the binding site). The treated cells were examined by the method of displacement of the label "pulse-chase", phase "pulse" was carried out for 2 h at 37°C, 4 times washed with 0.5 ml of medium and then spent phase "chase" in 1 ml of fresh medium in a period of time up to 70 hours Emaciated medium was aspirated from all wells and replaced with fresh medium containing 5 mccoury/ml3H-thymidine. After a further 2 h incubation at 37°C the cells 3 times amywali 0.5 ml PBS and then treated with ice-cold 5% trichloroacetic acid in the amount of 0.5 ml per well. After 15 minutes of trichloroacetic acid was aspirated and has solubilizers cellular material by the addition of 0.5 ml 0,25N sodium hydroxide for 15 minutes four Hundred and fifty µl of each solubilizing sample was transferred into scintillation vials containing 3 μl of scintillation mixture Ecolume, and then counted in a scintillation counter liquids. The final results are placed in the table, expressed as percentage3H-thymidine relative to untreated control samples.

As shown in the attached graphs, dose-dependent cytotoxicity was measured, and in most cases the values of the IC50(the concentration of the drug conjugate, is required to reduce the inclusion of3H-thymidine into newly synthesized DNA by 50%) were in the lower part of the nanomolar range. In addition, the values of cytotoxicity of these conjugates was reduced in the presence of an excess of folic acid, indicating that the observed cell killing was mediated by binding to folate receptors. The following table presents data for selected compounds for cytotoxicity against KB cells and against RAW264.7 cells.

tr>
Room
conjugate
The original medicinal product (money)Cells KBCells RAW264.7
IC50(nm)The ability to compete with an excess of folateIC50(nm)The ability to compete with an excess of folate
EC0234DAVLBH56Yes
EC0246DAVLBHYes
EC0258DAVLBH8,4Yes
EC0262cryptophycin4Yes
EC0263DAVLBH11Yes
EC0409DAVLBH7Yes
EC0525Timorese68Yes
EC0543tubulysin-1,6Yes
EC0551aminopterin1Yes
EC0552rapamycin100Yes
EC0561paclitaxel53Yes
EC0563Timorese+
rapamycin
387Yes
EC0582Timorese+
everolimus
51Yes
EC0592α-amanitin~3Yes5Yes
EC0595bis-Timorese4Yes
EC0598verrucarin33Yes
EC0605bis-verrucarin14Yes
EC0610didemnin B4Yes
EC0647bis-aminopterin 0,3Yes

Test for activity against different lines of cancer cells in vitro.Cells abundantly sown in 24-hole tablets Falcon and gave them to form almost confluently monolayers during the night. Thirty minutes before adding the test compounds emaciated medium was aspirated from all wells and replaced with fresh RPMI solution, not containing folate (FFRPMI). In the subgroup of planned holes have introduced a medium containing 100 μm folic acid. The cells in these wells were used to determine the specificity of targeting. Not limited to any theory, it is possible to assume that the cytotoxic activity, develop the test compounds in the presence of an excess of folic acid, i.e. when they are in competition for binding to FR, corresponds to that part of the total activity is not associated with the FR-specific delivery of the conjugate. After one wash with 1 ml fresh FFRPMI containing 10% fetal calf serum inactivated by heating, to each well was added 1 ml of medium containing the test compounds at increasing concentrations (4 wells per sample) in the presence or absence of 100 μm of free folic acid, as indicated in the results of the experiment. The treated cells were examined by the method of displacement of the label "pulse-chase", where the phases of the pulse was carried out for 2 h at 37°C, 4 times washed with 0.5 ml of medium and then spent phase "chase" in 1 ml of fresh medium in a period of time up to 70 hours Emaciated medium was aspirated from all wells and replaced with fresh medium containing 5 mccoury/ml3H-thymidine. After a further 2 h incubation at 37°C the cells 3 times washed with 0.5 ml PBS and then treated with ice-cold 5% trichloroacetic acid in the amount of 0.5 ml per well. After 15 minutes of trichloroacetic acid was aspirated and has solubilizers cellular material by the addition of 0.5 ml 0,25N sodium hydroxide for 15 minutes 450-µl aliquot of each solubilizing sample was transferred into scintillation vials containing 3 μl of scintillation mixture Ecolume, and then counted in a scintillation counter liquids. The final results are placed in the table, expressed as percentage3H-thymidine relative to untreated control samples.

Suppression of tumor growth in mice. Mice aged from four to seven weeks (Balb/c ornu/nu) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Ordinary food of rodents contains folic acid in high concentrations (6 mg/kg / meal); respectively used mice were kept on a diet, not including folate (Harlan diet # TD00434) within 1 week before implantation of tumors, to achieve concentrations of folate in serum, close diapason normal human serum. For the purpose of making tumor cells, 1×106the M109 cells (line Balb/c) or 1×106the KB cells (linenu/nu) in 100 μl were injected with subcutaneous layer in the region of the middle of the back. Tumor size was measured in two perpendicular directions every 2-3 days using a caliper, and tumor volumes were calculated by the formula of 0.5×L×W2where L=the size of the longest axis in mm, and W=the size of the axis, perpendicular to L, in mm, Then calculated values of Log destruction of cells (LCK) and the excess treated tumors over control (T/C), according to published methods (see, for example, Lee et al., “BMS-247550: a novel epothilone analog with a mode of action similar to paclitaxel but possessing superior antitumor efficacy”Clin Cancer Res7: 1429-1437 (2001); Rose , “Taxol-based combination chemotherapy and other in vivo preclinical antitumor studies”J Natl Cancer Inst Monogr47-53 (1993)). Each day, prepared fresh solutions for injection in PBS and injected through the lateral tail vein of mice. The introduction of drugs began when the subcutaneous tumors were purchased by the average amount in the range of 50-100 mm3(t0), usually within 8 days after injection of tumor cells (PTI) for tumors KB and 11 PTI for M109 tumors.

General research methodology tumors KB.Antitumor activity of the compounds described in this invention, when administered intravenously (i.v.) animals native tumor was evaluated in micenu/nuhaving subcutaneous swollen what does KB. Approximately 8 days after injection of 1×106tumor cells KB in the subcutis of the right axilla (average tumor volume at time t0=50-100 mm3), mice (5 animals/group) three times per week (TIW) for 3 weeks was carried out by i.v. the injection of 5 mmol/kg of the drug conjugate or an equivalent volume of PBS (control), unless otherwise noted. Tumor growth was measured with a caliper via 2-day or 3-day intervals in each group. The volume of tumor was calculated using the equation V=a×b2/2, where “a” represents the length of the tumor and b is the width, and all values are expressed in millimeters.

General research methodology M109 tumors.Antitumor activity of the compounds described in this invention, when administered intravenously (i.v.) animals native tumor, was evaluated in Balb/c mice that had subcutaneous M109 tumors (syngeneic lung carcinoma). After approximately 11 days after administration of 1×106tumor cells M109 in the subcutis of the right axilla (average tumor volume at time t0=60 mm3) mice (5 animals/group) three times per week (TIW) for 3 weeks was carried out by i.v. injection 1500 nmol/kg of the drug conjugate or an equivalent volume of PBS (control). Tumor growth was measured with a caliper via 2-day or 3-day prom is terrible in every group. The volume of tumor was calculated using the equation V=a×b2/2, where “a” represents the length of the tumor and b is the width, and all values are expressed in millimeters.

General research methodology tumors 4T-1.Mice at the age of six-seven weeks (females of Balb/c) were obtained from Harlan, Inc., Indianapolis, IN. Mice were kept at the stern Harlan''s free of folate, for a total of three weeks prior to and during the described experiment. Tumor cells 4T-1, not bearing receptors of folate (1×106cells per animal)were injected into the subcutaneous layer of the right armpit of the animal. Approximately 5 days after injection of tumor cells, when the average tumor volume 4T-1 was ~100 mm3the mice (5 animals/group) three times per week (TIW) for 3 weeks was carried out by i.v. the injection of 3 µmol/kg conjugate of a drug or an equivalent volume of PBS (control), if the proposal does not specify otherwise. Tumor growth was measured with a caliper via 2-day or 3-day intervals in each group. The volume of tumor was calculated using the equation V=a×b2/2, where “a” represents the length of the tumor and b is the width, and all values are expressed in millimeters.

Data on figa, 4A, 5A, 6A, 7A, 8A and 10A indicate that described in the invention conjugates in the treatment of tumors is her demonstrate the effectiveness, which exceeds the efficiency of the corresponding unconjugated compounds. Treatment of Balb/c mice with subcutaneous tumors M the conjugates EC0396 and EC145 (figa) led to complete remission in all animals who injected drugs (3/3 for EC0396 and 5/5 for EC145). In addition, approximately 70 days were not observed recurrence of the disease. Similarly, the introduction EC0400 (figa) resulted in complete remission and no recurrence of the disease in about 70 days. Treatment conjugated compounds described in this application, which included the hydrophilic spacer linker (for example, EC0436), was more successful compared to conjugates that had no hydrophilic spacer linker (for example, EC0305), indicating increased efficiency (figa). Conjugate EC0436 led to complete remission in 5 animals 5, in the absence of disease recurrence after 90 days.

Determination of the toxicity of drugs.Long-term toxicity of drugs was assessed by sampling blood from the puncture of the heart and an independent analysis of serum blood urea nitrogen (BUN), creatinine, total proteins, AST-SGOT, ALT-SGPT and standard hematological cell panel Ani-Lytics, Inc. (Gaithersburg, MD). In addition, conducted histopathological assessment fixed in formalin preparations of the heart, lungs, liver,spleen, kidney, intestine, skeletal muscle and bone (large tibia/fibula) involving certified pathologists in Animal Reference Pathology Laboratories (ARUP; Salt Lake City, Utah).

Determination of toxicity by measuring the weight loss of the body.Percentage change in body mass was determined in mice (5 mice/group) in certain selected days after injection of tumor (PTI) compared with the control animals and built schedule. As shown in figv, 4B, 5B, 6B, 7B, 8B and 10B, conjugated compounds described in this invention, showed equal or lesser toxicity compared to unconjugated compounds according to the percentage loss of body weight.

MTDappwhen single and multiple administrations in mice.Compounds described in this application can show a positive correlation between the number of hydrophilic spacer linker included in the conjugate, and the maximum tolerated dose of single administration in mice. For example, the following table placed data for conjugates of vinblastine described in this application, compared with the control conjugate.

ConnectionThe number of hydrophilic linkersMTDappfor one to the s (µmol/kg)
EC145015
EC0234112*
EC02462<20**
EC02633>20
* Dose limited by solubility; **1/3 of the mice died at 20 µmol/kg

EC0436 and conjugate comparative example EC0305 also injected i.v. mice Balb/c TIW within one week. The obtained values MTD with a repeated accounted for EC0305 (6 mmol/kg) and for EC0436 (9 mmol/kg). These data indicate that the dosage EC0436 can be 50% higher than EC0305.

Binding of serum.Binding of serum folate conjugates-DAVLBH, including hydrophilic spacer linker, in comparison with the conjugate of comparative example EC145, which has no hydrophilic spacer linker, when the content of the compound in the serum of 50 mm with 30K NMWL by filtration and the determination result by using HPLC with UV-detection (n=3).

The mouse serum (% binding)
ConnectionSerum human
(% binding)
SDSD
EC14554,31,667,32,6
EC039642,74,472,25,2
EC040061,11,975,51,4

Excretion of bile.Comparison of the excretion of bile (% ID) unconjugated drug, drug conjugate with a linker that does not contain a hydrophilic spacer linker, and conjugates of the present invention

EC0246
ConnectionThe presence of hydrophilic fragmentsExcretion of bile (%ID)
DAVLBHno58,0
EC145there is no hydrophilic spacer8,7
EC0234monorail10,6
bis-ribosylthe 4.7
EC0258three-ribosyl3,2
EC0434Tetra-ribosyl2,8
EC0400monoglucuronide6,3
EC0423bis-glucuronidea 3.9
EC0409PEG127,9
EC0429piperazine/Asp8,6

The results, shown in 11 and 13 indicate 76% decrease in excretion through the liver conjugate EC0434, which includes hydrophilic spacer linker, described in this application, compared to the standard conjugate EC145. Not limited to any theory, it can be assumed that these results are consistent with non-specific excretion through the liver, and, accordingly, assumes that you can enter significantly lower doses of those conjugates that include a hydrophilic structural spacer linker, described in the present invention, compared to the same is conjugate, which do not include such linkers. Further, not limited to a particular theory, I believe that the hepatic clearance leads to limiting the dosage of GI-related cytotoxicity that is observed in some conjugates.

Western blotting.Data on Fig show that the conjugate EC0565 (folate-sugar-everolimus) may cause dose-dependent and specific defeat targets mTOR, a downstream path of the transmission signal (intracellular targets for everolimus). Not limited to any theory, believed that folate contributes to the delivery of everolimus inside cells, where it inhibits mTOR, which is the target of rapamycin in mammals, and ser/thr kinase. Inhibition of subsequent targets mTOR (P70 S6 kinase and ribosomal S6) leads to the results shown in the Western band.

EXAMPLES of COMPOUNDS

Example. Methyl ether (3,4),(5,6)-betacatenin-D-gluconic acid. In a dry 250-ml round bottom flask in an argon atmosphere suspended δ-gluconolactone (4,14 g, 23,24 mmol) in a mixture of acetone-methanol (50 ml). To this suspension was added dimethoxypropane (17,15 ml, 139,44 mmol) and then a catalytic amount of p-toluensulfonate acid (200 mg). The resulting solution was stirred at room temperature for 16 hours TLC (50% EtOAc in petroleum EF is d) showed all source connections are entered into the reaction, and the formed reaction product. Acetone and methanol were removed under reduced pressure. The remainder of the reaction mixture was dissolved in EtOAc and washed with water. The organic layer was washed with saturated salt solution, dried over Na2SO4and concentrated to dryness. The obtained substance was introduced into a column with SiO2and chromatographically (30% EtOAc in petroleium ether)to give pure methyl ether (3,4),(5,6)-betacatenin-D-gluconic acid (3.8 g, 56%) and its Regio-isomer methyl ether (2,3),(5,6)-betacatenin-D-gluconic acid (0.71 g, 10%). Data1H NMR consistent with the desired product structure. C13H22O7; mol. weight 290,31; exact mass: 290,14.

Example. Methyl ether (3,4),(5,6)-betacatenin-2-OTf-D-gluconic acid. In a dry 100-ml round bottom flask in an argon atmosphere methyl ether (3,4),(5,6)-betacatenin-D-gluconic acid (3.9 g, 13,43 mmol) was dissolved in methylene chloride (40 ml) and cooled to a temperature of -20 -25°C. To this solution was added pyridine (3,26 ml, 40,29 mmol) and then anhydride triftormetilfullerenov acid (3,39 ml, 20,15 mmol). The obtained white turbid solution was stirred at -20°C for 1 h TLC (25% EtOAc in petroleum ether) showed that all of the original substance has entered the reaction and the formed reaction product. actionnow the mixture was poured into crushed ice and was extracted with diethyl ether. The organic layer was washed with water, saturated salt solution, dried over Na2SO4and concentrated, obtaining methyl ether (3,4),(5,6)-betacatenin-2-OTf-D-gluconic acid (5.5 g, 97%). The resulting substance was used in the subsequent syntheses without further purification. C14H21F3O9S; mol. weight 422,37; exact mass: 422,09.

Example.Methyl ether (3,4),(5,6)-betacatenin-2-deoxy-2-azido-D-mononova acid. In a dry 100-ml round bottom flask in an argon atmosphere was dissolved methyl ether (3,4),(5,6)-betacatenin-2-OTf-D-gluconic acid (5.5 g, 13,02 mmol) in DMF (20 ml). To this solution was added NaN3(0,93 g, 14,32 mmol). The resulting solution was stirred at room temperature for 1 h TLC (8% EtOAc in petroleum ether, triple pass) showed that all of the original substance has entered the reaction and the formed reaction product. DMF was removed under reduced pressure. The reaction mixture was diluted with saturated salt solution and was extracted with EtOAc. The organic layer was washed with water, saturated salt solution, dried over Na2SO4and concentrated to dryness. The crude product was then placed in a column with SiO2and purified by chromatography (20% EtOAc in petroleum ether)to give pure methyl ether (3,4),(5,6)-betacatenin-2-deoxy-2-azido-D-is annonomous acid (3.8 g, 93%). Data1H NMR consistent with the desired product structure. C13H21N3O6; mol. weight 315,32; exact mass: 315,14.

Example.Methyl ether (3,4),(5,6)-betacatenin-2-deoxy-2-amino-D-mononova acid. In the vessel Parra for hydrogenation of methyl ether (3,4),(5,6)-betacatenin-2-deoxy-2-azido-D-mononova acid (3.5 g, 11,10 mmol) was dissolved in methanol (170 ml). To this solution was added 10% Pd on coal (800 mg, 5% mole.). The hydrogenation was carried out using the unit for Parr hydrogenation at a pressure of 25 psi for 1 h TLC (10% methanol in methylene chloride) showed that all of the original substance has entered the reaction and the formed reaction product. The reaction mixture was filtered through a layer of celite and concentrated to dryness. Then the crude substance was placed in a column with SiO2and purified by chromatography (2% methanol in methylene chloride)to give pure methyl ether (3,4),(5,6)-betacatenin-2-deoxy-2-amino-D-mononova acid (2,61 g, 81%). Data1H NMR consistent with the desired product structure. C13H23NO6S; mol. weight 289,32; exact mass: 289,15.

Example.(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid. In a dry 100-ml round bottom flask was dissolved methyl ether (3,4),(56)-betacatenin-2-deoxy-2-amino-D-mononova acid (1.24 g, the 4.29 mmol) in a mixture of THF/MeOH (20 ml/5 ml). To this solution was added LiOH·H2O (215,8 mg, 5,14 mmol) in water (5 ml). The obtained pale-yellow solution was stirred at room temperature for 2 hours TLC (10% methanol in methylene chloride) showed that all of the original substance has entered the reaction and the formed reaction product. THF/MeOH was removed under reduced pressure. The aqueous phase resuspendable in a saturated solution of NaHCO3(10 ml). To this suspension was added Fmoc-Osu (1,74 g, 5,14 mmol) in 1,4-dioxane (10 ml). The obtained heterogeneous solution was stirred at room temperature for 18 hours TLC (10% methanol in methylene chloride) showed that a large part of the original substances entered into the reaction, and the formed reaction product. The dioxane was removed under reduced pressure. The aqueous layer was extracted with diethyl ether to remove less polar impurities. Then the aqueous layer was acidified to pH 6 using 0,2N HCl and again extracted with EtOAc. The EtOAc layer was washed with saturated salt solution, dried over Na2SO4and concentrated, obtaining (3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononofu acid (1.6 g, 76%). The resulting substance was used in the subsequent syntheses without further purification. Data1H NMR consistent with the desired product structure. C27H31NO8; mol. weight 497,54; exact mass: 497,20.

Example.EC0233 synthesized using PPPS (standard solid-phase peptide synthesis) in three stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,561.0 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,71,25497,540,348 g
Fmoc-Glu-OtBu1,122425,50,477 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,701,254080,286 g
2,244129,25 (d=0,742)0,390 ml
PyBOP1,1225200,583 g

Stage combination. In the vessel for the synthesis of peptides were added to the resin solution was added amino acids, DIPEA and PyBOP. Was barbotirovany argon for 1 h and 3 times washed with DMF and IPA. Before each stage combination to remove the Fmoc protection was used by 20% piperidine in DMF, 3×(10 min). In these conditions made all three stages combined. At the end of the washed resin with 2% hydrazine in DMF 3×(5 min) for removal of protective groups of paloemeu acid, i.e. TFA.

Stage removal. Reagent for cleavage: 92,5% (50 ml) TFA, 2.5% of (1,34 ml) H2O, and 2.5% (of 1.34 ml) triisopropylsilane, 2,5% (of 1.34 ml) acondition. Added 25 ml of a chip off the reagent and barbotirovany argon for 20 minutes, dried, and three times washed the remainder of the reagent. Was evaporated on a rotary evaporator to a residual volume of 5 ml and precipitated with ethyl ether. Centrifuged and dried.

Phase HPLC purification. Column: Waters NovaPak C18300×19 mm; buffer A=10 mm ammonium acetate, pH 5; B= ACN; method: 1% B To 20% B over 40 min at a speed of 15 ml/min; output ~202 mg, 50%. C28H35N9O12S; m is L. weight 721,70; exact mass: 721,21.

Example.The linker bis-sugar-folate EC0244. EC0244 synthesized using PPPS in five stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,561.0 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,71,25497,540,348 g
Fmoc-Asp(OtBu)-OH1,122411,50,461 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,71,25497,54 0,348 g
Fmoc-Glu-OtBu1,122425,50,477 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,701,254080,286 g
DIPEA2,244129,25 (d=0,742)0,390 ml
PyBOP1,1225200,583 g

Stage combination, the stage of detachment, a reagent for removal and cleaning stage HPLC corresponded to the above-described methods; output ~284 mg, 50%. C38H51N11O20S; mol. weight 1013,94; exact mass: 1013,30.

Example.EC0257 synthesized using PPPS in six stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

Mol. weight
ReagentsmmolEQ.Qty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,2of 0.333 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Asp(OtBu)-OH0,42411,50,165 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu-OtBu0,42425,50,170 g
N10TFA-peroia acid (dissolved in 10 ml of EBM is About) 0,251,254080,119 g
DIPEA0,84129,25 (d=0,742)0,139 ml
PyBOP0,425200,208 g

Stage combination, the stage of detachment, a reagent for removal and cleaning stage HPLC corresponded to the above-described methods; output ~170 mg, 71%. C44H62N12O25S; mol. weight 1191,09; exact mass: 1190,37.

Example.EC0261 synthesized using PPPS in seven stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,2 of 0.333 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Asp(OtBu)-OH0,42411,50,165 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Asp(OtBu)-OH0,42411,50,165 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu-OtBu0,42425,50,170 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,251,25 4080,119 g
DIPEA0,84129,25 (d=0,742)0,139 ml
PyBOP0,425200,208 g

Stage combination, the stage of detachment, a reagent for removal and cleaning stage HPLC corresponded to the above-described methods; output ~170 mg, 65%. C48H67N13O28S; mol. weight 1306,18; exact mass: 1305,39.

Example.The linker Tetra-sugar-Tris-Asp-folate EC0268. EC0268 synthesized using PPPS in nine stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,10.17 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540,062 g
Fmoc-Asp(OtBu)-OH0,22411,50,082 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540,062 g
Fmoc-Asp(OtBu)-OH0,22411,50,082 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540,062 g
Fmoc-Asp(OtBu)-OH0,22411,50,082 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,54 0,062 g
Fmoc-Glu-OtBu0,22425,5of 0.085 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,1251,254080,059 g
DIPEA0,44129,25 (d=0,742)0,070 ml
PyBOP0,225200.104 g g

Stage combination, the stage of detachment, a reagent for removal and cleaning stage HPLC corresponded to the above-described methods; output ~100 mg, 63%. C94H125N19O37S2; mol. weight 2177,24; exact mass: 2175,79.

The following illustrative examples can be obtained by the method of synthesis EC0268

Example.The linker Tetra-sugar-Asp-folate EC0463. EC0463 synthesized using PPPS in seven stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,10.167 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540,062 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540,062 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540,062 g
Fmoc-Asp(OtBu)-OH0,22411,50,082 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid 0,1251,25497,540,062 g
Fmoc-Glu-OtBu0,22425,5of 0.085 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,1251,254080,059 g
DIPEA0,44129,25 (d=0,742)0,070 ml
PyBOP0,225200.104 g g

Stage combination, the stage of detachment, a reagent for removal and cleaning stage HPLC corresponded to the above-described methods; output ~63 mg, 46%. C50H73N13O30S; mol. weight 1368,25; exact mass: 1367,43.

Example.The linker Tetra-sugar-bis-α-Glu-Arg-folate EC0480. EC0480 synthesized using PPPS in nine stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,2of 0.333 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2501,25497,540.124 g
Fmoc-Glu(OtBu)-OH0,42425,50,170 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2501,25497,540.124 g
Fmoc-Arg(Pbf)-OH0,42648,78is 0.260 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2501,25 497,540.124 g
Fmoc-Glu(OtBu)-OH0,42425,50,170 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1251,25497,540.124 g
Fmoc-Glu-OtBu0,42425,50,170 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,2501,254080,119 g
DIPEA0,84129,25 (d=0,742)0,139 ml
PyBOP0,425200,208 g

Stage combination, the stage of detachment, a reagent for removal and cleaning stage HPLC corresponded to the above-described methods; output ~100 mg, 33%. C62H94N18O20S; mol. weight 1667,58; the exact value of mA is si: 1666,59.

Example.The linker Tetra-sugar-bis-Asp-folate EC0452. EC0452 synthesized using PPPS in nine stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.6 mmol/g)0,150,250 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1881,25497,540,094 g
Fmoc-Asp(OtBu)-OH0,32411,50,123 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1881,25497,540.09 g
Fmoc-4-(2-amino-ethyl)-1-carboxymethyllysine the dihydrochloride0,32482,420,145 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1881,25497,540,094 g
Fmoc-Asp(OtBu)-OH0,32411,50,123 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,1881,25497,540,094 g
Fmoc-Glu-OtBu0,32425,50,128 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,1881,254080,077 g
DIPEA0,64129,25 (d=0,742)0.10 ml
PyBOP0,325200.156 g

Stage combination, the stage of detachment and reagent for cleavage corresponded to the above-described methods; phase HPLC purification. Column: Waters NovaPak C18300×19 mm; buffer A=10 mm ammonium acetate, pH 5; B= ACN; method: 1% B To 20% B over 40 min at a speed of 25 ml/min; output ~98 mg, 40%. C62H93N17O34S; mol. weight 1652,56; exact mass: 1651,58.

Example.The linker Tetra-sugar-bis-Asp-folate EC0457. EC0457 synthesized using PPPS in eight stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.6 mmol/g)0,20of 0.333 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-m is nonova acid 0,251,25497,540.124 g
Fmoc-Asp(OtBu)-OH0,301,5411,50,123 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Asp(OtBu)-OH0,301,5411,50,123 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu-OtBu0,301,5425,50,128 g
N10TFA-peroia acid (dissolved is 10 ml DMSO) 0,251,25408is 0.102 g
DIPEA2 EQ. Rel. amino acids129,25 (d=0,742)87 μl or
105 ál
PyBOP2 EQ. Rel. amino acids520260 mg or
312 mg

Stage combination, the stage of detachment and reagent for cleavage corresponded to the above-described methods; phase HPLC purification. Column: Waters NovaPak C18300×19 mm; buffer A=10 mm ammonium acetate, pH 5; B= ACN; method: 0% B To 20% B over 40 min at a speed of 25 ml/min; output ~210 mg, 71%. C54H78N14O33S; mol. weight 1483,34; exact mass: 1482,46.

Example.The linker Tetra-sugar-Tris-Glu-folate EC0477. EC0477 synthesized using PPPS in nine stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.M is L. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.6 mmol/g)0,20of 0.333 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu(OtBu)-OH0,301,5425,50,128 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu(OtBu)-OH0,301,5425,50,128 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu(OtBu)-OH0,30 1,5425,50,128 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,251,25497,540.124 g
Fmoc-Glu-OtBu0,301,5425,50,128 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,251,25408is 0.102 g
DIPEA2 EQ. Rel. amino acids129,25 (d=0,742)87 μl or 105 ál
PyBOP1 EQ. Rel. amino acids520130 mg and 156 mg

Stage combination, the stage of detachment and reagent for cleavage corresponded to the above-described methods; phase HPLC purification. Column: Waters NovaPak C18300×19 mm; buffer A=10 mm ammonium acetate, pH 5; B= ACN; method: 0% B To 20% B over 40 min at a speed of 25 ml/min; output ~220 mg, 67%. C61H89N 15O36S; mol. weight 1640,50; exact mass: 1639,53.

Example.EC0453 synthesized using PPPS according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Say.
weight
Qty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)rate £ 0.1620,290 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2031,25497,540,101 g
Fmoc-Asp(OtBu)-OH0,3242411,5of 0.133 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2031,25497,54 0,101 g
Fmoc-Asp(OtBu)-OH0,3242411,5of 0.133 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2031,25497,540,101 g
Fmoc-Asp(OtBu)-OH0,3242411,5of 0.133 g
(3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid0,2031,25497,540,101 g
Fmoc-Glu-OtBu0,3242425,5was 0.138 g
N10TFA-peroia acid (dissolved in 10 ml DMSO)0,2031,254080.083 g
DIPEA2 EQ. Rel. amino acids129,25 (d=0,742)71 μl or 85 ál
PyBOP1 EQ. Rel. amino acids520211 mg or 253 mg

Stage combination. In the vessel for the synthesis of peptides were added to the resin solution was added amino acids, DIPEA and PyBOP. Was barbotirovany argon for 1 h and 3 times washed with DMF and IPA. Before each stage combination to remove the Fmoc protection was used by 20% piperidine in DMF, 3×(10 min). In these conditions made all nine stages of the combination. At the end of the treated resin with 2% hydrazine in DMF 3× (5 min) for removal of protective groups of paloemeu acid, i.e. TFA, washed resin DMF (3×), IPA (3×), MeOH (3×) and barbotirovany through the resin argon for 30 minutes

Stage removal. Reagent for cleavage: 92,5% TFA, 2.5% of H2O, 2.5% triisopropylsilane, 2,5% acondition. Processed resin chip off the reagent 3 times (15 min, 5 min, 5 min) by passing argon, dried, collected and combined solutions. Was evaporated on a rotary evaporator to a residual volume of 5 ml and precipitated in diethyl ether (35 ml). Centrifuged, washed with diethyl ether, and dried. The crude solid was purified HPLC.

Stage purification HPLC. Column: Waters Xterra Prep MS C1810 μm 19×250 mm; solvent A=10 mm ammonium acetate, pH 5; B= ACN; method: 5 min 0% B to 40 min 20% B 25 ml/is in; fractions containing the product were collected and subjected to freeze drying, getting ~60 mg EC0453 (yield 23%). Data1H NMR and LC/MS were consistent with the desired product structure.

C58H83N15O36S; mol. weight 1598,43; exact mass: 1597,48. C, 43,58; H, 5,23; N, 13,14; O 36,03; S 2,01.

Example. (3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid-diazoketone. In a dry round-bottom 100 ml flask was dissolved (3,4),(5,6)-biatlone-2-deoxy-2-Fmoc-amino-D-mononofu acid (1.0 g, a 2.01 mmol) in THF (10 ml, dissolution was not completely) in an argon atmosphere. The reaction mixture was cooled to -25°C. To this solution was added NMM (0,23 ml, 2,11 mmol) and ethylchloride (228,98 mg, 2,11 mmol). The resulting solution was stirred at -20°C within 30 minutes of the Obtained white suspension was allowed to warm to 0°C was added a solution of diazomethane in the air until a steady yellow. Mixing continued until the mixture was warmed to room temperature. After 2 h stirring, the excess diazomethane was dissolved in few drops of acetic acid with vigorous stirring. The resulting mixture was diluted with ether, washed with saturated aqueous NaHCO3, saturated aqueous NH4Cl, saturated salt solution, dried over Na2SO4and concentrated to dryness. who received the crude substance was injected into the column with SiO 2and purified by chromatography (30% EtOAc in petroleum ether)to give pure (3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid-diazoketone (0.6 g, 57%). Data spectroscopy1H NMR consistent with the desired product structure. C28H31N3O7; mol. weight 521,56; exact mass: 521,22.

Example.(3R,4R,5S,6R)-(4,5),(6,7)-betacatenin-3-Fmoc-aminoheptanoic acid. In a dry 25-ml round bottom flask was dissolved (3,4),(5,6)-betacatenin-2-deoxy-2-Fmoc-amino-D-mononova acid-diazoketone (0.15 g, 0.29 mmol) in THF (1.6 ml) in an argon atmosphere. To this solution in the dark was added triptorelin silver (6.6 mg, 0.03 mmol) in water (0.4 ml). The resulting mixture was stirred at room temperature for 16 hours TLC (10% MeOH in methylene chloride) showed that all of the original substance has entered the reaction and the formed reaction product. The solvent (THF) was removed under reduced pressure, the residue was diluted with water (pH was 3.5 to 4.0) and was extracted with EtOAc. The organic layer was washed with saturated salt solution, dried over Na2SO4and concentrated to dryness. Then the crude substance was injected into the column with SiO2and purified by chromatography (gradient elution from 1% MeOH in methylene chloride to 5% MeOH in methylene chloride)to give pure (3R,4R,5S,6R)-(4,5),(6,7)-betacatenin-3-Fmoc-amine the heptane acid (0.10 g, 68%). Data spectroscopy1H NMR consistent with the desired product structure. C28H33NO8; mol. weight 511,56; exact mass: 511,22.

Example.The spacer Tetra-gameshare-Tris-α-Glu-folate EC0478. EC0478 synthesized using PPPS in nine stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,10.167 g
Homosexal0,121,2511,560,061 g
Fmoc-Glu(OtBu)-OH0,22425,5of 0.085 g
Homosexal0,121,2 511,560,061 g
Fmoc-Glu(OtBu)-OH0,22425,5of 0.085 g
Homosexal0,121,2511,560,061 g
Fmoc-Glu(OtBu)-OH0,22425,5of 0.085 g
Homosexal0,121,2511,560,061 g
Fmoc-Glu-OtBu0,22425,5of 0.085 g
N10TFA-peroia acid·TFA (dissolved in 10 ml DMSO)0,121,24080,049 g
DIPEA0,44129,25 (d=0,742)0,070 ml
PyBOP0,2 25200.104 g g

Stage combination, the stage of detachment and reagent for cleavage corresponded to the above-described methods; phase HPLC purification. Column: Waters NovaPak C18300×19 mm; buffer A=10 mm ammonium acetate, pH 5; B=ACN; method: 100% a for 5 min, then from 0% to 20% B over 20 minutes at a speed of 26 ml/min; output ~88 mg, 52%. C65H97N15O36S; mol. weight 1696,61; exact mass: 1695,59.

Example.Amide (3,4),(5,6)-betacatenin-D-gluconic acid. 20 g of methyl ester was dissolved in 100 ml of methanol, cooled using a mixture of dry ice/acetone in a vessel for carrying out reactions under high pressure, was introduced into the vessel 100 ml of liquid ammonia was heated to room temperature and was heated at 160°C/850 psi for 2 hours. The reaction vessel was cooled to room temperature and reset the pressure. Evaporation of the solvent was allowed to get a brownish syrup, to which was added a minimum amount of isopropyl alcohol to obtain a homogeneous solution while boiling under reflux. This solution was cooled to -20°C and the resulting solid was filtered, receiving of 8.3 g of solid substance. The mother solution was evaporated and to the obtained residue was added ether and boiled until the obrazovaniya homogeneous solution. Then the solution was cooled to -20°C and released in this solid was filtered, obtaining 4.0 g of product. The solid products were combined and recrystallized from isopropyl alcohol, getting 11.2 g (59%) of amide white. C12H21NO6; mol. weight 275,30; exact mass: 275,14.

Example. (3,4),(5,6)-betacatenin-1-deoxy-1-amino-D-glucitol. In a dry 100-ml round bottom flask in an argon atmosphere was dissolved LiAlH4(450 mg, up 11,86 mmol) in THF (10 ml) and cooled to 0°C. To this suspension is very slowly over 15 min was added to the solution of amide (3,4),(5,6)-betacatenin-D-gluconic acid (1,09 g of 3.96 mmol) in THF (30 ml). The resulting mixture was boiled under reflux for 5 hours TLC (10% MeOH in methylene chloride) showed that all of the original substance has entered the reaction and the formed reaction product. The reaction mixture was cooled to room temperature and then cooled to the temperature of the ice bath, diluted with diethyl ether (40 ml), was slowly added 0.5 ml water, 0.5 ml of 15% aqueous NaOH solution and then added 1.5 ml of water. The reaction mixture was heated to room temperature and was stirred for 30 minutes was Added MgSO4was stirred for another 15 min and filtered. The organic layer was concentrated to dryness, obtaining (3,4),(5,6)-betacatenin-1-deoxy-1-amino-D-glucitol. Data spec is roscopy 1H NMR consistent with the desired product structure. C12H23NO5; mol. weight 261,31; exact mass: 261,16.

Example.EC0475. O-allyl protected Fmoc-Glu (2.17 g, 1 EQ.), PyBOP (2,88 g, 1 EQ.) and DIPEA (1,83 ml, 2 EQ.) was added to a solution of (3,4),(5,6)-betacatenin-1-deoxy-1-amino-D-glucitol (1.4 g, 5.3 mmol) in dry DMF (6 ml) and the mixture was stirred at RT in the atmosphere of Ar for 2 h the resulting solution was diluted with EtOAc (50 ml), washed with saturated salt solution (10 ml × 3), the organic layer was separated, dried (MgSO4), filtered and concentrated, obtaining a residue that was purified column flash chromatography (silica gel, 60% EtOAc/petroleum ether)to give 1,72 g (50%) protected allyl EC0475 in the form of solids. To a solution of protected allyl EC0475 (1,72 g of 2.81 mmol) in NMM/AcOH/CHCl3(2 ml/4 ml/74 ml) was added Pd(Ph3)4(300 mg, 0.1 EQ.). The obtained yellow solution was stirred at RT in the atmosphere of Ar for 1 h and then added to it the second portion of Pd(Ph3)4(300 mg, 0.1 EQ.). After stirring for a further 1 h the mixture was washed with 1N HCl (50 ml × 3) and saturated salt solution (50 ml), the organic layer was separated, dried (MgSO4), filtered and concentrated, obtaining a yellow foamy solid, which was purified by chromatography (silica gel, 1% MeOH/CHCl3and then 1% MeOH/CHCl3 )to give 1.3 g (81%) EC0475 in the form of solids. Mol. weight 612,67; exact mass: 612,27.

Example.The spacer Tetra-SharePoint-bis-α-Glu-folate EC0491. EC0491 synthesized using PPPS in eight stages according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

EC0475
ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.56 mmol/g)0,10.167 g
EC04750,131,3612,670,080 g
Fmoc-Glu(OtBu)-OH0,22425,5of 0.085 g
EC04750,131,3612,670,080 g
0,131,3612,670,080 g
Fmoc-Glu(OtBu)-OH0,22425,5of 0.085 g
EC04750,131,3612,670,080 g
Fmoc-Glu-OtBu0,22425,5of 0.085 g
N10TFA-peroia acid·TFA (dissolved in 10 ml DMSO)0,224080,105 g
DIPEA0,44129,25 (d=0,742)0,070 ml
PyBOP0,225200.104 g g

Stage combination, the stage of detachment and reagent for cleavage corresponded to the above-described methods; phase HPLC purification: Column: Waters NovaPak C18300×19 mm; buffer A=10 mm acetate is monia, pH 5; B=ACN; method: 100% a for 5 min, then from 0% To 20% B over 20 minutes at a speed of 26 ml/min; output ~100 mg, 51%. C76H118N18O41S; mol. weight 1971,91; exact mass: 1970,74.

Example.EC0479 synthesized using PPPS according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

0,082 g 122 mg or
147 mg
ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.6 mmol/g)0,0940.16 g
EC04750,131,4612,670,082 g
Fmoc-Glu(OtBu)-OH0,192,0425,470,080 g
EC04750,131,4612,67
Fmoc-Arg(Pbf)-OH0,192,0648,770.12 g
EC04750,131,4612,670,082 g
Fmoc-Glu(OtBu)-OH0,192,0425,470,080 g
EC04750,131,4612,670,082 g
Fmoc-Glu-OtBu0,192,0425,470,080 g
N10TFA-peroia acid·TFA (dissolved in 10 ml DMSO)0,161,7408,29of 0.066 g
DIPEA2.0 EQ. AA41 µl or
49 ál
PyBOP1,0 EQ. AA

Stage combination. In the vessel for the synthesis of peptides were added to the resin solution was added amino acids, DIPEA and PyBOP. Was barbotirovany argon for 1 h and 3 times washed with DMF and IPA. Before each stage combination of amino acids for removal of the Fmoc protection was used by 20% piperidine in DMF, 3×(10 min). In these conditions made all nine stages of the combination. At the end of the treated resin with 2% hydrazine in DMF 3× (5 min) for removal of protective groups of paloemeu acid TFA, washed resin DMF (3×), IPA (3×), MeOH (3×) and the resin was barbotirovany argon for 30 minutes

Stage removal. Reagent for cleavage: 92,5% TFA, 2.5% of H2O, 2.5% triisopropylsilane, 2,5% acondition. Processed resin chip off the reagent 3 times in 15 min by passing argon, dried, washed resin decomposing reagent another time and combined solution. Was evaporated on a rotary evaporator to a residual volume of 5 ml and precipitated in diethyl ether (35 ml). Centrifuged, washed with diethyl ether, and dried. The crude solid was purified HPLC.

Stage purification HPLC. Column: Waters Atlantis Prep T3 10 μm OBD 19×250 mm; solvent A: 10 mm ammonium acetate, pH 5; solvent B: ACN; method: 5 min 0% B to 20 min 20% B 26 ml/min; fractions containing the product were collected and subjected to freeze drying, getting ~70 mg EC0479 (yield 35%). Data1 H NMR and LC/MS were consistent with the desired product structure.

Mol. weight 2128,10; exact mass: 2126,84.

EC0488.This compound was synthesized using PPPS according to the General methods of peptide synthesis described in this application, on the basis of H-Cys(4-methoxytrityl)-2-chlorotrityl-resin and the following PPPS reagents:

0,19
ReagentsmmolEQ.Mol. weightQty
H-Cys(4-methoxytrityl)-2-chlorotrityl-resin (content of active substance of 0.6 mmol/g)0,100.17 g
EC04750,131,3612,670,082 g
Fmoc-Glu(OtBu)-OH0,191,9425,470,080 g
EC04750,131,3612,670,082 g
Fmoc-Glu(OtBu)-OH1,9425,470,080 g
EC04750,131,3612,670,082 g
Fmoc-Glu-OtBu0,191,9425,470,080 g
N10TFA-peroia acid·TFA (dissolved in 10 ml DMSO)0,161,6408,29of 0.066 g
DIPEA2.0 EQ. AA
PyBOP1,0 EQ. AA

Stage combination. In the vessel for the synthesis of peptides were added to the resin solution was added amino acids, DIPEA and PyBOP. Was barbotirovany argon for 1 h and 3 times washed with DMF and IPA. Before each stage combination of amino acids for removal of the Fmoc protection was used by 20% piperidine in DMF, 3×(10 min). In these conditions made all nine stages of the combination. In conclusion, processed, see the Lu 2% hydrazine in DMF 3×(5 min) for removal of protective groups of paloemeu acid TFA, washed the resin DMF (3×), IPA (3×), MeOH (3×) and the resin was barbotirovany argon for 30 minutes

Stage removal. Reagent for cleavage: 92,5% TFA, 2.5% of H2O, 2.5% triisopropylsilane, 2,5% acondition. Processed resin chip off the reagent 3 times (10 min, 5 min, 5 min) by passing argon, dried, washed resin chip off the reagent another time and combined solutions. Was evaporated on a rotary evaporator to a residual volume of 5 ml and precipitated in diethyl ether (35 ml). Centrifuged, washed with diethyl ether, and dried. About half of the crude solids were purified HPLC.

Stage purification HPLC. Column: Waters Xterra Prep MS C18 10 μm, 19×250 mm; solvent A: 10 mm ammonium acetate, pH 5; solvent B: ACN; method: 5 min 0% B to 25 min 20% B 26 ml/min; fractions containing the product were collected and subjected to freeze drying, getting ~43 mg EC0488 (51%yield). Data1H NMR and LC/MS (exact mass value 1678,62) corresponded to the desired product structure.

Mol. weight 1679,63; exact mass: 1678,62.

As described in this application techniques received the following examples of intermediate compounds binding the ligand-linker: EC0233, EC0244, EC0257 and EC0261

EC0233; C28H35N9O12S; mol. weight 721,70; exact mass: 721,21

EC024; C38H51N11O20S; mol. weight 1013,94; exact mass: 1013,30

EC0257; C44H62N12O25S; mol. weight 1191,09; exact mass: 1190,37

EC0261; C48H67N13O28S; mol. weight 1306,18; exact mass: 1305,39

The following examples are illustrative of the intermediate compounds were obtained as described in this application.

Huisgen azide to obtain 1,2,3-triazole; (a) NaN3; (b) Ag2CO3, DCM, molecular sieve; (c) LiOH, MeOH, H2O.

EC0501 Timorese

EC0536 Intermediate compound conjugate

EC0632 Intermediate compound conjugate. C52H72N14O28S; mol. weight 1373,27; exact mass: 1372,44 derived from the corresponding tert-butylamine carboxylates.

EC0669 Intermediate compound conjugate. C49H71N13O24S; mol. weight 1258,23; exact mass: 1257,45

Example.Synthesis of cross-linking reagent EC0311. DIPEA (of 0.60 ml) was added to a suspension of HOBt-OCO2-(CH2)2-SS-2-pyridine·HCl (685 mg, 91%) in anhydrous DCM (5.0 ml) at 0°C, stirred in an atmosphere of ar is she in for 2 minutes and to the mixture was added anhydrous hydrazine (0.10 ml). The reaction mixture was stirred in argon atmosphere at 0°C for 10 minutes and at room temperature for another 30 min, filtered and the filtrate was purified flash chromatography (silica gel, 2% MeOH in DCM)to give EC0311 in the form of a clear thick oil (371 mg)which solidified on standing.

Example.Vinblastine pyridinyl disulfide. 2-[(benzotriazol-1-yl-(oxycarbonate)tildesley)]pyridine·HCl (601 mg) and 378 μl DIPEA sequentially added to a solution of desacetyl vinblastine hydrazide (668 mg) in 5 ml of DCM at 0°C. the Reaction mixture was allowed to warm to room temperature and was stirred for 3 hours. TLC (15% MeOH in DCM) showed complete conversion of the starting materials. The resulting mixture was purified by chromatography on silica gel (1:9 MeOH/DCM). Combined fractions were evaporated, re-dissolved in DCM and washed with a 10% solution of Na2CO3, a saturated solution of salt, dried (MgSO4) and was evaporated, getting 550 mg (80%) of product; HPLC RT 12,651 min, purity 91%,1H NMR spectrum consistent with the structure assigned to the product, MS(ESI+): 984,3, 983,3, 982,4, 492,4 491,9, 141,8. For more information about the methodology described in published application US patent # US 2005/0002942 A1.

Example.Getting hydrazides tubulysin. Shown at the example of a EC0347. N,N (DIPEA, 6,1 μl) and isobutylparaben (3,0 µl) were co-added with a syringe to a solution of tubulysin B (0.15 mg) in anhydrous EtOAc (2.0 ml) at -15°C. After stirring for 45 minutes at -15°C in argon atmosphere, the reaction mixture was cooled to -20°C and to it was added anhydrous hydrazine (5,0 ál). The reaction mixture was stirred in argon atmosphere at -20°C for 3 hours, extinguished 1.0 mm sodium-phosphate buffer (pH 7.0, 1.0 ml) was injected into the instrument for preparative HPLC to clean. Column: Waters Xterra Prep MS C1810 μm 19×250 mm; mobile phase A: 1.0 mm sodium-phosphate buffer, pH 7.0; mobile phase B: acetonitrile; method: 10% B to 80% B over 20 min, flow rate 25 ml/min Fraction 15,14-15,54 min assembled and liofilizovane getting EC0347 in the form of a white solid (2.7 mg). The above procedure is applicable also to obtain hydrazides other tubulysins an appropriate source tubulysins.

Example.Getting disulfides tubulysin (stepwise method). Shown at the example of a EC0312. DIPEA (36 μl) and isobutylparaben (13 μl) together was added via syringe to the solution tubulysin B (82 mg) in anhydrous EtOAc (2.0 ml) at -15°C. After stirring for 45 min at -15°C in an atmosphere of argon to the reaction mixture solution was added EC0311 in anhydrous EtOAc (1.0 ml). The resulting solution paramesh the Wali in the atmosphere of argon at -15°C for 15 minutes and at room temperature for a further 45 minutes, concentrated and the residue was purified flash chromatography (silica gel, 2-8% MeOH in DCM)to give EC0312 in the form of a white solid (98 mg). The above method is applicable also to obtain derivatives other tubulysins with appropriate choice of initial tubulysin.

Example.General methods of synthesis of disulfide-containing conjugates tubulysin. Shown at the example of a EC0312. The intermediate connection linking the ligand-linker containing Tilney group, was mixed with deionized water (approximately 20 mg/ml, through which before use missed argon for 10 minutes) and the pH of the suspension was made by adding a saturated solution of NaHCO3(before using missed argon for 10 minutes) to the value of approximately 6,9 (suspension can move in solution with increasing pH). If necessary, to the solution was added an additional amount of deionized water (approximately 20-25%) and to the resulting aqueous solution was immediately added to the solution EC0312 in THF (approximately 20 mg/ml). The reaction mixture quickly became homogeneous. After stirring in an argon atmosphere, for example, within 45 minutes, the reaction mixture was diluted with 2.0 mm sodium phosphate buffer (pH 7.0 to about 150 volume percent) and THF was removed in vacuum. is received, the suspension was filtered, and the filtrate was clear preparative HPLC (as described in this application). The desired fraction was liofilizovane to highlight conjugates. The above method is applicable also to obtain conjugates other tubulysins with appropriate choice other source tubulysins.

Comparative example. Obtaining a conjugate of vinblastine EC145, which lacks the hydrophilic spacer linker. Pipidinny fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH (example 13) in THF was treated with either thiosulfonate-activated vinblastine, or vinblastine pyridinyl by disulfide as a yellow solution obtained by dissolving in 0.1 M NaHCO3at pH>6.5 in argon atmosphere. Lyophilization and HPLC was possible to obtain a conjugate with the release of 70%; the fragments of the spectrum1H NMR (D2O) δ 8,67 (s, 1H, folic acid N-7), 7,50 (users, 1H, VLB, H-11'), 7,30-7,40 (users, 1H, VLB, H-14'), 7,35 (d, 2H, J=7.8 Hz, folic acid N-12 and 16), 7,25 (m, 1H, VLB, H-13'), 7,05 (users, 1H, VLB, H-12'), 6,51 (d, 2H, J=8.7 Hz folic acid H-13 and 15), 6,4 (c, 2H, VLB H-14 and 17)and 5.7 (m, 1H, VLB olefin) the 5.65 (m, 1H, VLB, H-7), and 5.5 (d, 1H, VLB olefin), and 5.5 (m, 1H, VLB, H-6), is 4.15 (m, 1H, VLB, H-8'), 3,82 (s, 3H, VLB C18'-CO2CH3), of 3.69 (s, 3H, VLB C16-OCH3), and 2.8 (s, 3H, VLB N-CH3), 1,35 (users, 1H, VLV H-3')and 1.15 (m, 1H, VLB, H-2'), and 0.9 (t, 3H, J=7 Hz, VLB, H-21'), 0,55 (t, 3H, J=6,9 Hz, VLB, H-21); LC-MS (ESI, m+H+) 1918.

Example. E0234 (conjugate mono-sugar-folate-vinblastine) comprising hydrophilic spacer linker. In polypropylene containers for centrifugation folate-containing linker (EC0233, 22 mg, 0,030 mmol) was dissolved in 2 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (27 mg, 0,028 mmol) in 2 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min-1 h the reaction course was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-sugar-folate (EC0234) was isolated after lyophilization for 48 h (34 mg, 76%). Data1H NMR consistent with the desired structure of the conjugate of folate. C74H93N15O21S2; mol. weight 1592,75; exact mass: 1591,61.

Example. EC0246 (conjugate bis-sugar-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0244, 30 mg, 0,030 mmol) was dissolved in ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (27 mg, 0,028 mmol) in 5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min-1 h the reaction course was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-bis-sugar-folate (EC0246) was isolated after lyophilization for 48 h (34 mg, 66%). Data1H NMR consistent with the desired structure of the conjugate of folate. C84H109N17O29S2; mol. weight 1884,99; exact mass: 1883,70.

Example. EC0258 (conjugate Tris-sugar-Asp-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0257, 37 mg, 0,031 mmol) was dissolved in 5 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution linker is gently increased to 6.9, using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (27.5 mg, 0,028 mmol) in 5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min-1 h the reaction course was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tris-sugar-Asp-folate (EC0258) was isolated after lyophilization for 48 h (36 mg, 62%). Data1H NMR consistent with the desired structure of the conjugate of folate. C90H120N18O34S2; mol. weight 2062,15; exact mass: 2060,77.

Example. EC0263 (conjugate Tris-sugar-bis-Asp-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0261, 37 mg, 0,029 mmol) was dissolved in 5 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine Piri is inil disulfide (25,5 mg, it was 0.026 mmol) in 5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min-1 h the reaction course was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tris-sugar-bis-Asp-folate (EC0263) was isolated after lyophilization for 48 h (36 mg, 64%). Data1H NMR consistent with the desired structure of the conjugate of folate. C94H125N19O37S2; mol. weight 2177,24; exact mass: 2157,79.

Example. EC0434 (conjugate Tetra-sugar-Tris-Asp-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0268, 20 mg, 0.012 mmol) was dissolved in 3 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (12 mg, 0.012 mmol) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred at which osphere argon for 15 min-1 hour Over the course of the reaction was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-sugar-Tris-Asp-folate (EC0434) was isolated after lyophilization for 48 h (26 mg, 62%). Data1H NMR consistent with the desired structure of the conjugate of folate. C104H141N21O45S2; mol. weight 2469,48; exact mass: 2467,88.

Example. EC0454 (conjugate Tetra-sugar-bis-Asp-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0452, 34 mg, 0.02 mmol) was dissolved in 3 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (20 mg, 0.02 mmol) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min-1 h the reaction course was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0, Aceto ITIL). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-sugar-bis-Asp-folate (EC0454) was isolated after lyophilization for 48 h (35 mg, 70%). Data1H NMR consistent with the desired structure of the conjugate of folate. C108H151N23O43S2; mol. weight 2523,62; exact mass: 2521,98.

Example. EC0455 (conjugate Tetra-sugar-bis-Asp-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0457, 20 mg of 0.013 mmol) was dissolved in 1.5 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (18 mg, 0.018 mmol) in 1.5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 30 minutes Over the course of the reaction was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column DL is preparative HPLC (column X-terra C 18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-sugar-bis-Asp-folate (EC0455) was isolated after lyophilization for 48 h (19 mg, 62%). Data1H NMR consistent with the desired structure of the conjugate of folate. C100H136N20O42S2; mol. weight 2354,39.

Example. EC0456. In polypropylene containers for centrifugation folate-containing linker (EC0453, 46 mg, 0,029 mmol) was dissolved in 3 ml of water, through which there barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 minutes in a saturated solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using the solution of NaHCO3. To the resulting solution was quickly added vinblastine pyridinyl disulfide (32 mg, 1.1 EQ.) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere. Over the course of the reaction was monitored by analytical HPLC (2 mm phosphate buffer, pH=7.0 and acetonitrile). After 30 min to the reaction mixture were added 12 ml of 2 mm phosphate buffer (pH 7), resulting turbid solution was filtered and the filtrate was injected into a column for preparative HPLC: column Waters Xterra Prep MS C18, 10 μm 19×250 mm; solvent A: 2 mm sodium phosphate pH 7; solvent B: ACN; method: 5 min 1% B to 40 min 80%B 25 ml/min Fractions containing EC0456, was collected and subjected to freeze drying, getting to 41.6 mg friable yellow solid containing 30 mg EC0456 (yield 42%) and 11.6 sodium phosphate salts. Data1H NMR and LC/MS were consistent with the desired product structure. C104H141N21O45S2; mol. weight 2469,48; exact mass: 2467,88. C, 50,58; H, USD 5.76; N, 11,91; O, 29,15; S 2,60.

Example. EC0481. In polypropylene containers for centrifugation folate-containing linker (EC0479, 12 mg, 0,0058 mmol) was dissolved in 2.5 ml of water, through which there barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 minutes in a saturated solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using the solution of NaHCO3. To the resulting solution was quickly added vinblastine pyridinyl disulfide (5.7 mg, 1.0 EQ.) in 2.5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere. Over the course of the reaction was monitored by analytical HPLC (2 mm phosphate buffer, pH=7.0 and acetonitrile). After 20 min to the reaction mixture were added 12 ml of 2 mm phosphate buffer (pH 7), resulting turbid solution was filtered and the filtrate was injected into a column for preparative HPLC: column Waters Atlantis Prep T3 10 μm OBD 19×250 mm; solvent A: 2 mm sodium phosphate pH 7; solvent B: ACN; method: 5 min 1% B to 25 min 50% B26 ml/min Fractions containing EC0481, was collected and subjected to freeze drying, getting to 15.5 mg friable yellow solid, containing 10.5 mg EC0481 (yield 60%) and 5.0 sodium phosphate salts. Data1H NMR and LC/MS were consistent with the desired product structure. Mol. weight 2999,15; exact mass: 2997,24.

Example. EC0484 (conjugate Tetra-sugar-bis-α-Glu-Arg-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0480, 15 mg, 0,009 mmol) was dissolved in 3 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (8,8 mg, 0,009 mmol) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min - 1H. Over the course of the reaction was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-sugar-bis-α-Glu-Arg-folate (EC0484) in delali after lyophilization for 48 h (16 mg, 70%). Data1H NMR consistent with the desired structure of the conjugate of folate. C108H152N24O43S2; mol. weight 2538,63; exact mass: 2536,99.

Example. EC0487 (conjugate Tetra-sugar-Asp-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0463, 21 mg, 0.015 mmol) was dissolved in 3 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (15 mg, 0.015 mmol) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min - 1 h the reaction course was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column Atlantis, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-sugar-Asp-folate (EC0487) was isolated after lyophilization for 48 h (28 mg, 84%). Data1H NMR consistent with the desired structure of the conjugate of folate. C96H131N19 O39S2; mol. weight 2239,30; exact mass: 2237,83.

Example. EC0489. In polypropylene containers for centrifugation folate-containing linker (EC0488, 26 mg, 0.015 mmol) was dissolved in 2.5 ml of water, through which there barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 minutes in a saturated solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using the solution of NaHCO3. To the resulting solution was quickly added vinblastine pyridinyl disulfide (15 mg, 1.0 EQ.) in 2.5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere. Over the course of the reaction was monitored by analytical HPLC (2 mm phosphate buffer, pH=7.0 and acetonitrile). After 20 min to the reaction mixture were added 12 ml of 2 mm phosphate buffer (pH 7), resulting turbid solution was filtered and the filtrate was injected into a column for preparative HPLC: column Waters Xterra Prep MS C1810 μm 19×250 mm; solvent A: 2 mm sodium phosphate pH 7; solvent B: ACN; method: 5 min 1% B to 25 min 50% B 26 ml/min Fractions containing EC0489, was collected and subjected to freeze drying, receiving 35 mg friable yellow solid containing 27.5 mg EC0489 (yield 71%) and 7.5 sodium phosphate salts. Data1H NMR and LC/MS were consistent with the desired product structure. Mol. weight 2550,68; exact mass: 259,01.

Example. EC0490 (conjugate Tetra-gameshare-Tris-αGlu-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0478, 22 mg of 0.013 mmol) was dissolved in 3 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was slowly added vinblastine pyridinyl disulfide (mg, mmol) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min - 1H. Over the course of the reaction was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-gameshare-Tris-Glu-folate (EC0490) was isolated after lyophilization for 48 h (15 mg, 45%). Data1H NMR consistent with the desired structure of the conjugate of folate. C111H155N21O45S2; mol. weight 2567,66; exact mass: 2565,99.

Example. EC0492 (conjugate Tetra-gameshare-the rice-αGlu-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0491, 26 mg of 0.013 mmol) was dissolved in 3 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was added vinblastine pyridinyl disulfide (13 mg, of 0.013 mmol) in 3 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 15 min - 1H. Over the course of the reaction was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH 7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-gameshare-Tris-Glu-folate (EC0492) was isolated after lyophilization for 48 h (22 mg, 60%). Data1H NMR consistent with the desired structure of the conjugate of folate. C122H176N24O50S2; mol. weight 2842,97; exact mass: 2841,14.

Example. EC0493 (conjugate Tetra-sugar-Tris-Glu-folate-vinblastine). In polypropylene containers for centrifugation folate-containing linker (EC0477, 25 mg, 0.015 g is mol) was dissolved in 1.5 ml of water and was barbotirovany argon for 10 minutes In another vessel was barbotirovany argon for 10 min in 0,1N solution of NaHCO3. the pH of the solution of the linker was carefully raised to 6.9 using 0,1N solution of NaHCO3. To the resulting solution was added vinblastine pyridinyl disulfide (20 mg, at 0.020 mmol) in 1.5 ml of tetrahydrofuran (THF). The obtained clear solution was stirred in an argon atmosphere for 30 minutes Over the course of the reaction was monitored by analytical HPLC (10 mm ammonium acetate, pH=7.0 and acetonitrile). THF was removed under reduced pressure, the aqueous solution was filtered and injected into the column for preparative HPLC (column X-terra C18, 19×300 mm). Elution 1 mm sodium phosphate pH=7.0 and acetonitrile was possible to obtain fractions containing pure product. Conjugate vinblastine-Tetra-sugar-Tris-Glu-folate (EC0493) was isolated after lyophilization for 48 h (23 mg, 61%). Data1H NMR consistent with the desired structure of the conjugate of folate. C107H147N21O45S2; mol. weight 2511,56; exact mass: 2509,93.

Example.EC0429. The compound of this example, including hydrophilic oligoamine the spacer, representing AMINOETHYLPIPERAZINE Asp-Asp-Cys, was obtained by the method described in this application.

The following illustrative examples glucuronide-containing compounds, namely EC0400 and EC0423, where the group, enabling the second saccharide, illustrative introduced with the use of “click chemistry”, also received, as described in this application.

The following illustrative examples in connection with PEG-spacer, namely EC0367 and EC0409 also received, as described in the present invention.

The following illustrative examples of the compounds including fragments Olkiluoto ether sulfuric acid, namely EC0418 and EC0428, where a fragment of sulfuric acid illustrative entered using “click chemistry”was received as described in this application.

The following illustrative examples of additional compounds, including oligoamine the spacer, where oligoimide includes a derived EDTE received, as described in this application.

EC0396 Conjugate DAVLBH

The following illustrative examples of the compounds including β-Alkylglucoside 2-diethoxyacetophenone, and connections with PEG linker can be obtained, as described in this application, with the use of “click chemistry” for attaching hydrophilic groups on the spacer linker.

conjugate DAVLBH, including β-alkyl 2-desoxyglucose

conjugate DAVLBH, including β-alkyl 2-deoxyglucosone

conjugate DAVLBH, including PEG

conjugate DAVLBH, including β-alkyl mannopyranoside

Comparative exampleconjugate tubulysin. EC0305, not containing hydrophilic spacers in the linker. EC89 (86 mg) was mixed with deionized water (4.0 ml before use was barbotirovany argon for 10 minutes) and the pH of the suspension was brought about to 6.9 (suspension turned into a solution after raising the pH) by adding a saturated solution of NaHCO3(before use was barbotirovany argon for 10 minutes). To the solution was added an additional amount of deionized water to bring the total volume to 5.0 ml, and the resulting aqueous solution was immediately added to the solution EC0312 (97 mg) in THF (5.0 ml). The reaction mixture quickly became homogeneous. After stirring in an argon atmosphere for 45 minutes the reaction mixture was diluted with 2.0 mm sodium phosphate buffer (pH 7.0, 15 ml) and THF was removed on the rotavapor rotary evaporator. The resulting suspension was filtered and the filtrate was injected into the preparative HPLC column for purification (column: Waters XTerra Prep MS C1810 μm 19×250 mm; mobile phase A: 2.0 mm sodium phosphate buffer pH 7.0; mobile phase B: acetonitrile; method: 5% B to 80% B over 25 min, flow rate 25 ml/min). Faction 10.04-11.90 minutes was collected and was liofilizovane getting EC0305 the form of pale yellow friable solid (117 mg).

Example.General method 2 to obtain conjugates comprising hydrophilic spacer linker (one vessel). The technique shown in the example of obtaining EC0543. DIPEA (7,8 μl) and isobutylparaben (3,1 µl) were co-added with a syringe into a solution tubulysin A (18 mg) in anhydrous EtOAc (0,50 ml) at -15°C. After stirring for 35 minutes at -15°C in argon atmosphere, the reaction mixture solution was added EC0311 (5.8 mg) in anhydrous EtOAc (0,50 ml). Stopped cooling and the reaction mixture was stirred in an argon atmosphere for 45 minutes, concentrated, kept in vacuum and the residue was dissolved in THF (2.0 ml). Meanwhile dissolved EC0488 (40 mg) in deionized water before use was barbotirovany argon for 10 minutes) and the pH of the aqueous solution was brought to 6.9 by addition of a saturated solution of NaHCO3. To a solution of EC0488 was added an additional amount of deionized water to obtain a total volume of 2.0 ml and the resulting solution was immediately added to the solution containing the activated tubulysin in THF. The reaction mixture, which quickly became homogeneous and was stirred in an argon atmosphere for 50 minutes and extinguished 2.0 mm sodium phosphate buffer (pH 7.0, 15 ml). The obtained turbid solution was filtered and the filtrate was injected into the preparative HPLC column for purification. Column: Wates XTerra Prep MS C 1810 μm 19×250 mm; mobile phase A: 2.0 mm sodium phosphate buffer pH 7.0; mobile phase B: acetonitrile; method: 1% B for 5 min, then 1% B to 60% B over the next 30 min, flow rate=26 ml/min Fraction 20.75-24.50 minutes was collected and was liofilizovane getting EC0543 in the form of a pale yellow friable solid (26 mg). The above technique is applicable also to obtain conjugates other tubulysins with appropriate choice of initial tubulysin.

The following additional illustrative examples of conjugates tubulysin, including hydrophilic spacer linker, was obtained with the application of methods and syntheses described in the present application is based on tubulysin.

EC0436 conjugate tubulysin

EC0444 conjugate tubulysin

EC0530 conjugate tubulysin

EC0531 conjugate tubulysin

EC0533 conjugate tubulysin

The following examples conjugates were also obtained, as described in the present invention.

EC0262 Conjugate cryptophycin-carbonate-CH2CH2-SS-Cys-sugar-Asp-sugar-Asp-sugar-folate. C87H115Cl2N15O38S2; mol. weight 2113,96; exact mass: 2111,63.

EC0278

Comparative examples of conjugates of bortezomib. Below are comparative examples of conjugates of bortezomib (Velcade) (EC0522 and EC0587), lacking the hydrophilic spacer linker, also received, as described in this application and the published patent application U.S. No. 2005/0002942.

EC0522 C56H69BN18O17S2C, 50,15; H, 5,19; B, 0,81; N, 18,80; O To 20.28; S, 4,78; mol. weight 1341,20; exact mass: 1340,46.

EC0587 C77H90B2N20O23S4; mol. weight 1813,55; exact mass: 1812,56.

The following examples of conjugates of bortezomib, comprising a hydrophilic spacer linker, also received, as described in the present invention.

EC0525 Conjugate of bortezomib (Velcade). C85H119BN20O36S2; mol. weight 2071,91; exact mass: 2070,76. Not limited to a particular theory, I believe that in the conjugate of bortezomib baronova acid and the linker can participate in intramolecular interactions with the side chains of carbohydrates. As an illustration, baronova acid forms complexes based on the formation of esters Bronevoy acid with one or two hydroxyl groups. Such ester complexes can be formed with vicinal the mi hydroxyl, and with 1,3-hydroxyl. Assume that ester complexes Bronevoy acid can be formed at the end of the carbohydrate fragment or in the middle part of the carbohydrate fragment. Further, it is understood that in an aqueous solution of the ester complexes Bronevoy acid can exist in equilibrium with Bronevoy acid.

EC0525 (hydrated) C85H119BN20O36S2; mol. weight 2071,91; exact mass: 2070,76. EC0525 (coordinated) C85H123BN20O38S2; mol. weight 2107,94; exact mass: 2106,78.

EC0595 bis-bortezomib conjugate. C108H145B2N25O39S4; mol. weight 2567,34; exact mass: 2565,92.

Comparative example conjugate α-amantina. Below is a comparative example of a conjugate of α-amantina, which lacks the hydrophilic spacer linker, also received, as described in this application and the published patent application U.S. No. 2005/0002942.

EC0323 not entered in competition with folic acid and showed the same value IC50in the presence and in the absence of an excess of folic acid.

The following examples of conjugates of α-amantina, including hydrophilic spacer linker, also the floor is made, as described in this application.

EC0592 Conjugate α-amantina. C107H154N26O50S3; mol. weight: 2700,71; exact mass: 2698,95. EC0592 demonstrated IC50~3 nm, and the conjugate can compete with an excess of folic acid against KB cells in the analysis to enable3H-thymidine.

The following examples are illustrative of the conjugates prepared as described in this application.

EC0535 conjugate of geldanamycin. C95H139N19O42S2, mol. weight: 2283,35, exact mass: 2281,88.

EC0568 conjugate of geldanamycin. C99H146N20O44S2, mol. weight: 2384,46, exact mass: 2382,92.

EC0539 conjugate comprising the lysine analogue of aminopterin

EC0544 conjugate comprising cysteine analogue of aminopterin. C83H116N24O37S2. (C, 47,33; H, to 5.55; N 15,96; O 28,11; S 3,05; mol. weight: 2106,08, exact mass: 2104,74.

EC0551 conjugate of aminopterin. C86H120N24O39S2. (C, 47,42; H, to 5.55; N 15,43; O 28,65; S 2,94; mol. weight: 2178,14, exact mass: 2176,76.

EC0543 conjugate tubulysin A. C111H67 N23O45S3. (C, 50,50; H, 6,38; N 12,20; O 27,27; S 3,64; mol. weight: 2639,84, m/z 2639,07 (100,0%), 2638,06 (80,8%), 2640,07 (79,6%).

EC0545 conjugate purvalanol. C87H125ClN22O37S2; mol. weight: 2170,63, exact mass: 2168,77.

EC0565 conjugate of everolimus. C121H183N17O50S2; mol. weight: 2739,96, exact mass: 2738,17.

Conjugate DAVLBH

EC0400 conjugate DAVLBH. Received by Huisgen cyclization of the corresponding alkyne and azidoanilide; 2 EQ. of Na ascorbate, 1 EQ. CuSO4·5H2O, THF/water (1:1); 5 EQ. of Na ascorbate, 2.5 EQ. CuSO4·5H2O, THF/water (9:1); (10 mg). C81H100N18O24S2. (C, 54,84; H, of 5.68; N 14,21; O 21,65; S 3,62; mol. weight: 1773,90, exact mass: 1772,66.

Conjugate DAVLBH. Obtained from EC0419. C82H97N17O21S2; mol. weight: 1720,88, exact mass: 1719,65 (90 mg).

EC0423 conjugate DAVLBH. Received by Huisgen cyclization; C98H123N23O35S2. (C, 52,38; H, 5,52; N 14,34; O 24,92; S 2,85; mol. weight: 2247,29, exact mass: 2245,80.

EC0637 conjugate DAVLBH. C98H130N20O37S2; mol. weight: 2244,32, exact mass: 2242,3.

Conjugate DAVLBH

Conjugate DAVLBH

Conjugate DAVLBH

EC0581 conjugate ispinesib. C98H133ClN20O38S2; mol. weight: 2298,80, exact mass: 2296,82.

EC0561 conjugate of paclitaxel. C124H159N19O53S2; mol. weight: 2827,82, exact mass: 2825,98.

EC0594 conjugate of docetaxel. C120H161N19O53S2; mol. weight: 2781,79, exact mass: 2779,99.

EC0598 conjugate Verrucaria. C95H134N16O45S2; mol. weight: 2284,29, exact mass: 2282,81.

EC0600 conjugate of budesonide. C93H134N16O42S2. (C, 50,49; H, 6,11; N, 10,13; O, 30.37 Per; S, 2,90; mol. weight: 2212,27, exact mass: 2210,83.

EC0610 conjugate didemnin B. C125H189N23O51S2; mol. weight: 2894,09, exact mass: 2892,23.

EC0631 conjugate of tyrosine kinase inhibitor 4-[(3-bromophenyl)amino]-6-[(2-hydroxyethyl)amino]pyrido[3,4-d]pyrimidine. C83H114BrN21O37S2; mol. weight: 2141,95, the exact value of the mass is: 2139,63.

EC0640: conjugate 4-[(3-bromophenyl)amino]-6-hydrazinophenyl[3,4-d]pyrimidine.

EC0663 conjugate of dasatinib. C90H126ClN23O38S3; mol. weight: 2269,74, exact mass: 2267,75.

EC0593 multiplikacionnoe intermediate connection for the two drugs. C68H103N17O35S3; mol. weight: 1782,77, exact mass: 1781,62.

EC0613 multiplikacionnoe intermediate connection for three drugs. C90H140N22O47S4; mol. weight: 2410,45, exact mass: 2408,81.

EC0542 multiplikacionnoe intermediate connection for the two drugs, with a choice of selectivity. C85H118N18O36S2. (C, 50,24; H, of 5.85; N, 12,41; O, Of 28.34; S, 3,16; mol. weight: 2032,08, exact mass: 2030,74.

EC0559 multiplikacionnoe intermediate connection for the two drugs, with a choice of selectivity. C90H121N19O36S3; mol. weight: 2141,22, exact mass: 2139,74.

EC0682 multiplikacionnoe intermediate connection for two drug medium is in, with a choice of selectivity. C95H132N20O42S2; mol. weight: 2290,30, exact mass: 2288,82.

EC0646 conjugate of aminopterin and intermediate connection for multirecording conjugate. C106H140N26O41S3; mol. weight: 2530,59, exact mass: 2528,88.

EC0555 conjugate of bortezomib (Velcade) and the intermediate connection for multirecording conjugate. C105H139BN22O38S3. (C, 52,02; H, 5,78; B, 0,45; N, 12,71; O, 25,08; S 3,97; mol. weight: 2424,36, exact mass: 2422,89.

EC0606 conjugate of everolimus and intermediate connection for multirecording conjugate. C141H203N19O52S3. (C, 54,76; H, 6,62; N, 8,61; O, 26,90; S, 3,11; mol. weight: 3092,42, exact mass: 3090,30.

EC0633 conjugate DAVLBH and intermediate connection for multirecording conjugate. C131H176N24O45S3; mol. weight: 2903,13, exact mass: 2901,14.

EC0661 conjugate 4-[(3-bromophenyl)amino]-6-aminopyrido[3,4-d]pyrimidine and the intermediate connection for multirecording conjugate. C145H209BrN24O55S4; mol. weight: 3376,51, exact mass: 3373,24.

EC0679 conjugate 4-[(3-bromophenyl)amino]-6-hydrazinophenyl[3,4-d]pyrimidine and the intermediate connection for multirecording conjugate. C101H131BrN24O38S3; mol. weight: 2465,36, exact mass: 2462,74.

EC0661 conjugate of doxorubicin and intermediate connection for multirecording conjugate. C115H149N19O49S3; mol. weight: 2677,71, exact mass: 2675,89.

EC0647 conjugate bis-aminopterin. C110H147N33O45S4; mol. weight: 2779,80, exact mass: 2777,9112, m/z: 2778,91 (100,00%), 2777,91 (74,4%), 2779,92 (62,2%).

EC0605 conjugate bis-verrucarin

EC0563 conjugate of bortezomib and rapamycin

EC0582 conjugate of bortezomib and everolimus. C147H214BN23O54S4. (C, 53,40; H, 6,52; B, 0,33; N, 9,74; O, 26,13; S, 3,88; mol. weight: 3306,47, exact mass: 3304,37.

EC0636 conjugate DAVLBH and everolimus. C173H251N25O61S4; mol. weight: 3785,23, exact mass: 3782,62.

EC0664 conjugate of everolimus and 4-[(3-bromophenyl)amino]-6-aminopyrido[3,4-d]pyrimidine. C145H209BrN24O55S4; mol. weight: 3376,51, that is the given value of the mass: 3373,24.

EC0680 conjugate of everolimus and 4-[(3-bromophenyl)amino]-6-hydrazinophenyl[3,4-d]pyrimidine. C143H206BrN25O54S4; mol. weight: 3347,47, exact mass: 3344,22.

EC0584 intermediate connection for possible non-directional delivery. C61H91N9O31S; mol. weight: 1478,48, exact mass: 1477,55.

EC0634 intermediate connection for possible non-directional delivery. C63H95N9O30S2; mol. weight: 1522,60, exact mass: 1521,56.

EC0586 intermediate connection for possible non-directional delivery. C48H83N9O30S; mol. weight: 1298,28, exact mass: 1297,50.

EC0588 intermediate compound conjugate of aminopterin for possible non-directional delivery. C69H105N17O35S2; mol. weight: 1769,79, exact mass: 1795,64.

EC0591 intermediate compound conjugate of rapamycin for possible non-directional delivery. C102H164N10O45S2. (C, 52,93; H, 7,14; N, 6,05; O, 31,11; S, 2,77; mol. weight: 2314,57, exact mass: 2313,03.

1. The compound of the formula
B-L-A
where a represents one or more of the LCO to bind to the receptor on the cell surface ligands, which bind to the receptor on the surface of target cells, L represents a polyvalent linker, which includes one or more hydrophilic spacer linker chosen from:
ororor their combination;
where R in each case independently selected from H, alkyl, cycloalkyl or arylalkyl; m in each case independently represents an integer from 1 to about 3; n1 in each case independently represents an integer from 2 to about 6; n2 in each case independently represents an integer from 1 to about 6; n3 in each case independently represents an integer from 1 to about 6; R in each case independently is an integer from 1 to about 5;
and a represents one or more diagnostic, therapeutic agents or renderers, which shall be delivered to the cell.

2. The compound according to claim 1, where at least one tool And a therapeutic agent.

3. The compound according to claim 1, where at least one tool And represents a therapeutic agent for cancer treatment.

4. The compound according to claim 1, where a is a set of therapeutic agents for cancer treatment.

5. The compound according to claim 1 where the binding ligand presented In yet a ligand, linking folate receptor.

6. The compound according to claim 1 where the binding ligand represents a folate.

7. The compound according to any one of claims 1 to 6, in which the linker L contains at least three polyhydroxylated group.

8. The compound according to any one of claims 1 to 6, in which at least one of the hydrophilic spacer linker is formed mainly by atoms of carbon, hydrogen and oxygen and the ratio of carbon/oxygen is about 3:1 or less.

9. The compound according to any one of claims 1 to 6, in which at least one of the hydrophilic spacer linker is formed mainly by atoms of carbon, hydrogen and nitrogen and the ratio of carbon/nitrogen is about 3:1 or less.

10. The connection according to claim 7, in which the linker L further comprises one or more aspartic acid residues, one or more glutamic acid residues, one or more arginine residues, or one or more residues aminoadenine, or a combination of both.

11. The connection according to claim 7, in which the linker L further comprises one or more residues of beta-aminoadenine.

12. The connection according to claim 7, in which the linker L further comprises one or more divalent residues of 1,4-piperazine, where at least part of the residues of 1,4-piperazine included in the chain of atoms connecting the at least one binding ligand (b) and for men is our least one means (A).

13. The connection according to claim 7, where the linker L further comprises at least one arginine residue.

14. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments representing polyhydroxylated groups associated residues of triazole.

15. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments representing polyhydroxylated groups associated amide bonds.

16. The compound according to any one of claims 1 to 6, where the linker L additionally contains one or more derivatives of EDTA.

17. The compound according to any one of claims 1 to 6, in which the linker L further comprises the fragment of the formula selected from the group consisting of


where m is an integer independently selected in each case from the integers from 1 to about 8; p indicates an integer selected in each case from the integers from 1 to about 10; and n means an integer independently selected in each case from the integers from 1 to about 3.

18. The compound according to any one of claims 1 to 6, in which the linker L comprises one or more groups of the formula selected from the group consisting of:

where m is an integer independently selected in each case from the integers from 1 to about 3; and r about the means integer, selected from the integers from 1 to about 3.

19. Connection p, in which the linker L comprises one or more groups of the formula selected from the group consisting of:


.

20. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of




where p means an integer selected from the integers from 1 to about 5.

21. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of


where n denotes an integer independently selected in each case from 2 to about 5; R indicates an integer selected from the integers from 1 to 5; and r is an integer independently selected in each case from the integers from 1 to about 4; provided that n is less than or equal to r+1.

22. The compound according to any one of claims 1 to 6, in which the linker L further comprises fragments displayed by a formula selected from the group consisting of

where each of n and r is CE is the first number, selected from the integers from 1 to about 3, provided that n is less than or equal to r.

23. The compound according to any one of claims 1 to 6, in which the linker L further comprises fragments displayed by a formula selected from the group consisting of

24. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where n, m and r are integers and each of them is in each case independently selected from the integers from 1 to about 5.

25. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where n and r are integers and each of them is in each case independently selected from the integers from 1 to about 5; and R is an integer selected in each case from the integers from 1 to about 4.

26. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where n is an integer selected in each case from the integers from 1 to about 3, and m is an integer selected from the integers from 1 to about 22.

27. Connection Liu the WMD one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where n and r are integers and each of them is in each case independently selected from the integers from 1 to about 5; and R is an integer selected from the integers from 1 to about 4.

28. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where r is an integer from 1 to about 3.

29. Connection p, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of


.

30. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where n1 and n2 are integers independently selected in each case from the integers from 0 to about 3; with the proviso that n1 and n2 are not simultaneously equal to 0; and n3 is an integer independently selected in each case from the integers from 1 to about 3.

31. The compound according to any one of claims 1 to 6, in which a link is L further comprises one or more fragments, displayed formulas selected from the group consisting of





where n is an integer independently selected in each case from the integers from 1 to about 3.

32. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where n is an integer independently selected in each case from the integers from 1 to about 3.

33. The compound according to any one of claims 1 to 6, in which the linker L further includes one or more fissile linkers.

34. The compound according to any one of claims 1 to 6, in which the linker L additionally contains one or more disulfide fissile linkers.

35. The compound according to any one of claims 1 to 6, in which the linker L further comprises one or more fragments displayed by a formula selected from the group consisting of

where r is an integer selected from the integers from 1 to about 3.

36. Connection p, in which the linker L contains one or more fragments displayed by a formula selected from the group consisting of




37. The pharmaceutical composition intended for directed delivery of one or more diagnostic, therapeutic agent or renderers to the cell that expresses or excessively expresses the receptor on the cell surface, containing a therapeutically effective amount of one or more compounds according to any one of claims 1 to 6, and optionally a carrier, diluent and/or excipient for these compounds or their combination.

38. The use of compounds according to any one of claims 1 to 6 or a pharmaceutical composition containing this compound, for visualization, treat, cure or diagnose diseases or conditions or combinations thereof, where imaging, treatment or diagnosis, or a combination of these include the targeting of cells that Express or redundantly Express the receptor on the cell surface capable of contacting at least one binding with the receptor ligand Century



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a diagnostic and therapeutic agent which is a bombesin analogue peptide antagonist conjugate having the general formula (I), [A-(B)n]x-C, wherein A represents a metal chelator containing at least one radionuclide metal, B represents a spacer bound to N terminal C, or a covalent bond, and C represents a bombasin analogue peptide antagonist, wherein additionally x represents an integer 1 to 3, and n represents an integer 1 to 6.

EFFECT: preparing the therapeutic agent representing the bombesin analogue peptide antagonist conjugate.

17 cl, 12 dwg, 1 tbl, 13 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel non-branched carbamate derivatives of some peptides Wnt-5a, in particular to N-butyloxycarbonyl derivative, their pharmaceutical compositions and their application for treatment of gastric melanoma and cancer.

EFFECT: obtaining novel non-branched carbamate derivatives of some peptides Wnt-5a.

7 cl, 9 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to method of degarelix obtaining. Claimed is stage-by-stage synthesis of degarelix, containing 0.3 wt % or less than analogue 4-([2-(5-gidantoyl)]acetylamino)-phenylalanine, on solid amino group-containing substrate, which includes the following stages: supply of amino acid or peptide solution, where α-aminogroup is protected by fluorenylmethyloxycarbonyl group (Fmoc); bringing substrate in contact with said solution in presence of reagent to create peptide bond between carboxyl group of amino acid or peptide and solid amino group-containing substrate, connected with said substrate; removal of Fmoc by bringing substrate in contact with organic base, in particular piperidine, in organic solvent.

EFFECT: increase of method efficiency.

10 cl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to application of peptide, which has sequence originating from amino acid sequence of protein SNAP-25, for treatment of pain and/or inflammation.

EFFECT: obtaining novel composition.

9 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to peptides, possessing ability to induce cytotoxic T-cells, which contain amino acid sequence from SEQ ID NO: 1, 2, 3, 4, 16, 17, 30, 31, 34, 36, 37, 40, 41, 45, 49, 55, 57 and 61, as well as peptides, containing said amino acid sequences, in which 1, 2 or several amino acids are substituted and/or added. Claimed invention also relates to medications for treatment or prevention of tumours, with medications containing said peptides.

EFFECT: peptides in accordance with claimed invention can be applied as vaccines.

14 cl, 5 dwg, 2 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a biotechnology industry, and namely to synthetic peptides having a non-narcotic type of analgetic action of a general formula: 1 H - XDL - L-Leu - D-His - L-Lys - L-Leu - L-Gln - L-Thr - R2 (I), where: H - hydrogen, XDL - absence of amino acid or L-Tyr, R2 - OMe or NH2, as well as peptides - retroinversions of formula (I), which have reverse sequence of amino acids with replacement of L-shape of amino acids with D-shape and D-shape of amino acids with L-shape, with the following general formula: 2 H - D-Thr - D-Gln - D-Leu - D-Lys - L-His - D-Leu - XDL1 - R2 (II), where: H - hydrogen, XDL1 - absence of amino acid or D-Tyr, R2 - OMe or NH2.

EFFECT: invention allows producing safe analgetic medical preparations with a non-narcotic type of analgetic action.

5 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a chromatographic method of purifying an insulin analogue selected from aspartate, lispro and glargine, atosiban or eptifibatide, from a mixture containing at least one parent admixture.

EFFECT: method employs agents for forming ionic pairs in OF-preparative linear chromatography, which enables to achieve high degree of purity of the end product.

7 cl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to molecular pharmacology and particularly to a peptide which is an interleukin-15 (IL-15) sequence derivative which is optimised for inhibiting biological activity of said compound. The invention shows that when bound with an alpha subunit of the receptor (IL-15Rα) the peptide inhibits T cell proliferation induced by IL-15, tumour necrosis factor α (TNFα) induction caused by IL-15, and expression of IL-8 and IL-6 caused by IL-15Rα. The invention also relates to use of the peptide in treating pathologies where anomalous expression of IL-15 or IL-15Rα is associated with the course of a disease such as rheumatoid arthritis (AR) and prostate cancer.

EFFECT: obtaining an interleukin-15 (IL-15) sequence derivative which is optimised for inhibiting biological activity of said compound.

18 cl, 6 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: disclosed is use of a heptapeptide of general formula Tyr-D-Ala-Phe-Gly-Tyr-X-Ser-NH2, where X is D-Pro or Dh-Pro, or Dh-D-Pro, where Dh-Pro is 3,4-dehydroproline, as an antispasmodic, anxiolytic, central anti-inflammatory or anti-alcohol agent.

EFFECT: obtaining an agent used as an antispasmodic, anxiolytic, central anti-inflammatory or anti-alcohol agent.

3 cl, 8 tbl, 26 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to peptides or peptide-like molecules of the following formula: CCLLCCLLC (I) (SEQ ID NO: 1) or CLLCCLLCC (III) (SEQ ID NO:3), where C is cationic aminoacid and L is aminoacid with lipophile group R, in which one of the aminoacids, which has lipophile group R, represents genetically uncoded aminoacid. This compound is probably in the form of pharmaceutically acceptable salt, ester or amide, and refers to their use in therapy, and namely as antitumour agents.

EFFECT: improvement of compound properties.

20 cl, 7 dwg, 12 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: disclosed is an agent which is one of the derivatives of N-substituted 1,4-diazabicyclo[2.2.2.]octane, which exhibits antiviral activity on DNA viruses. The disclosed agent can be used in veterinary and healthcare.

EFFECT: higher antiviral activity towards DNA viruses.

6 dwg, 3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a pharmaceutical composition showing stress-protective action which contains peptide R1-Lys1-Arg2-Pro3-R2 [SEQ ID NO:1] or R1-Lys1-Arg2-Arg3-Pro4-R2 [SEQ ID NO:2] wherein R1=NH2 or CH3CO and R2=OH or NH2 and a method for prevention and/or treatment functional or stress-induced disorders caused by extreme factors.

EFFECT: producing the pharmaceutical composition exhibiting stress-protective action.

3 cl, 9 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: agent is an N- and C-substituted peptide selected from n-decyl ether (1-tetradecyl-1,4-diazoniabicyclo[2.2,2.]octan-4-yl)-acetyl-glutamyl-glycyl lysyl-glycine (1), n-decyl ether (1-tetradecyl-1,4-diazoniabicyclo[2.2,2.]octan-4-yl)-acetyl-glutamyl-β-alanyl-arginyl-glycine (2) and n-decyl ether glutamyl-β-alanyl-lysyl-glycine (3).

EFFECT: high antiviral activity of the agent.

3 dwg, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to bioorganic chemistry and specifically to synthesis of peptides which inhibit attraction to morphine in the period for quitting narcotic drugs. This compound can be used as a medicinal agent which inhibits attraction to morphine during a prolonged period for quitting a narcotic drug, which facilitates achieving prolonged remission of one form of opium addiction - morphine addiction and prevent disease recurrence.

EFFECT: wide range of agents which inhibit attraction to morphine during a period for quitting narcotic drugs, which is achieved through use of the chlorohydrate of tetrapeptide Trp-Nle-Asp-PheNH-CH(CH3)2.

4 dwg, 2 tbl, 2 ex

Rgd-like peptides // 2396271

FIELD: chemistry.

SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.

EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.

2 tbl, 1 ex

FIELD: chemistry; medicine.

SUBSTANCE: invention relates to derivatives of 2-hydroxytetrahydrofurane , of general formula (I) , which possess ability to inhibit calpaines and/or ability to catch active oxygen forms and can be used to obtain medication, intended for inhibiting calpaines and/or lipid peroxidation.

EFFECT: medications possess higher efficiency.

9 cl, 64 ex

FIELD: medicine.

SUBSTANCE: invention can be used for medical treatment of secondary hypothyroid state accompanied by low synthesis of thyrotrophic hormone by hypophysis and of iodine hormone by thyroid gland. Substance of invention implies application of peptide Lys-Glu-Asp-Gly as a medicine stimulating synthesis of thyrotrophic hormone by hypophysis and of thyroid hormone by thyroid gland.

EFFECT: high specific activity of introduced peptide and decrease of side effect risk.

4 tbl, 1 ex

FIELD: medicine; pharmacology.

SUBSTANCE: releasing peptides of growth hormone are described with formula (I): R112345-R2, where:А1 designates Aib, Apc or Inp; А2 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl) or D-Тrp; А3 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-2Ser(Bzl) or D-Trp; А4 designates 2Fua, Orn, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, 3Thi, Thr(Bzl); А5 designates Apc, Dab, Dap, Lys, Orn or deleted; R1 designates hydrogen; and R2 designates NH2; and their pharmaceutically acceptable salts.

EFFECT: pharmaceutical compositions and the methods of their application are presented.

25 cl, 1 tbl, 2 ex

The invention relates to new compounds of General formula I

R1-A-B-D-En-R2 (I)

in which R1 represents R12C(O), and R12 is selected from the group consisting of alkenyl, alkenylacyl or alkenylamine; And is a group A1-A2-A3, where A1 represents NH, A2 is a CHR93 in which R93 is 4-amidinophenoxy; A3 represents C(O); is a group B1-B2-B3, where B1 represents NH; B2 is a CHR97 where R97 represents ethyl, which is substituted in position 2 by hydroxycarbonyl or allyloxycarbonyl; B3 represents C(O); D represents a group D1-D2-D3, where D1 represents NH, D2 represents CR81R82 where R81 and R82 are independently selected from the group consisting of hydrogen and unsubstituted or substituted residues of alkyl, aryl, arylalkyl, heteroallyl; D3 represents C(O); Enis a (E1-E2-E3)nin which n is 0 or 1; E1 represents NR70, where R70 is H; E2 represents CR71R72, where R71 and R72 include independently selected from the group consisting of hydrogen and unsubstituted or substituted residues of alkyl, aryl, arylalkyl, heteroallyl; E3 represents C(O); R2 is a NR21R22 where R21 of iillil and geterotsiklicheskikh, moreover, the alkyl contains from 1 to 13 carbon atoms, alkenyl contains from 2 to 13 carbon atoms, aryl and heteroaryl contain from 5 to 13 ring carbon atoms, where in the rest of heteroaryl one or more carbon atoms are replaced by heteroatoms selected from the group consisting of N, O and S; heteroseksualci contains from 3 to 8 ring carbon atoms, of which from one to three carbon atoms are replaced by heteroatoms selected from the group consisting of N, O and S; in any stereoisomeric forms or their mixtures in any ratio, and their pharmaceutically acceptable salts; the method of obtaining compounds of General formula I, including linking protected amino acids; to pharmaceutical compositions which are able to exert an antithrombotic effect by activated factor VII(FVIIa) blood coagulation

The invention relates to oligopeptides derivative containing amino acid D-2-alkyltrimethyl, which is capable of releasing growth hormone (GH) from the somatotropic cells and active when administered orally

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine and deals with a pharmaceutical composition for enhancing the efficiency of treatment of liver cancer by Sorafenib, with the said composition containing an anti-glypican 3-antibody as an active ingredient.

EFFECT: invention provides an improved anti-cancer effect and reduction of the side effect, in particular loss of the body weight.

14 cl, 9 dwg, 2 tbl, 6 ex

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