Chemical linkers and conjugates thereof

FIELD: chemistry.

SUBSTANCE: medicine-ligand conjugates are powerful cytotoxins in which the medicine is bonded to the ligand through a peptide, hydrazine or disulphide linker.

EFFECT: agents are highly effective.

63 cl, 8 ex

 

This application claims the priority benefit of provisional patent applications US 60/572667, US 60/661174 and US 60/669871, which are incorporated herein as references.

The SCOPE of the INVENTION

The present invention relates to linkers that are attached to the drug and ligand and hatshepsuts in vivo. The linkers used in obtaining prodrugs and conjugates of the cytotoxins of the present invention, as well as other diagnostic and therapeutic components.

BACKGROUND of the INVENTION

Many therapeutic agents, especially those that are particularly effective in cancer chemotherapy, often exhibit high toxicity in vivo, mainly toxicity to the brain, and mucous membranes, as well as chronic cardiac and neurological toxicity. This high toxicity may limit their application. For greater effectiveness against tumor cells and reducing the number and severity of side effects (toxicity, destruction of non-neoplastic cells, and others) require the development of new more secure specific therapeutic agents, particularly anti-tumor agents.

Another difficulty of using some of the available therapeutic agents is smaller (compared to the optimal stability in plasma. In the add function the social groups to stabilize these compounds have significant activity decrease. Thus, it is desirable to identify ways of stabilizing compounds, while maintaining acceptable levels of therapeutic activity.

The search for more selective cytotoxic agents has been extremely active for many decades, with one of the main causes of failure in the treatment of cancer is DLT (dose limiting toxicity (i.e. unwanted effect of cytotoxins on normal tissue). For example, it is known that CC-1065 and duocarmycin are extremely potent cytotoxins.

CC-1065 was first isolated from Streptomyces zelensis in 1981 decision Upjohn Company (Hanka and others, J. Antibiot. 31: 1211 (1978); Martin and others, J. Antibiot. 33: 902 (1980); Martin and others, J. Antibiot. 34: 1119 (1981)) and, as found, has a strong antitumor and antimicrobial activities in vitro and in experimental animals (Li and others, Cancer Res. 42: 999 (1982)). CC-1065 binds to double-strand B-DNA minor groove (Swenson and others, Cancer Res. 42: 2821 (1982)) with a predominant sequence 5'-d(A/GNTTA)-3' and 5'-d(AAAAA)-3' and alkiliruet N3-position of the 3'-adenine on his left CPI-link present in the molecule (Hurley and others, Science 226: 843 (1984)). Despite its strong and broad antitumor activity, CC-1065 cannot be used on people, because he is the cause of delayed death in experimental animals.

Experts know many analogues and derivatives of CC-1065 and duocarmycin. The ima is conducted surveys on the studies of the structure, synthesis and properties of many compounds. See, for example, work Boger and others, Angew. Chem. Int. Ed. Engl. 35: 1438 (1996) and Boger and others, Chem. Rev. 97: 787 (1997).

A group of researchers from Kyowa Hakko Kogya Co., Ltd. received a number of derivatives of CC-1065. See, for example, U.S. patents№№ 5101038; 5641780; 5187186; 5070092; 5703080; 5070092; 5641780; 5101038 and 5084468; published PCT application WO 96/10405 and published European application 0537575 A1.

Company decision Upjohn (Pharmacia decision Upjohn) was actively working on obtaining derivatives of CC-1065. See, for example, U.S. patent No. 5739350; 4978757; 5332837 and 4912227.

This study focused on the development of new therapeutic agents, which have the form of prodrugs, i.e. compounds which are capable of developing into drugs (active therapeutic compounds) in vivo under certain chemical or enzymatic modifications of their structure. To reduce the toxicity of this conversion preferably limit the impact site or target tissue and does not conduct through the bloodstream or non-target tissue. However, even prodrugs are problematic, as many are characterized by low stability in blood and serum due to the presence of enzymes that destroy or activate prodrugs before they reach the designated desired effect in the patient's body.

Bristol-Myers Squibb describes specific lysosomal destroy the enzyme is m conjugates of anticancer drugs. See, for example, U.S. patent No. 6214345. This patent relates to aminobenzenesulphonyl.

Company Seattle Genetics published patent application U.S. 2003/0096743 and 2003/0130189 that describe the pair-aminoaniline ethers agents in drug delivery. Described in these applications linkers limited to compositions aminoaniline esters.

Other groups of people also describe the linkers. See, for example, the work of de Groot and others, J. Med. Chem. 42, 5277 (1999); de Groot and others, J. Org. Chem. 43, 3093 (2000); de Groot and others, J. Med. Chem. 66, 8815, (2001); WO 02/083180; Carl and others, J. Med. Chem. Lett. 24, 479, (1981); Dubowchik and others, Bioorg &Med. Chem. Lett. 8, 3347 (1998). These linkers include a spacer in the form of aminobenzene simple ether, systems with extended electronic cascade and cyclizes spacer elements of the system, the spacers that are resolved by cyclization, for example w-aminocarbonyl and para-aminobenzeneboronic the linker.

Stability cytotaxonomic medicines, including in vivo stability is another important issue that requires consideration. In addition, the toxicity of many compounds makes them less suitable, so that the required composition that will reduce the toxicity of drugs such as split prodrugs. Therefore, despite advances in this field, there remains a need for the development of improved therapeutic agents for the treatment of melicope the surrounding and people, in particular, a more specific cytotoxins, demonstrating the high specificity of action, reduced toxicity and improved stability in the blood in comparison with the known compounds of similar structure. The present invention is directed to these purposes.

SUMMARY of INVENTION

The present invention relates to conjugates of a drug-ligand, in which a drug and a ligand bound peptide, or hydrazine powered by a disulfide linker. These conjugates are potent cytotoxins that can be selectively delivered to a specific site of action in the active form, and then split with the release of the active drug. New linker sites on this invention can be chipped off from cytotoxic drugs, for example, enzymatic or rehabilitation methods in vivo, releasing the active drug of the derived prodrugs. In addition, the invention includes conjugates of the linker sites and cytotoxins of the present invention and the linker conjugates areas, cytotoxin and directing agent, such as antibodies or peptide.

The invention also applies to groups that are suitable for the stabilization of therapeutic agents and markers. The stabilizing group is selected, for example, in such a way as to limit the clearance and metabol the ZM therapeutic agent or marker enzymes, which may be present in blood or non-target tissue. Stabilizing group can serve to lock the decomposition of the agent or marker and can also act providing other physical characteristics of the agent or marker, for example, increase the solubility of the compound or decreased aggregation properties of the connection. Stabilizing group may also improve the stability of the agent or marker when stored in the form of the preparative form or by itself.

The first aspect of the invention relates to cytotoxic compounds the drug-ligand having a structure corresponding to any of the formulas 1-3

in which the symbol D denotes the medicinal part, has suspended its main chain chemically reactive functional group, with the specified functional group selected from the group comprising primary or secondary amine, hydroxyl, sulfhydryl, carboxyl, aldehyde and ketone.

The symbol L1indicates auto-disable spacer, where m is an integer from 0, 1, 2, 3, 4, 5 or 6.

X4indicates a component selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides the agents.

The symbol L4denoted by the AET of the linker component, and p is 0 or 1. L4is a piece that gives the conjugates increased solubility or decreased aggregation properties. Examples of components of L4include substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl or an unsubstituted heteroalkyl, each of which can represent a linear, branched or cyclic, positively or negatively charged such amino acid polymer, as polylysine or polyalanine, or another polymer, for example polyethylene glycol.

The symbols F, H and J denote the linkers described herein below. In one embodiment, the invention relates to a conjugate with a peptide linker structure

where

D denotes the medicinal part, has suspended its main chain chemically reactive functional group, with the specified functional group selected from the group comprising primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde and ketone;

L1indicates auto linker;

m is an integer 0, 1, 2, 3, 4, 5 or 6;

F denotes a linker containing structure

where

AA1indicates one or more components, independently selected from the group comprising natural is minamikata and artificial α-amino acid;

c is an integer from 1 to 20;

L2indicates auto linker;

L3denotes a spacer elements group containing a primary or secondary amine or a carboxyl functional group; in which there is L3, m is 0, and the amine group of L3forms an amide bond with dangling carboxyl functional group of D or carboxyl from L3forms an amide bond with dangling amine functional group of D;

o is 0 or 1;

L4denotes a linker component, in which L4is not directly attached to the N-end component (AA1)ccarboxylic acyl group;

p is 0 or 1; and

X4indicates a component selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides the agents.

In one embodiment, the conjugate with a peptide linker has the following structure:

In another embodiment, a conjugate with a peptide linker has the following structure:

In a preferred embodiment, L3includes an aromatic group. For example, L3may include benzoic acid group, an aniline group or indole group. Neogranichena the examples of-L 3-NH - include a structure selected from the following groups:

where Z denotes a component selected from O, S and NR23and

where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl.

In preferred embodiments, the peptide linker (AA1)withrepresents a peptide sequence that is cleaved by a protease expressed in tumor tissue. A preferred protease is a lysosomal protease. In preferred embodiments, c is an integer from 2 to 6 or equal to 2, 3 or 4. In some embodiments, the amino acid (AA1)withlocated closest to the medicinal part selected from the group including: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. In preferred variants (AA1)withrefers to a peptide sequence selected from the group comprising Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2) and Gly-Phe-Leu-Gly (SEQ ID NO: 3). In a particularly preferred variants (AA1)withdenotes Val-Cit or Val-Lys.

In some preferred embodiments, the peptide linker F has the structure

where

R24selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl;

each K denotes a component that is independently selected from the group including substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR21R22, NR21COR22, OCONR21R22, OCOR21and OR21,

where

R21and R22independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci; and

a is an integer of 0, 1, 2, 3, or 4.

In other preferred embodiments,- F-(L1)m has the structure

where

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl.

Another aspect of the invention relates to conjugates with hydrazine powered by linkers patterns

where

D denotes the medicinal part, has suspended its main chain chemically reactive functional group, with the specified functional group selected from the group comprising primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde and ketone;

L1indicates auto linker;

m is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

X4indicates a component selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides agents;

L4denotes a linker component;

p is 0 or 1;

H denotes a linker comprising the structure

where

n1an integer from 1 to 10;

n2is 0, 1 or 2;

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl; and

I stands for either communication or

where n3is 0 or 1 provided that when n3is 0, n2not equal to 0 and n4is 1, 2 or 3,

where if I denotes a bond, n1equal to 3 and n2is 1, D could not be

where R is Me or CH2-CH2-NMe2.

In some preferred embodiments, the substitution in the phenyl ring is para-substitution. In some preferred embodiments, n1is 2, 3 or 4, or n1equal to 3, or n2is 1.

In some embodiments, the I denotes the relationship. In other embodiments, n30 and n4equal to 2.

Various aspects of the invention relate to hydrazine powered the linkers H, which may form a 6-membered linker, self-destructive decomposition, or two 5-membered linker, self-destructive decomposition, or one 5-membered linker, self-destructive decomposition, or one 7-membered linker, self-destructive decomposition, or 5-membered auto linker and 6-membered linker, self-destructive decomposition.

In the preferred embodiment, H is genialne dimethyl substitution.

In the preferred embodiment, H has the structure

Preferably n1is 2, 3 or 4, more preferably n1equal to 3. Preferably each R24independently selected from CH3and H. In some preferred embodiments, each R24denotes H.

In another preferred variant of H has the structure

Preferably n1equal to 3. Preferably the each R 24independently selected from CH3and H.

In other preferred embodiments, H has the structure

Preferably each R24independently represents H or substituted or unsubstituted alkyl.

Another aspect of the invention relates to conjugates with hydrazine powered by linkers patterns

where

D denotes the medicinal part, has suspended its main chain chemically reactive functional group, with the specified functional group selected from the group comprising primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde and ketone;

L1indicates auto linker;

m is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

X4indicates a component selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides agents;

L4denotes a linker component;

p is 0 or 1, and

H has the structure

where q is 0, 1, 2, 3, 4, 5 or 6 and

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl.

the school is one aspect of the invention relates to conjugates with by a disulfide linkers patterns

where

D denotes the medicinal part, has suspended its main chain chemically reactive functional group, with the specified functional group selected from the group comprising primary or secondary amine, hydroxyl, thiol, carboxyl, aldehyde and ketone;

L1indicates auto linker;

m is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

X4indicates a component selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides agents;

L4denotes a linker component;

p is 0 or 1;

J denotes a linker having the structure

where

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl;

each K denotes a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2 , NR21R22, NR21COR22, OCONR21R22, OCOR21and OR21,

where

R21and R22independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci and the unsubstituted heteroseksualci;

a is an integer of 0, 1, 2, 3 or 4 and

d is an integer from 0, 1, 2, 3, 4, 5 or 6.

In various embodiments, J may have one of the following structures:

In all previous the linker conjugates of D preferably represents a cytotoxic drug. In preferred embodiments, D is chemically reactive functional group selected from the group comprising primary or secondary amine, hydroxyl, sulfhydryl and carboxyl. Non-restrictive examples of preferred drugs D include duocarmycin and analogues and derivatives duocarmycins, CC-1065, analogs of duocarmycin on the basis of CBI analogues of duocarmycin based MCBI, analogs of duocarmycin on the basis of the CCBI, doxorubicin, conjugates of doxorubicin, morpholino doxorubicin, cyanomethane doxorubicin, dolastatin, dolastatin-10, combretastatin, calicheamicin, maytansine, analogues is of atensina, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethylaniline E (MMAE), AE-ester 5-benzoylamino acid (AEVB), tubulysin, decorazon, epothilone, paclitaxel, docetaxel, SN-38, topotecan, rhizoxin, economizing, colchicine, vinblastine, vindesine, estramustin, cemadotin, eleutherobin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, Larsen, leirosidin, actinomycin, daunorubicin, conjugates of daunorubicin, mitomycin C, mitomycin A, karminomitsin, aminopterin, talisayan, depapillated, derivatives depapillation, etoposide, etoposide, vincristine, Taxol, Taxotere-retinoic acid, butyric acid, N8-acetylspiramycin and camptothecin.

In a preferred embodiment, D represents an analogue or derivative duocarmycin, which has the structure

where cyclic system denotes A component selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geteroseksualnoe group;

E and G are components that independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, heteroatoms, simple communication, or E and G are combined, forming a cyclic system, select the NUU from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geterotsiklicheskie;

X denotes a component selected from O, S and NR23;

R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R3indicates a component selected from the group comprising (=O)SR11, Other11and OR11where

R11indicates a component selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, diphosphate, triphosphates, acyl, C(O)R12R13C(O)OR12C(O)NR12R13, P(O)(OR12)2C(O)CHR12R13, SR12and SiR12R13R14,

in which

R12, R13and R14denote components that are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, where R12and R13together with the nitrogen atom or carbon to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe ring system having from 4 to 6 elements, optionally containing two or more heteroatoms;

R4, R4', R5and R5' denote the components independently selected the s group, including H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, SR15, OR15, CR15=NR16and O(CH2)nN(CH3)2where

n is an integer from 1 to 20;

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie and substituted or unsubstituted of peptidyl, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe ring system having from 4 to 6 elements, optionally containing two or more heteroatoms;

R6indicates a simple relationship that is present or absent and, if present, R6and R7United, forming cyclopropyl ring; and

R7denotes CH2-X1or-CH2-United in the specified cyclopropyl ring with R6where

X1denotes a leaving group which,

where at least one of R11, R12, R13, R15or R16associates the specified drug with L1if present, or to F, H, or J.

In a preferred embodiment, D has the structure

where Z denotes a component selected from O, S and NR23,

where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8where R8indicates a component selected from the group including substituted alkyl, unsubstituted alkyl, NR9R10, NR9Other10and OR9,

in which R9and R10are components that independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl; and

R2denotes H, substituted alkyl or unsubstituted lower alkyl;

in which at least one of R11, R12, R13, R15or R16associates the specified drug with L1if present, or to F, H or J.

In the above preferred embodiment, R2denotes unsubstituted lower alkyl.

In another preferred embodiment, D has the structure

where Z denotes a component selected from O, S and NR23,

where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8where R8indicates a component selected from NR9R10and OR9in which

R9and R10denote components that are independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R1represents H, substituted or unsubstituted lower alkyl or C(O)R8where R8indicates a component selected from NR9R10and OR9in which

R9and R10denote components that are independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R2denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl, or cyano, or alkoxy; and

R2'denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl,

in which at least one of R11, R12, R13, R15or R16associates the specified drug with L1if present, or to F, H or J.

All pre is idusa structures of the linker conjugates of L 4preferably contains an acyclic fragment. L4preferably increases the solubility of the compounds in comparison with the connection, not having L4and/or L4reducing the aggregation of connections compared to the connection that does not have L4. In a preferred embodiment, L4contains polietilenglikolya fragment. Polietilenglikolya fragment can contain, for example, 3-12 repetitive units, or between 2 to 6 repeating units, or, more preferably, 4 duplicate link.

Another aspect of the invention relates to cytotoxic compounds the drug-ligand having a structure corresponding to the following formula:

where the symbol L1is a self-destroying a spacer, where m is an integer 0, 1, 2, 3, 4, 5 or 6.

X4indicates an element selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides the agents.

The symbol L4denotes a linker component, and p is 0 or 1. L4indicates the component that gives the conjugates increased solubility or decreased aggregation properties. Examples of components of L4include substituted alkyl, unsubstituted alkyl, substituted aryl, nez is displaced aryl, substituted heteroalkyl or an unsubstituted heteroalkyl, each of which may be linear, branched or cyclic, positively or negatively charged amino acid polymer, such as polylysine or polyalanine, or other polymers, for example polyethylene glycol.

The symbol Q denotes ottsepleny linker, including but not limited to these, any peptide, hydroconone and disulfide linkers described herein. Otsepleniya linkers include linkers that can selectively be chipped off chemical or biological manner and subject to removal to separate the drug D1from the X4.

The symbol D1refers to a drug having the following formula:

where X and Z denote the components independently selected from O, S and NR23;

R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8,

R1'represents H, substituted or unsubstituted lower alkyl or C(O)R8,

where R8indicates a component selected from NR9R10and OR9and R9and R10denote components that are independently selected from H, substituted or nezamedin the th of alkyl and substituted or unsubstituted heteroalkyl;

R2denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl, or cyano, or alkoxy;

R2'denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl,

R3indicates a component selected from the group comprising SR11, Other11and OR11where R11indicates a component selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, diphosphate, triphosphates, acyl, C(O)R12R13C(O)OR12C(O)NR12R13, P(O)(OR12)2C(O)CHR12R13, SR12and SiR12R13R14in which R12, R13and R14denote components that are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, where R12and R13together with the nitrogen atom or carbon to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 elements, optionally containing two or more heteroatoms;

where at least one of R11, R12and R13associates the specified drug with L1if present, or Q

R6hereafter which includes a simple link, which is present or absent and, if present, R6and R7United, forming cyclopropyl ring; and

R7denotes CH2-X1or-CH2-combined in the specified cyclopropane ring with R6where

X1denotes a leaving group,

R4, R4', R5and R5' denote the components independently selected from the group consisting of H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, SR15, OR15, CR15=NR16and O(CH2)nNR24R25where n is an integer from 1 to 20;

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie and substituted or unsubstituted of peptidyl, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 to the components, optional comprising two or more heteroatoms;

and R24and R25independently selected from unsubstituted alkyl, and,

where at least one of R4, R4', R5and R5' denotes O(CH2)nNR24R25.

Another option is a compound having a structure corresponding to formula 1

in which X and Z are independently selected from O, S and NR23where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8,

R1'represents H, substituted or unsubstituted lower alkyl or C(O)R8,

each R8indicates a component that is independently selected from NR9R10and OR9and R9and R10denote components that are independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R2represents H, substituted or unsubstituted lower alkyl, unsubstituted heteroalkyl, cyano or alkoxy;

R2'represents H, substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl,

R3indicates a component selected from the group comprising SR11, Other11OR 11where R11indicates a component selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, diphosphate, triphosphates, acyl, C(O)R12R13C(O)OR12C(O)NR12R13, P(O)(OR12)2C(O)CHR12R13, SR12and SiR12R13R14in which R12, R13and R14denote components that are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, or R12and R13together with the nitrogen atom or carbon to which they are attached, join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 elements, optionally containing two or more heteroatoms;

R6indicates a simple relationship that is present or absent, and if present, R6and R7United, forming cyclopropyl ring; and

R7denotes CH2-X1or-CH2United in the specified cyclopropane ring with R6where X1denotes a leaving group,

R4, R4', R5and R5' denote the components independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted aryl, nezamescennych, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, SR15, OR15, CR15=NR16and O(CH2)nNR24R25where n is an integer from 1 to 20;

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie and substituted or unsubstituted of peptidyl, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 elements, optionally containing two or more heteroatoms;

and R24and R25independently selected from unsubstituted alkyl, and

where at least one of R4, R4', R5and R5' denotes O(CH2)nN R24R25.

Another aspect of the invention relates to pharmaceutical preparations. Such preparations usually contain a compound, the conjugate according to this invention and a pharmaceutically acceptable carrier.

Another aspect of the invention is asaeda methods of using compounds of the conjugates according to this invention. For example, the invention relates to a method of destroying cells, in which the compound-conjugate according to this invention is injected into the cell in sufficient quantity to her death. In a preferred embodiment, the cell is a tumor cell. In another embodiment, the invention relates to a method of slowing or stopping tumor growth in a patient is a mammal, in which the compound-conjugate according to this invention is administered to the patient in a quantity sufficient to slow or stop tumor growth.

Other aspects, advantages and objectives of the invention will be clear from a review of the following detailed description.

DETAILED description of the INVENTION

Reduction

Used herein, the abbreviation "Ala" refers to alanine.

"Boc" refers to tert-butyloxycarbonyl.

"CPI" refers to cyclopropylamino.

"Cbz" refers to carbobenzoxy.

Used herein, the abbreviation "CHM" refers to dichloromethane.

"DDQ" refers to 2,3-dichloro-5,6-dicyano-l,4-benzoquinone.

"DIPEA" means diisopropylethylamine

"DMDA" means N,N'-dimethylethylenediamine

"RBF" refers to a round bottom flask.

"DMF" means N,N-dimethylformamide.

"HATU" means N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-yl]methylene]-N-methylmethanesulfonate N-oxide.

Used herein, the symbol "E" denotes fer entative tsepliaeva group.

"EDCI" indicates 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

Used herein, the abbreviation "FMOC" represents 9-fluorenylmethoxycarbonyl.

"FMOC" represents 9-fluorenylmethoxycarbonyl.

"HOAt" refers to 7-Aza-1-hydroxybenzotriazole.

"Leu" denotes leucine.

"PABA" means para-aminobenzoic acid.

"PEG" means polyethylene glycol.

"PMB" means para-methoxybenzyl.

"TBAF" means tetrabutylammonium.

The abbreviation "TBSO" means tert-butyldimethylsilyloxy ether.

Used herein, the abbreviation "TEA" refers to triethylamine.

"TN" refers to triperoxonane acid.

The symbol "Q" refers to a therapeutic agent, diagnostic agent or a detectable label.

Definition

If not specified differently, all used here is the technical and scientific terms generally have the same meaning as commonly understood by experts in the field, belongs to this invention. Generally, the nomenclature used here and described below laboratory techniques for the cultivation of cells, molecular genetics, organic chemistry and chemistry of nucleic acids and hybridization are well known and are commonly used in this field. For the synthesis of nucleic acids and peptides using standard techniques. Usually the enzymatic reaction stage and the cleaning is carried out according to the manufacturers ' instructions. Methods and procedures are typically performed under generally accepted in the field of methods and various links (in most cases see the work Sambrook and other, Molecular Cloning: A LABORATORY MANUAL, 2d ed. (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which is contained in this document. The nomenclature used here and described below laboratory techniques in analytical chemistry and organic synthesis are well known and are commonly used in this field. For chemical synthesis and chemical analysis using standard methods or their modifications.

The terms "therapeutic agent" is intended to denote a compound that, when present in a therapeutically effective amount that produces the desired therapeutic effect in the mammal. For the treatment of carcinoma preferably, therapeutic agent was also able to enter into the target cell.

The expression "cytotoxin" is intended to refer to a therapeutic agent, providing the desired cytotoxic effect on cancer cells. The terms "cytotoxic" means that the agent inhibits the growth or destroys cells. Typical cytotoxins include, as examples, not limitations) combretastatin, duocarmycin, the antitumor antibiotic CC-1065, anthracyclin and related compounds. Other cytotoxins include mycotoxins, ricin and its analogues, calicheamicin, doxirubicin and maytansinoid.

The term "prodrug" and the expression "conjugate drugs" are used here interchangeably. Both expressions refer to a connection that is relatively safe for the cells being in the form of a conjugate, but which selectively breaks down to a pharmacologically active form in certain conditions, for example, enzymes that are located within or near the target cells.

The term "marker" is intended to refer to compounds useful in characterizing tumors or other medical condition, such as diagnosis, tumor development and in the study of factors secreted by tumor cells. Markers are a subset of "diagnostic agents".

The term "selective"as used in connection with enzymatic means decomposition, means that the rate of decomposition of the linker fragment is greater than the rate of decomposition of a peptide having a random sequence of amino acids.

The expression "guide group" and "smart agent" is intended to refer to a fragment, which: (1) capable of directing the object to which it is attached (e.g., therapeutic agent or marker)in the target cell, e.g. in a tumor cell specific the IPA, or (2) preferably is activated in the target tissue, such as tumors. Guided group or a smart agent can be a small molecule, which, as implied, includes dipeptide and peptides. Guide the group may also be a macromolecule that includes sugars, lectins, receptors, ligands for receptors, proteins such as BSA, antibodies, etc. In a preferred embodiment of the present invention, guide the group represents the antibody or fragment of the antibody, preferably a monoclonal antibody or fragment monoclonal antibody.

The expression "self-destroying spacer" refers to a bifunctional chemical fragment that is capable of covalently bind two chemical component in a normally stable molecule. Auto-disable spacer is able to spontaneously separate from the second fragment, if destroyed the relationship with the first fragment.

The expression "detected label" is intended to denote the fragment with detective physical or chemical property.

The expression "tsepliaeva group" is intended to refer to a fragment, which is unstable in vivo. Preferred "tsepliaeva group" allows activation of the marker or therapeutic agent for the removal of marker or agent of the remainder of the conjugate. Quickly determined that the linker is preferably cleaved in vivo in a biological environment. Decomposition can result from any process without restrictions, such as enzymatic, rehabilitation, pH, etc. is Preferable to choose tsepliaeva group so that activation occurred in the place of the required impact, which may be within or near the target cells (for example, carcinoma cells or tissues, for example, at the place of therapeutic action or activity of the marker. This decomposition can be enzymatic, and typical otsepleniya groups include natural amino acids or peptide sequences that have at the end of the natural amino acid and is attached to the linker at its carboxyl end. Although the degree of increase of the degradation rate is not critical to the invention, preferred examples tseplyaesh of linkers are those in which at least about 10% tseplyaesh groups hatshepsuts in the blood stream for 24 h after injection, most preferably at least about 35%.

The term "ligand" refers to any molecule that specifically binds or reactive way associated or complexesa with a receptor, substrate, antigenic determinant, or other binding site n is the target cell or the target tissue. Examples of ligands include antibodies and fragments thereof (e.g., monoclonal antibody or its fragment), enzymes (e.g., fibrinolytic enzymes, biological response modifiers (e.g., interleukins, interferons, erythropoietin or colony-stimulating factors), peptide hormones and their antigen-binding fragments.

The expression "hydrazine powered linker" and "semiclassical hydrazine powered linker" are used here interchangeably. These terms apply to the linker fragment that when conditions change, for example the shift of pH, undergoes cyclization and forms one or more loops. Hydrazine powered fragment is transformed into a hydrazone, when attached. This connection can occur, for example, by interacting with the ketone group on the fragment of L4. Therefore, for the description of the linker of the present invention can also use the expression "gerasoulis linker" is due to the conversion of the hydrazone when joining.

The expression "five-membered hydrazine powered linker" or "5-membered hydrazine powered linker" refers to a hydrazine-containing molecular fragments that, when changed conditions, such as pH shift, undergo a cyclization reaction to form one or more 5-membered cycles.

Differently, this five-membered linker can similarly be described as paticle the hydrated gerasoulis linker, or 5-membered gerasoulis the linker.

The expression "six-membered hydrazine powered linker"or "6-membered hydrazine powered linker"refers to a hydrazine-containing molecular fragments, which, when conditions change, for example the pH shift, subjected to cyclization reaction to form one or more 6-membered cycles. This six-membered linker can similarly be described as a six-membered gerasoulis linker, or 6-membered gerasoulis the linker.

The expression "reaction of cyclization"if it belongs to the cyclization of the peptide, hydrazine powered or disulfide linker, denotes the cyclization of this linker in the ring and initiating separation of complex drug-ligand. The degree of reaction can be determined ex situ and to complete the reaction in the formation of at least 90%, 95% or 100% of the product.

The terms "polypeptide", "peptide" and "protein" are used here interchangeably to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding natural amino acids, as well as natural amino acid polymers and non-natural amino acid polymers. These terms also include the term "antibody".

The term "amino acid" refers to natural and synthetic AMI is ocelotl, as well as analogs of amino acids and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are the amino acids encoded by the genetic code, as well as those amino acids that are later subjected to modification, for example, hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Similar amino acids are compounds that have the same basic chemical structure as natural amino acid, i.e. the atoms of the α-carbon, which are connected with hydrogen atoms, carboxyl group, amino group and a group R, for example, homoserine, norleucine, methanesulfonic, metonymically. Such analogs have modified group R (for example, norleucine) or modified peptide main chain, but retain the same basic chemical structure as natural amino acid. In particular, one amino acid that can be used is citrulline, which is a precursor of arginine and included in the formation of urea in the liver. The amino acid mimetics refers to chemical compounds that have a structure different from the General chemical structure of amino acids, but are similar to the natural amino acid. The expression "synthetic amino acid" is intended to denote stereochemical form "D" twenty when adnych amino acids, described above. In addition, it is clear that the expression artificial amino acid includes homologues of natural amino acids and synthetically modified forms of natural amino acids. Synthetically modified forms include, but are not limited to, amino acids, having alkylene chain, shortened or elongated by two carbon atoms, amino acids, including optionally substituted aryl groups, and amino acids containing halogenated groups, preferably halogenated alkyl and aryl groups. If the amino acid is attached to the linker or the conjugate according to this invention, it is present in the form of "amino acid side chain", where the carboxylic acid group of the amino acids replaced by a keto group (C(O)). Thus, for example, alanine side chain represents-C(O)-CH(NH2)-CH3and so on.

Amino acids and peptides can be protected by blocking groups. The blocking group is an atom or chemical fragment, which protects the N-end amino acid or peptide against undesirable reactions, and can be used for the synthesis of a conjugate of the drug-ligand. It should remain attached to the N-end during synthesis and can be removed upon completion of the synthesis of the conjugate drugs through chemical or other conditions, in the which achieved its selective removal. The blocking group suitable for protecting the N-end, well-known in the field of peptide chemistry. Typical blocking groups include, but are not limited to, hydrogen, D-amino acid, carbobenzoxy(Cbz) chloride.

The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers in the form of single - or double-stranded form. The term encompasses nucleic acids, including known nucleotide analogs or modified residues of the main chain, or compounds that are synthetic, natural and unnatural, which have similar binding properties as this one nucleic acid, and which undergo metabolism similarly considered nucleotides. Examples of such analogues include (without limitation) phosphorothioate, phosphoramidate, methyl phosphonates, chiral methylphosphonate, 2-O-methylribonucleotide, peptide-nucleic acid (PNA).

If not specified differently, the specific sequence of nucleic acids is also fully embraces its conservatively modified variants (for example, replacement of degenerative codon) and complementary sequences, as well as the explicitly specified sequence. Specifically, the replacement of degenerative codon can be performed by generating sequences in which the third position of one or NESCO is gcih selected (or all) codons is substituted mixed primary and/or deoxyinosine residues (Batzer and others, Nucleic Acids Res. 19:5081 (1991); Ohtsuka, etc., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini and others, Mol. Cell. Probes 8: 91-98 (1994)). The term “nucleic acid” is used interchangeably with the terms gene, cDNA, mRNA, oligonucleotide and polynucleotide.

The symbolused to indicate connection or location perpendicular to the connection, indicates the point at which the display portion is attached to the remainder of the molecule, the solid substrate and other

If nothing else is stated, the term "alkyl", by itself or as part of another substituent, refers to a linear or branched chain, or cyclic carbohydrate radical, or combination thereof, which may be fully saturated, mono - or polyunsaturated and can include di - and multivalent radicals, having the indicated number of carbon atoms (namely C1-C10means from one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, such groups as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentile, n-hexyl, n-heptyl, n-Attila and the like. Unsaturated alkyl group represents a group having one or more double bonds or triple bonds. Examples of unsaturated alkyl GRU is p include, but not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethinyl, 1 - and 3-PROPYNYL, 3-butynyl and higher homologs and isomers. While not noted differently, it is understood that the term "alkyl" also includes derivatives of alkyl defined in more detail below, for example, "heteroalkyl." Alkyl groups that are restricted to the carbohydrate groups are referred to as "gomolka".

The term "alkylene", by itself or as part of another substituent, refers to a bivalent radical derived alkane, an example of which is (but not limited to) -CH2CH2CH2CH2and, in addition, include groups described below as "heteroalkyl". Typically, an alkyl (or Allenova) group has 1 to 24 carbon atoms, despite the fact that in this invention the preferred group having 10 or less carbon atoms. The expression "lower alkyl" or "lower alkylene" denote alkyl or alkilinity group with a shorter chain, generally having eight or less carbon atoms.

If nothing else is stated, the term "heteroalkyl", by itself or in combination with another term, means a stable linear or branched chain, or cyclic carbohydrate radical, or combinations thereof, containing the indicated number of carbon atoms and at least one heteroa the Ohm, selected from the group comprising O, N, Si and S, where the nitrogen, carbon and sulfur may not necessarily be in the oxidized state, and the nitrogen heteroatom may optionally be quaternity. Heteroatom(s) O, N, S and Si may be placed at any interior position heteroalkyl group or at the position at which the alkyl group attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3and-CH=CH-N(CH3)-CH3. Up to two heteroatoms can be consecutive, such as-CH2-NH-OCH3and-CH2-O-Si(CH3)3. Similarly, the term "heteroalkyl", by itself or as part of another substituent, refers to a bivalent radical derived heteroalkyl, such as (but not limited to, -CH2-CH2-S-CH2-CH2- and-CH2-S-CH2-CH2-NH-CH2-. For heterouncinata groups, heteroatoms can also occupy either end or both ends of the chain (for example, alkylene, alkylenedioxy, alkylamino, alkylenediamine and the like). The terms "heteroalkyl and heteroalkyl" cover poly(ethylenglycol is) and its derivatives (see, for example, Shearwater Polymers Catalog, 2001). In addition, for alkilinity and heteroarenes binder groups does not mean orientation of the linking group in the direction in which you draw the formula of the linking group. For example, the formula-C(O)2R'represents both provisions, -C(O)2R'and R C(O)2-.

The term "lower" in combination with the terms "alkyl" or "heteroalkyl" refers to a fragment having from 1 to 6 carbon atoms.

The terms "alkoxy", "alkylamino", "alkylsulfonyl" and "alkylthio" (or dialkoxy) are used in their usual meaning and denote alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, a group of SO2or sulfur atom, respectively. The term "arylsulfonyl" refers to an aryl group attached to the remainder of the molecule through a group of SO2and the term "sulfhydryl" refers to a group of SH.

In General, the "acyl Deputy" is also selected from the group defined above. Used herein, the term "acyl Deputy" refers to groups that are attached and fill the valence carbonyl carbon, which directly or directly attached to the polycyclic nuclei of the compounds of the present invention.

If nothing else is stated, the terms "cycloalkyl and heteroseksualci", by themselves or in combination with other terms, qi denote the symbolic variants substituted or unsubstituted "alkyl" and substituted or unsubstituted "heteroalkyl" respectively. In addition, for geterotsiklicheskie heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to this, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl and the like. Examples of heterocyclization include, but are not limited to, 1-(1,2,5,6-tetrahydropyridine), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothieno-2-yl, tetrahydrothieno-3-yl, 1-piperazinil, 2-piperazinil and the like. Heteroatoms and carbon atoms of the cyclic structures are not necessarily oxidized.

The terms "halo" or "halogen" by themselves or as part of another substituent mean, if nothing else is stated, a fluorine atom, chlorine, bromine or iodine. In addition, it is understood that such terms as "halogenated include monohalogenated and POLYHALOGENATED. For example, it is understood that the term "halogen(C1-C4)alkyl" includes, but is not limited to this, trifluoromethyl, 2,2,2-triptorelin, 4-chlorobutyl, 3-bromopropyl and the like.

If nothing else is stated, the term "aryl" means substituted or unsubstituted polyunsaturated aromatic carbohydrate Deputy, which may constitute one cycle or several cycles (preferably from 1 to 3 cycles), which is s fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or cycles)that contain from one to four heteroatoms selected from N, O and S, where the atoms of nitrogen, carbon and sulfur are not necessarily oxidized, and the atom(s) nitrogen optional quaternity. Heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-restrictive examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4 pirimidil, 5-benzothiazolyl, purines, 2-benzimidazolyl, 5-indolyl, 1-ethanolic, 5-ethanolic, 2-quinoxalinyl, 5-quinoxalinyl, 3-chinolin and 6-chinolin. Substituents for each of the above aryl and heteroaryl cyclic system selected from the group of acceptable substituents described below. "Aryl" and "heteroaryl also cover a cyclic system in which one or more non-aromatic cyclic systems condensed or linked in any other way, forming an aryl or heteroaryl system.

For brevity, the term "aryl", used in combination with other terms (for example, aryloxy, arylthiols, arylalkyl)includes both aryl and heteroaryl cycles that defined above. Thus, it is assumed that the term "arylalkyl" includes radicals in which the aryl group attached to the alkyl group (such as benzyl, phenetyl, pyridylmethyl and the like)including those alkyl groups in which a carbon atom (for example, a methylene group) has been replaced by, for example, an oxygen atom (for example, phenoxymethyl, 2-pyridyloxy, 3-(1-naphthyloxy)propyl and the like).

Each of the above terms (e.g., "alkyl," "heteroalkyl", "aryl" and "heteroaryl") include substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are listed below.

Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkyl, heteroalkyl, quinil, cycloalkyl, heteroseksualci, cycloalkenyl and geteroseksualen) is usually specified as substituents for alkyl, and the substituents for heteroalkyl" respectively, and they can be one or more of a variety of groups selected from (but not limited to): -OR', =O, =NR', =N-OR', -NR'r R", -SR', -halogen, -R SiR'"R'", -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)R NR'r", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'r R"R'")=NR"", -NR-C(NR'r R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'r R", -NRSO2R', -CN and-NO23and-CH2CF3) and acyl (e.g.,- C(O)CH3, -C(O)CF3, -C(O)CH2OCH3and the like).

Similar to the substituents described for the alkyl radical, substituents for the aryl and alternates for heteroaryl usually referred to as the substituents for aryl" and "Deputy for heteroaryl" respectively, and in rerout and choose for example, from: halogen, -OR', =O, =NR', =N-OR', -NR'r R", -SR', halogen, -SiR'R"R'", -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)R NR'r", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O)2R', -NR-C(NR'r R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'r R", -NRSO2R', -CN and-NO2, -R', -N3, -CH(Ph)2, fluorine(C1-C4)alkoxy, and fluoro(C1-C4)alkyl in the range from zero to the total number of free valences of the aromatic cyclic system; and where R', R", R'" and R""each preferably independently selected from a hydrogen atom, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl. For example, if the connection of the invention includes more than one group R, then each of the R groups is independently selected as are each R', R", R'" and R""if there is more than one of these groups.

Two of the substituents for aryl with adjacent atoms of aryl or heteroaryl cycle may not necessarily be replaced by the Deputy of the formula-T-C(O)-(CRR')q-U, where T and U independently represent-NR-, -O-, -CRR'- or a simple bond, and q is an integer from 0 to 3. In another, two of the substituents at adjacent atoms of aryl or heteroaryl cycle may not necessarily be replaced by the Deputy of the formula-A-(CH2)r-B-, where A and B independently represent-CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O) 2NR'- or a simple bond, and r is an integer from 1 to 4. One of the simple relationships formed thus a new cycle may not necessarily be replaced by a double bond. In another, two of the substituents for aryl with adjacent atoms of aryl or heteroaryl cycle may not necessarily be replaced by a Deputy of the formula -(CRR')S-X-(CR"R"')d-, where s and d are independently equal integers from 0 to 3 and X represents-O-, -NR'-, -S-, -S(O)-, -S(O)2- or-S(O)2NR'-. The substituents R, R', R" and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C1-C6) alkyl.

Used herein, the term "diphosphate" includes, but is not limited to this, ether phosphoric acid containing two phosphate groups. The term "Tris" includes, but is not limited to this, ether phosphoric acid containing three phosphate groups. For example, specific medicines containing diphosphate or triphosphate, include:

Used herein, the term "heteroatom" includes oxygen atom (O), nitrogen (N), sulfur (S) and silicon (Si).

The symbol "R" is a General designation, which is replacing a group that is selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted gets reallyh and substituted or unsubstituted heterocyclyl groups.

Used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition or media, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, included in the chemical carrier or transporting the chemical agent. Pharmaceutically acceptable carriers include pharmaceutically acceptable salts, where the expression "pharmaceutically acceptable salt" includes salts of the active compounds, which are derived from relatively nontoxic acids or bases, depending on the particular substituents described in this connection. If the compounds of the present invention contain relatively acidic functionalities, additive salts of the bases can be obtained by the interaction of the neutral form of such compounds with a sufficient amount of the desired base, pure or in a suitable inert solvent. Examples of pharmaceutically acceptable additive salts of bases include salts of sodium, potassium, calcium, ammonium, organic amine or magnesium, or a similar salt. If the compounds of the present invention contain relatively basic functionalities, it is possible to obtain additive salts of acids by the interaction of the neutral form of such compounds with sufficient quantities of the m desired acid, pure or in a suitable inert solvent. Examples of pharmaceutically acceptable additive salts of acids include salts derived from such inorganic acids as hydrochloric, Hydrobromic, nitric, carbonic, monocytopenia, phosphoric, managerfactory, dihydrotestorone, sulfur, monohydrogen, uudistoodetena or phosphorous acid and the like, and salts, derivatives relatively nontoxic organic acids, such as acetic, propionic, somalina, maleic, malonic, benzoic, succinic, cork, fumaric, lactic, almond, phthalic, benzolsulfonat, para-tolilsulfonil, citric, tartaric, methanesulfonate and the like. Also included are salts of amino acids such as arginate and the like, and salts of such organic acids like glucuronic or galacturonic acids and the like (see, for example, Berge and others, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Some specific compounds of the present invention contain both basic and acidic functionality that allows you to turn the data connection in additive salts of bases or acids.

When the interaction of the salt with a base or acid preferably is restored to the neutral form of the compound and is the original connection in the usual way. The original form of connection is different from the decomp is cnyh salt forms in certain physical properties, such as solubility in polar solvents, in other respects salt equivalent to the original form of connection for the purposes of the present invention.

In addition to salt forms, the present invention relates to compounds, which are proletarienne form. Described here proletarienne compounds are compounds that readily undergo chemical changes under physiological conditions, providing compounds of the present invention. Additionally, prodrugs can be converted to compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to compounds of the present invention, when placed with a suitable enzyme or chemical reagent in the reservoir of the patch for transdermal application.

Some compounds of the present invention may exist in resolutiony forms as well as solvated forms, including hydrated forms. In General, the solvated forms are equivalent nonsolvated forms and the scope of the present invention. Some compounds of the present invention can exist in many crystalline or amorphous forms. In General, all physical forms are equivalent for applications provided by the present invented the eat, and it is assumed that they are included in the scope of the present invention.

Some compounds of the present invention have asymmetric carbon atoms (optical active centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are included in the scope of the present invention.

Compounds of the present invention may also contain artificial atomic ratio of isotopes of one or more of the atoms that constitute such compounds. For example, the compounds can be labeled with radioactive isotopes, such as tritium (3H), iodine-125 (125I) or carbon-14 (14C). It is assumed that all isotopic variations of the compounds of the present invention, a radioactive or nonradioactive included in the scope of the present invention.

The expression "attached fragment" or "fragment to attach the directing group" refers to a fragment, which allows connection of the directing group to the linker. Usually join groups include (for example and not for limitation) alkyl, aminoalkyl, aminocarbonyl, carboxyethyl, hydroxyalkyl, alkyl-maleimide, alkyl-N-hydroxysuccinimide, poly(ethylene glycol)-maleimide and poly(ethylene glycol)-N-hydroxysuccinimide, each of which may be optionally substituted. The linker mo the et also have attached a snippet, actually suspended to guide the group.

Used herein, the term "leaving group" refers to a part of the substrate, which is cleaved from the substrate by interaction.

Used herein, the term "antibody" includes antibodies in General and any antigen binding fragment (i.e., "antigen-binding portion") or single chains. The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion. Each heavy chain consists of changing plot heavy chain (VHand permanent part of the heavy chain. The constant region of the heavy chain consists of three domains, a CH1CH2and CH3it may represent the mu, Delta, gamma, alpha, or Epsilon-isotype. Each light chain consists of a variable area light chain (VLand permanent part of the light chain. Permanent plot the light chain consists of a single domain CLthat can be a Kappa or lambda isotype. Plots of VHand VLcan be further divided into plots of hypervariability, called plots, complementarity determining (CDR), interspersed among areas that are more conservative, called frame sections (FR). Each VH and VLconsists of three CDRs and four FR, placed as follows from the amino end to the carboxy-end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Changing areas of heavy and light chains contain a binding domain that interacts with the antigen. Permanent sections of the antibodies may mediate the binding of immunoglobulin with tissues or factors of media, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical system of a compliment.

Used here, the term "antibody fragment" or "antigen-binding portion" of an antibody (or simply "antibody") refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen. It is shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments encompassed by the term "antibody fragment" or "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of domains VLVHand CLand CH1; (ii) the fragment F(ab')2, a bivalent fragment comprising two Fab fragment linked by a disulfide bridge in the area of the loop; (iii) a Fd fragment consisting of domains VHand CH1; (iv) a Fv fragment consisting of domains VLand VHone of the groups of the antibody, (v) a dAb fragment (Ward and others, (1989) Nature 341:544-546), which consists of domain VH; and (vi) the selection, complementarity determining (CDR). In addition, although the two domains of the VLand VHthe Fv fragment encoded by individual genes, they can be combined, using recombinant methods, by a synthetic linker that enables to receive them in the form of a single protein chain in which the plots of VLand VHform a couple, giving monovalent molecules (known as single-chain Fv (scFv); see e.g., Bird and others (1988) Science 242: 423-426; and Huston and others (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883. It is also expected that such single-chain antibodies covered by the expression "antigen-binding portion" of an antibody. These fragments of antibodies receive, using conventional techniques known to experts in this field, and these fragments are selected based on the suitability similar to whole antibodies.

Used herein, the terms "monoclonal antibody" relates to the production of antibody molecules of single molecular composition. The composition of monoclonal antibodies demonstrates a single binding specificity and affinity for a particular epitope.

To obtain monoclonal or polyclonal antibodies can be used any method known in the art (see, for example, Kohler &Milstein, Nature 256: 495-497 (1975); Kozbor and others, Immunology Today 4: 72 (193); Cole and others, pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985)).

Methods of obtaining polyclonal antibodies known to specialists in this field. Inbred strain of mice (e.g., BALB/C mice or rabbits subjected to immunization with the protein using a standard adjuvant, such as adjuvant Freind, and a standard immunization Protocol. The immune response of the animals to immunogenic drug control, taking blood samples and determining the titer of reactivity relative to beta-pojedinici. If you get an acceptable high titers of antibodies against the immunogen, collect the blood of an animal and prepare antisera. In addition, if you want, you can perform the fractionation of the antisera to enrich antibodies, relatively reactive protein.

Monoclonal antibodies can be obtained by various techniques known to experts in this field. The spleen cells of an animal, immunising desired antigen, immortalized usually by fusion with the cell myeloma (see the work of Kohler &Milstein, Eur. J. l. 6: 511-519 (1976)). Alternative methods of immortalization include transformation using virus Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the field. In a preferred embodiment, the antibody is a chimeric, or humanized, antibody. Chimeric or PTS is lovescene antibodies of the present invention can be obtained, based on the sequence of rat monoclonal antibodies. DNA encoding the immunoglobulin heavy and light chain can be obtained from the considered rat hybridoma and constructed so that it contains not a rat (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody rat-changing areas can be linked to human constant plots, using methods known in the art (see, for example, U.S. patent No. 4816567 (Cabilly and others)). To create a humanized antibody of rat plots CDR can be inserted into a human framework using methods known in the art (see, for example, U.S. patent No. 5225539 (Winter) and U.S. patent No. 5530101; 5585089; 5693762 and 6180370 (Queen and others).

In another preferred embodiment, the antibody is a human antibody. Such human antibodies can be obtained by immunization of transgenic or transamazonic mice in which the endogenous mouse immunoglobulin genes inactivated and introduced exogenous human immunoglobulin genes. Such mice are known in the art (see, for example, U.S. patents№№ 5545806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429 (all Lonberg and Kay); U.S. patent No. 5939598; 6075181; 6114598; 650584 and 6162963 (Kucherlapati and others) and PCT publication WO 02/43478 (Ishida and others)). Human antibodies can also be obtained using the methods of isolation of phages for screening libraries of human immunoglobulin genes. Such methods of isolation of phages for the selection of human antibodies are also known in the art (see, for example, U.S. patent No. 5223409; 5403484 and 5571698 (Ladner and others); U.S. patent No. 5427908 and 5580717 (Dower and others); U.S. patent No. 5969108 and 6172197 (McCafferty and others) and U.S. patent№№ 5885793; 6521404; 6544731; 6555313; 6582915 and 6593081 (Griffiths and others).

Used herein, the terms "solid support" refers to a substance which is substantially insoluble in the selected solvent system or which can be easily separated (e.g., deposition) of the selected solvent system in which it is dissolved. Solid carriers suitable for use in practice of the present invention, can include groups that are activated or can be activated, allowing the selected types to establish a connection with the solid carrier. Solid media can also be a substrate, such as a chip, a substrate or a cell, which is associated with an individual connection or more than one connection.

Used herein, the expression "reactive functional group" refers to groups, including (but not limited to) olefins, acetylene, alcohols, phenols, ethers, oxides, halides, aldehydes is, ketones, carboxylic acids, esters, amides, cyanate, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diezani, nitro, NITRILES, mercaptans, sulfides, disulfides, sulfoxidov, sulfones, sulfonic acid, sulfinate acids, acetals, ketals, anhydrides, sulphates, isonitrile Sultanovich acids, amidine, imides, imidate, Nitron, hydroxylamine, oximes, hydroxamic acids, digitoxigenin acid, alley, ortho-esters, sulfites, enamines, inamine, urea, pseudoceramides, semicarbazides, carbodiimide, carbamates, imine, azides, azo-compounds, azoxy-compounds and nitroso compounds. Reactive functional groups also include groups used to obtain bioconjugate, for example, N-hydroxysuccinimide esters, maleimides and the like (see, e.g., Hermanson, Bioconjugat Methods, Academic press, San Diego, 1996). Methods of obtaining each of these functional groups are well known in this field, and their application or modification for a particular purpose are included in the scope of the capabilities of the person skilled in the art (see, e.g., Sandler and Karo, eds., Organic Funktional Group Preparations, Academic Press, San Diego, 1989). Reactive functional groups may be protected or unprotected.

Compounds of the invention receive in the form of a single isomer is (for example, enantiomers, CIS-TRANS, positional, diastereoisomer) or in the form of a mixture of isomers. In a preferred embodiment, the connection get essentially in the form of a single isomer. Ways to get essentially isomere-pure compounds known in the field. For example, enantiomerically-enriched mixture, and pure enantiomeric compounds can be obtained by using synthetic intermediates that are enantiomerically pure, in combination with reactions that are either left unchanged the stereochemistry at the chiral center, or give her a full inversion. Differently, along with the method of synthesis, the final product or intermediate products can be separated into individual stereoisomers. Methods of inversion or maintain a particular stereocenter and methods of separating mixtures of stereoisomers are well known in this area, and specialist in this area has plenty of opportunities to choose and to determine the method for a specific situation. In a broad sense, see the work Furniss and others (eds.),Vogel''s Encyclopedia of Practical Organic Chemistry 5th Ed., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816; and Heller, Ace. Chem. Res. 23: 128 (1990).

LINKERS

The present invention relates to conjugates of a drug-ligand, where the drug is linked to the ligand via a chemical linker. This linker is a peptide, hydrazine powered or disulfide linker is depicted here as (L 4)p-F-(L1)m, (L4)p-H-(L1)mor (L4)p-J-(L1)mrespectively. In addition to the linkers that are attached to the medication, the present invention also relates to tseplyaesh linker groups which are suitable for connection of essentially any molecular types. The aspect of the invention relating to the linker groups, illustrated here by reference to their adherence to therapeutic fragment. However, the person skilled in the art it is clear that the linkers can be attached to various types, including, but not limited to, diagnostic agents, analytical agents, biomolecules, guides, agents, detected labels and the like.

One aspect of the present invention relates to linkers that are suitable for attaching the guide to therapeutic agents and markers. Another aspect of the invention relates to linkers that attach to the connection stability, reduce their in vivo toxicity or, in other words, a favorable impact on their pharmacokinetics, bioavailability and/or pharmacodynamics. Usually preferably, in such embodiments, the linker was tsalala, releasing the active drug as soon as the medication is delivered to the place of his actions. Thus, in one embodiment of the invention, the linkers of danagoulian are complete, so when you remove the linker from therapeutic agent or marker (as in activation) leaves no traces of his presence.

In another embodiment of the invention, the linkers are characterized by their ability to be chipped off on site or near the cell target, for example, on the site of therapeutic action or activity of the marker. This decomposition can be enzymatic in nature. This feature contributes to the reduction of systemic activation of therapeutic agent or marker, reducing toxicity and systemic side effects. Preferred enzymatic decomposition of degradable groups include peptide bond, ester and disulphide bonds. In other embodiments, the linkers are sensitive to pH and hatshepsuts change in pH.

An important aspect of the current invention is the ability to adjust the speed at which hatshepsuts linkers. For example, a particularly suitable is described here hydrazine powered linkers, so as, depending on the specific structure, it is possible to vary the speed at which the linker cichlisuite and it cure ligand. WO 02/096910 with respect to some specific complexes ligand-medication with hydrazine powered by the linker. However, there is no way to "adapt" linker composition, dependent on the required speed cyclization, and specific op the sledge connection otscheplaut ligand from drugs at a slower rate than the preferred speed for many of conjugates of a drug-linker. In contrast, hydrazine powered linkers of the present invention to provide a speed range cyclization from very fast to very slow, thereby allowing the selection of a specific hydrazine powered linker based on the desired speed of the cyclization. For example, very rapid cyclization can be carried out with hydrazine powered by linkers, which result in the decomposition of one 5-membered cycle. Preferred speeds cyclization for the targeted delivery of the cytotoxic agent in the cells reach using hydrazine powered linkers, which give the decomposition or two 5-membered cycle, or one 6-membered cycle, resulting from a linker having two methyl in genialnom position. It is shown that the gem-dimethyl effect increases the reaction rate of the cyclization compared with one 6-membered cycle in the absence of two metrov in genialnom position. This is the result of tension that is released in the loop. Sometimes, however, the substituents can slow reaction instead of speed it up. Often the slowdown may be steric hindrance. As shown in example 2.4, gem-dimethyl substitution allows a much more rapid reaction of cyclization compared with the case where genialny carbon p is outstay in the form of CH 2.

However, it is important to note that in some embodiments, the linker that is cleaved more slowly, may be preferable. For example, in the preparation of sustained-release or in the preparation of both components, for fast and slow release, it may be useful to provide a linker that is cleaved more slowly. In some embodiments, the low speed cyclization achieved using hydrazine powered linker, which produces decomposition of one or 6-membered cycle without gem-dimethyl substitution, or one 7-membered cycle.

Linkers also serve to stabilize therapeutic agent or marker from decomposition in the circulating blood. This feature provides a significant advantage as this stabilization results in a prolongation of half-life in the circulation of the attached agent or marker. The linker also serves to weaken the activity of the attached agent or marker, so the conjugate is relatively soft in circulation and has the desired effect, for example, is toxic after activation in a desirable place. For conjugates of therapeutic agents this feature of the linker is used to improve therapeutic index of the agent.

The stabilizing group is preferably selected to limit the clearance and IU is abolism therapeutic agent or marker enzymes, which may be present in blood or non-target tissue and, in addition, choose to limit transport of the agent or marker in cells. Stabilizing groups serve to lock the decomposition of the agent or marker and can also act providing other physical characteristics of the agent or marker. Stabilizing group may also improve the stability of the agent or marker when stored in the form of the drug or preception.

In the ideal case, the stabilizing group is suitable for the stabilization of therapeutic agent or marker, if it contributes to the protection of the agent or marker from decomposition in the study during storage of the agent or marker in human blood at 37°C for 2 h and gives less than 20%, preferably less than 10%, more preferably less than 5% and even more preferably less than 2% decomposition of the agent or marker enzymes present in human blood in these terms of research.

The present invention also relates to conjugates containing these linkers. More specifically, the invention relates to prodrugs that can be used for the treatment of diseases, especially for cancer chemotherapy. More precisely, the application described here linkers provides prodrugs, demonstrating the high specificity of action, low toxicity of the improved stability in the blood relative to the prodrugs of similar structures.

The linkers of the present invention, which is described here, may be present in any position inside the cytotoxic conjugate.

Thus, provided the linker, which may contain any of a variety of groups as part of their chain that is cleaved in vivo, for example, in the blood stream, at a speed which is increased compared to the rate for structures that do not have such groups. Provided also conjugates of the linker group to therapeutic and diagnostic agents. Linkers suitable for the formation of analogues of prodrugs of therapeutic agents and reversible binding of a therapeutic or diagnostic agent with the directing agent, detectable label or solid carrier. Linkers can be incorporated into complexes that contain the cytotoxins of the invention.

In addition tsepliaeva peptide, hydrazine powered or disulfide groups between the cytotoxin and the directing agent optionally enter one or more auto linker groups L1. These linker group can also be described as a spacer elements of the group and contain at least two reactive functional groups. Usually one chemical functionality of the spacer elements of the group associated with the chemical functionality of a therapeutic agent, such as a cytotoxin, while the other hee is practical functionality spacer elements group is used for communication with the chemical functionality of the sending agent or tsepliaeva linker. Examples of spacer elements groups include hydroxy, mercapto, carbonyl, carboxy, amino, ketone, and mercapto-group.

Auto linkers, denoted as L1usually represent a substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heteroalkyl group. In one embodiment, the alkyl or aryl groups can contain from 1 to 20 carbon atoms. They may also contain polietilenglikolya fragment.

Typical spacer elements groups include, for example, 6-aminohexyl, 6-mercaptohexanol, 10-hydroxydecanoic acid, glycine and other amino acids, 1,6-hexanediol, β-alanine, 2-aminoethanol, the group probably facilitates (2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid, 3-maleimidomethyl acid, phtalic, α-substituted phthalide, carbonyl group, animals esters, nucleic acids, peptides and the like.

The spacer can serve for the introduction of additional molecular weight and chemical functionality in complex cytotoxin-directing agent. Usually the additional weight and functionality affect the half-life in serum and other properties of the complex. Thus, by careful selection of the spacer elements of the group can be obtained cytotaxonomy complexes with installation set the Ohm of half-life in serum.

The spacer(s)located in close proximity to drug fragment, denoted (L1)mwhere m is an integer selected from 0, 1, 2, 3, 4, 5 or 6. If there is a lot of spacers L1it is possible to use an identical or different spacers. L1can be any self-destructive group.

In one embodiment, the L1preferably denotes a substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl, the unsubstituted heteroseksualci and substituted heteroseksualci. If conjugate the drug-ligand contains hydrazine powered linker, L1does not contain disulfide bonds.

L4denotes a linker fragment, which tells enhanced solubility or decreased aggregation properties of the conjugates using a linker that contains this fragment. The linker L4must not be self-destructive. In one embodiment, the fragment of L4represents a substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl or an unsubstituted heteroalkyl, any of them may be linear, branched or cyclic. The Deputy may be, for example, low (C1-C6)alkyl, alkoxy, alkylthio, alkylamino or dialkylamino. In some embodiments, L4contains the end of Klionsky fragment. In another embodiment, L4contains any positively or negatively charged such amino acid polymer, as polylysine or polyalanine. L4may contain a polymer, such as polietilenglikolya fragment. In addition, the linker L4contains, for example, and a polymer component, and a small chemical fragment.

In a preferred embodiment, L4contains polietilenglikolya (PEG) fragment. PEG is part of the linker L4can be between 1 and 50 units in length. Preferably, if the PEG has 1 to 12 repeating units, more preferably 3 to 12 repeating units, more preferably 2 to 6 repeating units, more preferably 3-5 repetitive units and most preferably 4 duplicate link. L4may consist solely of PEG-fragment, or may also contain additional substituted or unsubstituted alkyl or heteroalkyl. It is useful to combine PEG as part of the fragment L4to increase the solubility of the complex in water. In addition, PEG-fragment reduces the degree of aggregation that can occur during conjugation of drugs and antibodies.

(1) Peptide linkers (F)

As discussed above, the peptide linkers of the invention can be represented General formula (L4)p-F-(L1)mwhere F denotes a linker part, including pepti the hydrated fragment. In one embodiment, the F-part contains optional auto-disable linker(s) L2and a carbonyl group. In the other variant of the F-part contains an amino group and an optional spacer elements group (s) L3.

Thus, in one embodiment, the conjugate comprising a peptide linker, contains a structure of formula 4

In this embodiment, L1indicates auto linker, which is described above, and L4denotes the fragment, which gives enhanced solubility or decreased aggregation properties, as described above. L2represents the auto-linker(s); m is 0, 1, 2, 3, 4, 5 or 6; o and p are independently 0 or 1. In one embodiment, m is 3, 4, 5 or 6. AA1represents one or more natural amino acids and/or synthetic α-amino acids; c is an integer from 1 to 20.

In the peptide linkers of the present invention having the above formula 4, AA1linked at its amino end directly with L4or, if L4no, directly with a group of X4(i.e. directing agent, detectable label, protected reactive functional group or unprotected reactive functional group). In some embodiments, L4if present, does not contain carboxy who enoy acyl group, directly attached to the N-end (AA1)c. Thus, in these embodiments is not necessary the presence of a carboxylic acyl unit directly between the L4or X4and AA1as required in the peptide linkers of U.S. patent No. 6214345.

In another embodiment, a conjugate comprising a peptide linker, contains a structure of formula 5

In this embodiment, L4denotes the fragment, which gives enhanced solubility or decreased aggregation properties, as described above; L3denotes a spacer elements group containing a primary or secondary amine or a carboxyl functional group, and amine linker L3forms an amide bond with dangling carboxyl functional group of D or carboxyl linker L3forms an amide bond with dangling amine functional group of D; and o and p independently are 0 or 1. AA1represents one or more natural amino acids and/or synthetic α-amino acids; c is an integer from 1 to 20. In this embodiment, L1absent (i.e. in the General formula m is 0).

In the peptide linkers according to this invention, having the above formula 5, AA1linked at its amino end directly with L4or, if L4no, directly with X4 group (i.e. directing agent, detectable label, protected reactive functional group or unprotected reactive functional group). In some embodiments, L4if present, does not contain a carboxylic acyl group directly attached to the N-end (AA1)c. Thus, in these embodiments is not necessary the presence of a carboxylic acyl unit directly between the L4or X4and AA1as required in the peptide linkers of U.S. patent No. 6214345.

Auto linker L2

Auto linker L2represents a bifunctional chemical fragment that is capable of covalently linking together two spaced chemical component in a normally stable molecule, consisting of three parts, releasing one of these spatially distinct chemical components of the molecule, consisting of three parts, through enzymatic decomposition; and after this enzymatic decomposition spontaneously chipped off from the remaining part of the molecule, releasing the other of these spatially distinct chemical components. According to the present invention, self-destructive spacer is covalently bonded at one of its ends with peptide the fragment and covalently linked at the other end with a chemically reactive site of drug fragment, the derivatization which inhibits pharmacological activity, therefore, to separate in space and covalently bind together the peptide fragment and drug fragment consisting of the three parts of the molecule that is stable and pharmacologically inactive in the absence of the target enzyme, but which are capable of enzyme to split this target enzyme for communications, covalently linking the spacer elements fragment and peptide fragment, thereby making the release of the peptide fragment consisting of the three parts of the molecule. Such enzymatic decomposition, in turn, activates the self-destructive nature of the spacer elements of the fragment and initiates spontaneous breakdown of communication, covalently linking the spacer elements snippet drug fragment, thereby making the drug release in a pharmacologically active form.

Auto linker L2can be any self-destructive group. Preferably L2represents a substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, the unsubstituted heteroseksualci, substituted heteroseksualci, substituted and unsubstituted aryl and substituted and unsubstituted heteroaryl.

One particularly preferred auto spacer L2can p is edstavit formula 6

Aromatic ring aminobenzene group may be substituted by one or more groups "K". Group "K" represents a substituent in the aromatic ring, which replaces the hydrogen atom, in another case attached to one of the four unsubstituted carbon atoms that are part of a cyclic structure. Group "K" may be a single atom such as halogen, or polyatomic group, such as alkyl, heteroalkyl, amino, nitro, hydroxy, alkoxy, halogenated and cyano. Each group K is independently selected from the group including substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR21R22, NR21COR22, OCONR21R22, OCOR21and OR21where R21and R22independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci and the unsubstituted heteroseksualci. Typical substituents K include, but are not limited to, F, Cl, Br, I, NO2, H, OCH3, NHCOCH3N(CH3)2, NHCOCF3and methyl. For a group of Ka" a is an integer of 0, 1, 2, 3 or 4. In one preferred embodiment, a is 0.

Etheric oxygen atom shown above structure is connected to the carbonyl group. Line from NR24functionality in the aromatic cycle indicates that the amine functionality can be associated with any of the five carbon atoms, which form the loop and not substituted by a group-CH2-O-. Preferably, if NR24functionality from X covalently linked to the aromatic cycle in para-position relative to group-CH2-O-. R24indicates a component selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl. In a specific embodiment, R24denotes hydrogen.

In a preferred embodiment, the invention relates to a peptide linker of the formula (4)above, where F has the structure

where

R24selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl;

each K denotes a component that is independently selected from the group including substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, resumes the config aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR21R22, NR21COR22, OCONR21R22, OCOR21and OR21where

R21and R2independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci; and

a is an integer of 0, 1, 2, 3, or 4.

In another embodiment, the peptide linker of the formula (4)above contains-F-(L1)m-which has the structure

where

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl.

Spacer elements group L3

Spacer elements group L3characterized in that it contains primary or secondary amino or carboxyl functional group and an amine group of L3forms an amide bond with dangling carboxyl functional group of D or carboxyl from L3forms an amide bond with dangling amine functional group of d L3can be selected from GRU the dust, including substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or substituted or unsubstituted heteroseksualci. In a preferred embodiment, L3contains an aromatic group. More preferably, if L3contains benzoic acid group, an aniline group or indole group. Non-restrictive examples of structures that can serve as a spacer-L3-NH-, include the following structures:

where Z denotes a component selected from O, S and NR23and

where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl.

After removal of the linker of the present invention, containing L3fragment L3remains attached to the medication D. Therefore, the fragment of L3is chosen so that its presence is attached to D did not significantly alter the activity of D. In another embodiment, the part D drugs itself functions as a spacer L3. For example, in one embodiment, the medicine D is a derivative duocarmycin, in which part of the drug functions as a spacer L3. Non-restrictive examples of such options, the ants include options, in which NH2-(L3)-D has a structure selected from the group including

and

where Z denotes a component selected from O, S and NR23,

where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl; and

where the group NH2each structure interacts with (AA1)withforming -(AA1)with-NH-.

Peptide sequence AA1

Group AA1represents a single amino acid or a set of amino acids that are combined together to form amide bonds. Amino acids may be natural amino acids and/or synthetic α-amino acids.

The peptide sequence (AA1)withfunctionally represents the balance of aedificatoria individual amino acids (if c=l) or many amino acids linked together by an amide bonds. The peptide of the present invention are chosen so that to send catalyzed by enzyme cleavage of the peptide to the desired location of the biological system. For example, conjugates, which are directed into the cell, using the smart agent, and then fixed cell, choose the peptide so that he tsalala one or more lysosomal what proteases, so that cleavage of the peptide occurred in the intracellular lysosome. The number of amino acids in the peptide can be from 1 to 20; but it is more preferable (AA1)withcomprising 2 to 8 amino acids, 2 to 6 amino acids or 2, 3 or 4 amino acids. Peptide sequences that are sensitive to cleavage by specific enzymes or classes of enzymes are well known in this field.

In this area there are many known peptide sequence, which is broken down by enzymes in the serum, liver, intestines and other Typical peptide sequence according to this invention includes a peptide sequence that is cleaved by the protease. The subsequent discussion focused on the use of protease-sensitive sequence for clarity of illustration and not to limit the scope of the present invention.

If the enzyme that cleaves the peptide is a protease, the linker typically includes a peptide containing identifiable sequence for a protease. The sequence of the cleavage recognition for protease is an amino acid sequence recognized by the protease during proteolytic decomposition. In this area there are many known sites proteasome decomposition, and these and other sites of decomposition can be included in interny fragment. See, for example, work Matayoshi and others, Science 247: 954 (1990); Dunn and others, Meth. Enzymol. 241: 254 (1994); Seidah and others, Meth. Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994); Weber and others, Meth. Enzymol. 244: 595 (1994); Smith and others, Meth. Enzymol. 244: 412 (1994); Bouvier and others, Meth. Enzymol. 248: 614 (1995), Hardy and others, Amyloid Protein Precursor in Development, Aging and Alzheimer's Disease, ed. Masters and others, pp. 190-198 (1994).

Amino acids of the peptide sequence (AA1)withchoose on the basis of their suitability for selective enzymatic degradation of specific molecules, for example, is associated with tumor protease. Used amino acids may be natural or unnatural amino acids. They can be in the L or D configuration. In one embodiment, the use of at least three different amino acids. In another embodiment uses only two amino acids.

In a preferred embodiment, the peptide sequence (AA1)withchoose on the basis of its ability to be broken down by lysosomal proteases, non-restrictive examples of which include cathepsins B, C, D, H, L and S. Preferably, if the peptide sequence (AA1)withable to split by cathepsin B in vitro, which can be verified by using in vitro studies proteasome decomposition, known in this field.

In another embodiment, the peptide sequence (AA1)withchoose on the basis of its ability to split PR is teapoy, associated with a tumor, such as a protease that is found outside of the cells close to the tumor cells, non-restrictive examples of which include time-oligopeptides (TOP) and CD10. The ability of the peptide to split through the TOP or CD10 can be checked by applying known in the field of in vitro studies proteasome decomposition.

Suitable, but non-restrictive examples of peptide sequences that are suitable for use in the conjugates of this invention include Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2) and Gly-Phe-Leu-Gly (SEQ ID NO: 3). Preferred peptide sequences are Val-Cit and Val-Lys.

In another embodiment, the amino acid located at the nearest position to medicinal fragment selected from the group including: Ala, Asn, Asp, Cit, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. In another embodiment, the amino acid located at the nearest position to medicinal fragment selected from the group including: Ala, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

Proteases involved in cancer metastasis. Increased synthesis of protease urokinase correlates with an enhanced ability metastasis in many cancers. Urokinase, activateplugin of plasminogen, located everywhere in the extracellular space, and its activation can cause the destruction of proteins in the extracellular matrix, through the defeat of metastatic tumor cells. The plasmin can also activate collagenase, promotora thus, the destruction of collagen in the base membrane surrounding the capillaries and lymph system, thereby allowing tumor cells to hit the target tissue (Dano, and others, Adv. The cancer. Res., 44: 139 (1985)). Thus, in the scope of the present invention includes the use of the linker peptide sequence that is cleaved by urokinase.

The invention also concerns the use of peptide sequences that are sensitive to cleavage by tryptase. Mast cells Express at least four different tryptase, denoted by α, βI, βII and βIII. These enzymes are not regulated by inhibitors of proteinases of the blood plasma and only break down some of the physiological substrates in vitro. Tryptase family semiprotect involved in a variety of allergic and inflammatory diseases, including fat cells, due to elevated levels of tryptase, detected in biological fluids of patients with these disorders. However, the exact role of tryptase in the pathophysiology of the disease requires vyyasnilost biological functions and physiological effects of tryptase is determined, mainly, their substrate specificity.

Tryptase is a potent activator prourokinase plasminogen activator (uPA), imagenow form of a protease associated with the metastasis and invasion of tumors. Activation plasminogen cascade, which results in degradation of the extracellular matrix due to cell extravasation and migration, may be a function tryptase activation prourokinase plasminogen activator in the sequence P4-P1 Pro-Arg-Phe-Lys (SEQ ID NO: 4) Stack and others, Journal of Biological Chemistry 269 (13): 9416-9419 (1994)). Vasoactive intestinal peptide, neuropeptide, which is included in the regulation of pronitsaemosti vessels, is also cleaved by tryptizol mainly on the sequence Thr-Arg-Leu-Arg (SEQ ID NO: 5) (Tarn and others, Am. J. Respir. Cell Mol. Biol. 3: 27-32 (1990)). Associated with G-protein receptor PAR-2 can be split and activate tryptizol sequence Ser-Lys-Gly-Arg (SEQ ID NO: 6) by proliferation of fibroblasts, whereas thrombin-activated receptor PAR-1 is deactivated by tryptizol the sequence Pro-Asn-Asp-Lys (SEQ ID NO: 7) (Molino and others, Journal of Biologic Chemistry 272(7): 4043-4049 (1997)). Taken together, these facts suggest a Central role for tryptase in the correction of tissue as a consequence of the disease. This is consistent with the profound changes observed in some disorders, mediated by fat cells. One item is inacom chronic asthma and other long-lasting respiratory disease is fibrosis and seal exposed to tissue, which can be the result of activation tryptase physiological targets. Similarly, the number of messages shows that angiogenesis associated with the density of mast cells, tryptase activity and poor prognosis in a variety of cancer (Coussens and others, Genes and Development 13(11): 1382-97 (1999)); Takanami and others, Cancer 88(12): 2686-92 (2000); Toth-Jakatics and other, Human Pathology 31(8): 955-960 (2000); Ribatti and others, the International Journal of Cancer 85(2): 171-5 (2000)).

In this area known methods for evaluation, splits any specific protease selected peptide sequence. For example, using 7-amino-4-methylcoumarine (AMC) fluorogenic peptide substrates is a well-known method to determine the specificity of the protease (Zimmerman, M., and others, (1977) Analytical Biochemistry 78:47-51). Specific cleavage anilides communication frees fluorogenic leaving group AMC, allowing easy determination of the rate of decomposition for individual substrates. Later used arrays (Lee, D., and others, (1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72) and libraries positional scanning (Rano, T.A., and others, (1997) Chemistry and Biology 4:149-55) libraries AMC-peptide substrates for rapid profiling of N-terminal specificity of proteases, in one experiment by taking samples of substrates in a wide range. Thus, the person skilled in the art can easily estimate the order of peptide sequences for measuring the perceptions of their suitability in the present invention, without resorting to undue experimentation.

(2) hydrazine powered linkers (H)

In the second embodiment, the conjugate according to this invention contains hydrazine powered auto linker, where the conjugate has the structure

where D, L1L4and X4such as defined above and described hereafter, and H denotes a linker comprising the structure

where

n1an integer from 1 to 10;

n2is 0, 1 or 2;

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl; and

I denotes either the link (i.e. the link between the carbon atom of the main chain and the adjacent nitrogen atom), or

where n3is 0 or 1, provided that if n3is 0, n2not equal to 0; and

n4is 1, 2 or 3,

where, if I denotes a bond, n1equal to 3 and n2equal to 1, then D could not be

,

where R is Me or CH2-CH2-NMe2.

In one embodiment, the substitution on phenyl ring is para-substitution. In preferred embodiments, n1is 2, 3 or 4, or n1equal to 3. In preferred embodiments, n2equal to 1. In site titeling options I indicates the relationship (i.e. the relationship between the carbon atom of the main chain and the adjacent nitrogen atom). In one aspect of hydrazine powered linker H can be formed by the decomposition of 6-membered auto linker, for example, if n30 and n4equal to 2. In another aspect of hydrazine powered linker H can be formed by decomposition of two 5-membered auto linker. In other aspects of H forms a decomposition of 5-membered auto linker, H forms a 7-membered auto linker or H forms a 5-membered auto linker and 6-membered auto linker. On the rate of decomposition is affected by the size formed by the decomposition cycle. Thus, depending on the desired degradation rate you can select a loop of suitable size, which will be obtained by the decomposition.

Five-membered hydrazine powered linkers

In one embodiment, the hydrazine powered linker represents a 5-membered hydrazine powered linker, where H has the structure

In a preferred embodiment, n1is 2, 3 or 4. In another preferred embodiment, n1equal to 3. In the above structure, each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl. In one embodiment, each R24independently denotes H or C1-C6alkyl. In another embodiment, b is th R 24independently denotes H or C1-C3alkyl, more preferably H or CH3. In another embodiment, at least one R24denotes a methyl group. In another embodiment, each R24denotes H. Each R24selected to provide compounds steric effects and for changes in solubility.

5-Membered hydrazine powered linkers can undergo one or more reactions of cyclization, which separate the drug from the linker, and can be described, for example, as follows:

Typical synthetic method to obtain five-membered linker according to this invention is

Spend interaction Cbz-protected DMDA b with 2,2-dimethyl-malonic acid in a solution with thionyl chloride, receiving Cbz-DMDA-2,2-dimethylmaleic acid c. Spend the interaction of compound c with Boc-N-methylhydrazino d in the presence of hydrogen, receiving DMDA-2,2-dimethylmaleic Boc-N-methylhydrazine e.

Six-membered hydrazine powered linkers

In another embodiment, hydrazine powered linker represents a 6-membered hydrazine powered linker, where H has the structure

In a preferred embodiment, n1equal to 3. In the above structure, each R24is a component that is independently selected from the group, who with H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl. In one embodiment, each R24independently denotes H or C1-C6alkyl. In another embodiment, each R24independently denotes H or C1-C3alkyl, more preferably H or CH3. In another embodiment, at least one R24denotes a methyl group. In another embodiment, each R24denotes H. Each R24selected to provide compounds steric effects and for changes in solubility. In the preferred embodiment, H has the structure

In one embodiment, H is genialne dimethyl substitution. In one embodiment, the above structure each R24independently represents H or substituted or unsubstituted alkyl.

6-Membered hydrazine powered linkers undergo the cyclization reaction, which separates the drug from the linker, and can be described as follows:

Typical synthetic method of obtaining six-membered linker according to this invention is

Spend interaction Cbz-protected dimethylalanine in solution with dichloromethane with HOAt and CPI, receiving Cbz-protected dimethylaminopropan b. Remove protection from hydrazine b, affecting the meta is and I getting connection c.

Other hydrazine powered linkers

I believe that the invention provides a linker having the seven components. The linker will probably not be cilitates as fast as five - or six-membered linkers, but it may be preferable for some conjugates of the drug-ligand. Similarly, hydrazine powered linker can contain two six-membered cycle or hydrazine powered linker having one six-membered cyclization product and one five-membered. Also consider five and semicolony linker, as well as six and semicolony the linker.

Other hydrazine powered structure H has the formula

where q is 0, 1, 2, 3, 4, 5 or 6; and

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl.

This hydrazine powered structure may also form a five-, six - or semicolonies cycles, and you can add additional components, receiving a variety of loops.

(3) Disulfide linkers (J)

In another embodiment, the linker contains enzymatic tsepliaeva disulfide group. In one embodiment, the invention relates to cytotoxic compounds the drug-ligand having a structure corresponding to formula 3

where D, L1L4and X 4such as defined above and described hereafter, and J represents a disulfide linker, including a group having the structure

where

each R24indicates a component that is independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl and the unsubstituted heteroalkyl;

each K denotes a component that is independently selected from the group including substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR21R22, NR21COR22, OCONR21R22, OCOR21and OR21where

R21and R22independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci and the unsubstituted heteroseksualci;

a is an integer of 0, 1, 2, 3 or 4; and

d is an integer from 0, 1, 2, 3, 4, 5 or 6.

Aromatic cycle disulfide linker may be substituted by one or more groups "K". Group "K" represents the substituent in ar is automatic cycle, which replaces the hydrogen atom, in another case attached to one of the four unsubstituted carbon atoms that are part of a cyclic structure. Group "K" may be a single atom such as halogen, or polyatomic group, such as alkyl, heteroalkyl, amino, nitro, hydroxy, alkoxy, halogenated and cyano. Typical substituents K independently include, but are not limited to, F, Cl, Br, I, NO2, OH, OCH3, NHCOCH3N(CH3)2, NHCOCF3and methyl. For a group of Ka", a is an integer of 0, 1, 2, 3 or 4. In one preferred embodiment, a is 0.

In a preferred embodiment, the linker contains enzymatic tsepliaeva disulfide group of the following formula:

In this embodiment, L4X4, p, and R24have the same meanings as described above, and d is equal to 0, 1, 2, 3, 4, 5 or 6. In a particular embodiment, d is 1 or 2.

More specific disulfide linker shown in the formula below

A specific example of this variant is the following:

Preferably d is 1 or 2.

Another disulfide linker shown in the formula below

A specific example of this variant is the following:

Preferably d is 1 or 2.

In various embodiments, the disulfides are in ortho-position to the amine. In another specific embodiment, a is 0. In preferred embodiments, R24independently selected from H and CH3.

Typical synthetic method of obtaining a disulfide linker according to this invention is as follows:

Spend interaction solution of 3-mercaptopropionic acid with altricial-2, getting the 3-methylbenzothiazolium b. Spend the interaction of 3-methylbenzothiazolium c sodium hydroxide, receiving the connection d. Then hold the interaction of a solution of compound d in methanol with compound b, when receiving the connection e. Remove protection from the connection e, influencing acetylchloride and methanol, receiving the connection f.

Conjugate the drug-ligand of the present invention may optionally contain two or more linkers. These linkers may be the same or different. For example, you can use a peptide linker to connect the drug with the ligand, and the second peptide linker can attach a diagnostic agent to the complex. Differently, any of the peptide, hydrazine powered and disulfide linkers can bind the complex of the drug and ligand, and any of the peptide, hydrazine powered and disulfide linkers may Pris is to merge the diagnostic agent to the complex. Other applications for additional linkers include linking analytical agents, biomolecules, guides, agents and detectable labels with complex drug-ligand.

In the scope of the present invention also includes compounds of the present invention, which are poly - or multivalent types, including, for example, types such as dimers, trimers, tetramer and higher homologues of the compounds of this invention or their reactive counterparts. Poly - and multivalent type may consist of a single type or several types of compounds in this invention. For example, dimeric structure can be "homodimers" or "heterodimeric". In addition, the scope of the present invention include poly - and multivalent constructs, in which the compound of this invention or its reactive counterpart attached to oligomeric or polymeric skeleton (for example, polylysine, dextran, gidroxiatilkrahmal and the like). The frame is preferably a polyfunctional (i.e. with reactive sites for attachment of compounds according to this invention). In addition, the frame can be subjected to derivatization with the use of compounds of a single type or several types according to this invention.

In addition, the present invention includes compounds in which call is received some functions for message compounds solubility in water, which is increased in comparison with similar compounds that do not have such functions. Thus, any of these substituents can be substituted with radicals that have a high solubility in water. For example, in the scope of the present invention includes the replacement of the hydroxyl group diola, or amine - quaternions Amin, hydroxyamino or similar, better water-soluble fragment. In a preferred embodiment, additional solubility in water reported by the substitution site not essential for the activity of these compounds relative to the ion channel, fragment, which increases the solubility of the parent compounds in water. Ways of increasing the water solubility of organic compounds known in the field. Such methods include, but are not limited to this, the functionalization of organic nuclei is constantly charged fragments, such as Quaternary ammonium or by a group that is charged at physiologically suitable pH, such as carboxylic acid, amine. Other methods include adding to the organic cores hydroxyl - or amine-containing groups, such as alcohols, polyols, polymeric ethers and the like. Typical examples include, but are not limited to this, polylysin, polyethylenimine, polyethylene glycol and propylenglycol. Suitable chemistry and functionalization strategy for these compounds are well known in this field. See, e.g., Dunn, R.L., and others, Eds. Polymeric Drugs and Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical Society, Washington, D.C., 1991.

DRUGS

This invention relates to drugs, here marked "D", as part of a conjugate of the drug-ligand, where the medication is associated with ligand through peptide, hydrazine powered or disulfide linker. Medicine must possess the desired biological activity and contain reactive functional group to communicate with the ligand. Desirable biological activity includes the diagnosis, treatment, alleviation, treatment, or prevention of disease in an animal, such as man. Thus, because of the presence of reactive functional groups, the term "drug" refers to chemical reagents, officially recognized as drugs in the USP (United States Pharmacopeia), the homeopathic Pharmacopoeia of the USA (Homeopathic Pharmacopeia of the United States) or national rules (National Formulary), or any of its amendments. Typical drugs set forth in Physician''s Desk Reference (PDR) and the Orange Book, supported by Management under the control over products and medicines of the U.S. Food and Drug Administration (FDA)). New drugs are continuously being discovered and developed the Xia, and the present invention provides that these new drugs also can be included in the complex of the drug-ligand in this invention.

Preferred functional groups include primary or secondary amines, hydroxyl, sulfhydryl, carboxy, aldehydes and ketones. More preferred functional groups include hydroxyl, primary or secondary amines, sulfhydryl and carboxylic acid functional groups. More preferred functional groups include hydroxyl, primary and secondary amines and carboxylic acid functional groups. The drug must have at least one reactive functional group, but may have 2, 3, 4, 5, 6 or more of these groups. In addition, between the reactive functional group of the drug and the peptide, or hydrazine powered by a disulfide linker, you can enable auto-disable spacer L1.

Conjugate the drug-ligand is effective for the ordinary purposes for which effective medications, but has superior efficiency due to the characteristic of the ligand's ability to transport the drug to the target cell, where it is particularly useful.

Typical drugs include medicines in the form of proteins, peptides and small molecules containing the functionality of the ing group for communication with the ligand. More specifically, these drugs include, for example, enzyme inhibitors, such as inhibitors digidrofolatreduktazy and inhibitors timedilation, DNA intercalator, agents, DNA cleavage, inhibitors topoisomerase, medications, family anthracycline, drugs Vinca, mitomycin, bleomycin, the cytotoxic nucleosides, drugs, family pteridine, deeney, depapillated, stimulants differentiation and taxali.

Preferred medicaments according to the present invention include cytotoxic drugs useful in the treatment of cancer, and other small molecules, proteins or polypeptides with desired biological activity, such as a toxin. The medicine can be chosen in such a way that it was activated in tumor cells by conjugation with a tumor-specific ligand. These tumor-specific conjugates the drug-ligand possess the specificity of the tumor arising from the specificity of the ligand. Examples of this are the conjugates of the drug-ligand, which are highly selective substrates for tumor-specific enzymes, these enzymes are present in the neighborhood of the tumor in sufficient quantities to generate cytotoxic levels of free drug near the tumor. One advantage of these tumor-specific is kompleksow drug-ligand is that they are stable with respect to random proteases in human serum. Another advantage of the complex drug-ligand is that they are less toxic than the corresponding free drug; in addition, the specificity of the complex may result in a lower total concentration compared with the free drug, as a result of increased specificity on the site of the tumor will be a higher concentration of the complex.

Cytotoxins

Cytotoxic drugs suitable for the present invention include, for example, duocarmycin, CC-1065 and its analogs, including analogs of duocarmycin and CC-1065 on the basis of CBI (1,2,9,9a-tetrahydrocyclopent[c]Benz[e]indol-4-it), on the basis of MCBI (7-methoxy-1,2,9,9a-tetrahydrocyclopent[c]Benz[e]indol-4-it) and on the basis of CCBI (7-cyano-1,2,9,9a-tetrahydrocyclopent[c]Benz[e]indol-4-it), doxorubicin and conjugates of doxorubicin, such as morpholino doxorubicin, cyanomethane doxorubicin, dolastatin, such as dolastatin-10, combretastatin, calicheamicin, maytansine, the analogues of maytansine, DM-1, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), AE-ether 5-benzoylamino acid (AEVB), tubulysin, decorazon, epothilone, paclitaxel, docetaxel, SN-38, topotecan, rhizoxin, economizing, colchicine, vinblastine, vindesine, estramustin, cemadotin, Aleut the Robin, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, citizenoriented, melphalan, Larsen, leirosidin, actinomycin, daunorubicin, and conjugates of daunorubicin, mitomycin C, mitomycin A, karminomitsin, aminopterin, talisayan, depapillated and derivatives depapillation, such as etoposide or etoposide, vincristine, Taxol, Taxotere-retinoic acid, butyric acid, N8-acetylspiramycin, camptothecin and their analogues. Other well-known drugs can be modified to provide a functional group for conjugation to the one described here by the linker. Such chemical modification known in this field.

Preferred cytotoxins for use in the present invention include: duocarmycin, CC-1065 and its analogs on the basis of CCBI and MCBI, morpholino doxorubicin, cyanomethane doxorubicin, dolastatin-10, combretastatin, calicheamicin, maytansine, DM-1, auristatin E, AEB, AEFP, MMAE, tubulysin a, decorazon, epothilone A and epothilone B.

Particularly preferred cytotoxins of the present invention are active, potent derivatives duocarmycin and CC-1065. The original agents are extremely effective antitumor antibiotics, biological effects which are the result of reversible stereoelectronic-regulated sequence is lnost-selective alkylation of DNA (Boger and others, J. Org. Chem, 55: 4499 (1990); Boger and others, J. Am. Chem. Soc. 112: 8961 (1990); Boger and others, J. Am. Chem. Soc. 113: 6645 (1991); Boger and others, J. Am. Chem. Soc. 115: 9872 (1993); Boger and others, Bioorg. Med. Chem. Lett. 2: 759 (1992)). After the initial disclosure of duocarmycin were undertaken comprehensive attempt to explain the selectivity of duocarmycin respect to the alkylation of DNA and their structural origin.

Particularly preferred aspect of the present invention relates to cytotoxic compounds having a structure corresponding to formula 7:

in the cyclic system A indicates a component that is selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geterotsiklicheskikh groups. Typical cyclic systems include phenyl and pyrrole.

The symbols E and G are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, heteroatoms, simple communication, or E and G are not necessarily United, forming a cyclic system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geterotsiklicheskie.

The symbol X represents a member selected from O, S and NR23. R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted is on heteroalkyl and acyl.

The symbol R3represents a member selected from (=O)SR11, Other11and OR11where R11represents H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, diphosphate, triphosphates, acyl, C(O)R12R13C(O)OR12C(O)NR12R13, P(O)(OR12)2C(O)CHR12Rl3, SR12or SiR12R13R14. The symbols R12, R13and R14independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, where R12and R13together with the nitrogen atom or carbon to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms. One or more of R12, R13and R14may contain in its structure tsepliaeva group.

R4, R4', R5and R5' denote the components independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, SR15, OR15, CR =NR16and O(CH2)nN(CH3)2where n is an integer from 1 to 20. R15and R16independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroseksualci and substituted or unsubstituted peptidyl, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms. One typical structure is an aniline.

R4, R4', R5, R5' R11, R12, R13, R15or R16optionally contain in its structure one or more tseplyaesh groups. Typical otsepleniya groups include, but are not limited to, peptides, amino acids, hydrazines and disulfides.

At least one of R11, R12, R13, R15and R16used to attach the drug to the linker of the present invention, as described here, for example, L1if present, or to F, H or J.

In another typical embodiment, at least one of R4, R4', R5, R5', R11, R12,R 13, R15or R16bears a reactive group suitable for conjugation of this connection. In another typical embodiment, R4, R4', R5, R5', R11, R12, R13, R15or R16independently selected from H, substituted alkyl and substituted heteroalkyl and have a reactive functional group at the free end of the alkyl or heteroalkyl fragment. One or more of R4, R4', R5, R5', R11, R12, R13, R15and R16you can konjugierte with other species, for example, the directing agent, detectable label, a solid carrier and other

As will be clear from the discussion here, if at least one of R15and R16contains a reactive functional group, this group can be a component to the relationship between medicine and the other molecule. In a typical embodiment, where at least one of R15and R16contains a relationship between medicine and other views, then at least one of R15and R16is a fragment that is cleaved by an enzyme.

In another typical embodiment, at least one of R4, R4', R5, R5' is a

In the formula 8 characters X2and Z1PR is astavliaut components, independently selected from O, S and NR23. Group R17and R18independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie, halogen, NO2, NR19R20, NC(O)R19, OC(O)NR19, OC(O)OR19C(O)R19, SR19or or19provided that at least one of R12, R13, R19or R20contains a linker of the present invention, which are described here.

The symbols R19and R20independently represent a substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroseksualci, substituted or unsubstituted peptidyl, where R19and R20together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms, provided that when Z1denotes NH, then both R17and R18are not H, and R17is not NH2. Throughout the present description, the symbols R19/sup> and R20also cover the groups mentioned for R4and R5. Thus, in the scope of the present invention included the provision of compounds having two or more of the following directly above the condensed phenyl-cyclic heterocyclic systems that are connected in series, or a condensed cycle in combination with the linker. In addition, in embodiments where the linker, it may be present as substituent R4, R4', R5or R5' or as substituent R17and R18.

R6indicates a simple relationship that is present or absent. If R6there is, R6and R7United, forming cyclopropyl ring. R7denotes CH2-X1or-CH2-. If R7denotes-CH2-it is a component cyclopropanol cycle. The symbol X represents a leaving group such as halogen, for example Cl, Br or F. the Combination of R6and R7interpret in a way that does not violate the principles of chemical valence.

A curve inside the six-membered cycle shows that the loop can have one or more degrees of unsaturation and may be aromatic. Thus, a cyclic structure, such as structure, described below, and related structures covered by the formula (9)

In a typical embodiment, the cyclic system A represents a substituted or unsubstituted phenyl cycle. The cyclic system A is preferably replaced by one or more substituents for the aryl group, as described here in the section definitions. In one preferred embodiment, the phenyl cycle is replaced by fragment CN or methoxy.

In another typical embodiment, the invention relates to compounds having a structure corresponding to formula 10

In this embodiment, the radicals R3, R4, R4', R5, R5', R6, R7and X is essentially the same as described above. The symbol Z denotes a component that is independently selected from O, S and NR23. The symbol R23indicates an element selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl. Each R23chosen independently. The symbol R1represents H, substituted or unsubstituted lower alkyl, or C(O)R8or CO2R8. R8indicates an element selected from NR9R10, NR9Other10and OR9, R9and R10independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

The radical R2denotes H or substituted or unsubstituted lower alkyl. Usually is about preferably if R2represents a substituted alkyl, it is different from perfluoroalkyl, for example, CF3. In one embodiment, R2represents a substituted alkyl where the Deputy is not a halogen. In another embodiment, R2denotes unsubstituted alkyl.

As discussed above, X1can be a leaving group. Suitable leaving groups include, but are not limited to, halides, azides, sulfonic esters (for example, alkylsulfonyl, arylsulfonyl), hydronium ions, alkylphenolate, esters ammoniumchloride, alkylphosphonate and fluorinated compounds (e.g., triflate, nonflat, cresylate) and the like. Specific halogen-free, suitable as leaving groups are F, Cl and Br. Data selection and other leaving groups suitable for a specific set of reaction conditions is in the range of abilities of the person skilled in the art (see, for example, J. March, Advanced Organic Chemistry, 2nd Edition, John Wiley and Sons, 1992; Sandier S.R., W. Karo, Organic Functional Group Preparations, 2nd Edition, Academic Press, Inc., 1983; and L.G. Wade, Compendium of Organic Synthetic Methods, John Wiley and Sons, 1980).

In a typical embodiment, R1denotes ester fragment such as CO2CH3. In another typical embodiment, R2denotes a lower alkyl group which may be substituted or unsubstituted. Currently, the preferred lower alkyl group which is CH 3. In another embodiment, R1denotes CO2CH3and R stands for CH3.

In another typical embodiment, R4, R4', R5, R5' components are independently selected from H, halogen, NH2, OMe, O(CH2)2N(Me)2and NO2.

In one embodiment, the drug is selected so that the leaving group X1is a component selected from the group comprising halogen, alkylsulfonyl, arylsulfonyl and azide. In another embodiment, Z represents O. In some embodiments, R1can be CO2CH3or R2maybe CH3; in addition, R1can be CO2CH3and R2maybe CH3. One of R4, R4', R5and R5' can represent C(O)R15and the other three of R4, R4', R5and R5' are H. in Addition, at least one of R4, R4', R5and R5' may differ from the item selected from H and OCH3. In one embodiment, R4, R4', R5and R5' are components that independently selected from H, halogen, NH2, O(CH2)2N(Me)2and NO2.

In a preferred embodiment, one of R4, R4', R5and R5' represents O(CH2)2N(Me)2and the other of R4, R4', R5and R5denote H. In another embodiment, R7denotes CH2-X1where X1represents F, Cl or Br and R6is missing.

In another typical embodiment, the invention relates to compounds having a structure corresponding to formula 11 and 12

In one embodiment, the above formula, X preferably represents O; and Z preferably represents O. In another embodiment, Z represents NR23or O. In another way, one of R4, R4', R5and R5' can represent O(CH2)2N(Me)2while the other three of R4, R4', R5or R5' are H. In one embodiment, R4, R4', R5or R5' can be selected from the group comprising R29, COOR29C(O)NR29and C(O)NNR29where R29selected from the group comprising H, OH, substituted alkyl, unsubstituted alkyl, substituted cycloalkyl, the unsubstituted cycloalkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, substituted cyclogeranyl, the unsubstituted cyclogeranyl, substituted heteroaryl and the unsubstituted heteroaryl.

In another embodiment of the above formula, X preferably represents O, Z preferably represents O, R1preferably denotes CO2CH3, R7preferably represents CH2-Cl, R2preferably the means CH 3, R3preferably denotes OH. Differently, one of R4, R4', R5and R5' can represent NHC(O)(C6H4)NH2while the other three of R4, R4', R5or R5' denote H.

In one embodiment, R29you can choose from a group including

In one embodiment, the medication one element selected from R4and R5represents a

where X2and Z1represent components, independently selected from O, S and NR23; R17and R18represent components, independently selected from the group comprising H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroseksualci, halogen, NO2, NR19R20, NC(O)R19, OC(O)NR19, OC(O)OR19C(O)R19, OR19and O(CH2)nN(CH3)2. In this embodiment, n is an integer from 1 to 20; R19and R20independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geterotsiklicheskie, where R19The R 20together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms, where one of R11, R12, R13, R15, R16, R19or R20associates the specified medication with L1if present, or to F. In one preferred embodiment, X2denotes O and Z1represents O or NR23.

Another preferred structure similar duocarmycin formula 7 is a structure in which the cyclic system A represents unsubstituted or substituted phenyl ring. Preferred substituents in the molecule described herein above drugs for a structure of formula 7, when the cyclic system A is a pyrrole, are also preferred substituents, if the cyclic system A represents unsubstituted or substituted phenyl ring.

For example, in a preferred embodiment, the drug (D) has the structure

In this structure, R3, R6, R7X are as described above for formula 7.

In addition, Z denotes a component selected from O, S and NR23where R23indicates a component selected from H, substituted the CSO or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8where R8indicates an element selected from NR9R10and OR9in which R9and R10denote components that are independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R1'represents H, substituted or unsubstituted lower alkyl or C(O)R8where R8indicates an element selected from NR9R10and OR9in which R9and R10denote components that are independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R2denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl, or cyano, or alkoxy; and

R2'denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl.

At least one of R11, R12, R13, R15and R16associates medicine for L1if present, or to F, H or J.

In a preferred embodiment, one of R4, R4', R5and R5' represents O(CH2)2N(Me)2and the other of R4, R4', R5and R5' are H. In another embodiment, R7 denotes CH2-X1where X1denotes F, Cl or Br and R6is missing.

In one embodiment, the invention relates to cytotoxic compounds the drug-ligand having a structure corresponding to the following formula:

in which the symbol L1indicates auto-disable spacer, where m is an integer from 0, 1, 2, 3, 4, 5 or 6.

X4indicates an element selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides the agents.

The symbol L4is the linker element and p is 0 or 1. L4is a piece that gives the conjugates increased solubility or decreased aggregation properties. Examples of components of L4include substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroalkyl or an unsubstituted heteroalkyl, any of which may be linear, branched or cyclic, positively or negatively charged amino acid polymer, such as polylysine or polyalanine, or other polymers, such as polyethylene glycol.

The symbol Q represents ottsepleny linker, including but not limited to these, any described despatie, hydroconone and disulfide linkers. Other suitable linkers include, but are not limited to, linkers described in U.S. patent No. 6214345; publication of patent applications U.S. No. 2003/0096743, 2003/0130189 and 2004/121940; publications PCT patent application no WO 03/026577 and WO 04/043493 and publication of European patent applications No. EP1243276 and EP1370298, which are all incorporated herein by reference. Otsepleniya linkers include linkers that can selectively be chipped off chemical or biological methods and decomposition to separate the drug D1from the X4. Decomposition can occur anywhere along the length of the linker or on any end of the linker.

The symbol D1refers to a drug having the following formula:

where X and Z denote the components independently selected from O, S and NR23;

R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8,

R1' represents H, substituted or unsubstituted lower alkyl or C(O)R8,

where R8indicates a component selected from NR9R10and OR9and R9and R10are components that independently selected from H, zameshannogo unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R2denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl, or cyano, or alkoxy;

R2'denotes H, or substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl,

R3indicates a component selected from the group comprising SR11, Other11and OR11where R11is a component selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, diphosphate, triphosphates, acyl, C(O)R12R13C(O)OR12C(O)NR12R13, P(O)(OR12)2C(O)CHR12R13, SR12and SiR12R13R14in which R12, R13and R14are components that independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, where R12and R13together with the nitrogen atom or carbon to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms;

where at least one of R11, R12and R13associates the specified drug with L1if there is, or Q,

R6indicates a simple relationship that is present or absent and, if present, R6and R7United, forming cyclopropyl ring; and

R7denotes CH2-X1or-CH2-combined in the specified cyclopropane ring with R6where X1denotes a leaving group,

R4, R4', R5and R5' are components that independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, SR15, OR15, CR15=NR16and O(CH2)nNR24R25where n is an integer from 1 to 20;

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie and substituted or unsubstituted of peptidyl, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted generalklauseln the cyclic system, having from 4 to 6 components, optionally containing two or more heteroatoms;

and R24and R25independently selected from unsubstituted alkyl and

where at least one of R4, R4', R5and R5' denotes O(CH2)nNR24R25.

In some embodiments, n is 2. In some embodiments, R24and R25represent methyl. In some embodiments, R4denotes O(CH2)nNR24R25and R4', R5and R5' are H. In some embodiments, R4denotes O(CH2)2N(CH3)2and R4', R5and R5' are H. In some embodiments, Q is a linker selected from F, H and J, which is described above. In some embodiments, R1, R1', R2and R2' are H.

A preferred formula for a drug D1is the following:

Other preferred drugs D1is the following:

Still more preferred versions of the medicines D1are as follows:

In another typical embodiment of the present invention the cytotoxic drug may be similar tubulysin or related compound, for example with the unity, the described structure corresponding to formula 13

where R1and R2denote H or lower alkyl, or, more specifically, isobutyl, ethyl, propyl or tert-butyl and R3denotes H or OH. Tubulysin and its application in the treatment of cancer are described, for example, in PCT publications WO 2004/005327 and WO 2004/005326. Obtaining compounds tubulysin described in DE10008089. Methods that can be used to link tubulysin with different linkers of the present invention, shown in the examples. Preferred analogs tubulysin are tubulysin A-F.

The CBI analogs

These specific compounds are analogs of the CBI, because they include 1,2,9,9a-tetrahydrocyclopent[c]Benz[e]indol-4-one (CBI)is an alkylating domain or alkylating polyadenine. These compounds can be used as medicines. Preferred medicaments according to the present invention include cytotoxic drugs suitable for the treatment of cancer. These compounds may be conjugated or not, or to include linkers as described above. Cytotoxic drugs that are suitable in the present invention include, for example, analogues on the basis of CBI (1,2,9,9a-tetrahydrocyclopent[c]Benz[e]indol-4-it), based counterparts MCBI (7-methoxy-1,2,9,9a-tetrahydrocyclopent[c]Benz[e]indol-4-it) and analogues on the basis of CCBI (7-cyano-1,2,9,9a-Tetra is erotica-disappear[c]Benz[e]indol-4-it).

In one embodiment, the compound of the invention has the following formula (14):

where X and Z are independently selected from O, S and NR23where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl;

R1represents H, substituted or unsubstituted lower alkyl, C(O)R8or CO2R8,

R1' represents H, substituted or unsubstituted lower alkyl or C(O)R8,

each R8is a component that is independently selected from NR9R10and OR9and R9and R10denote components that are independently selected from H, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;

R2represents H, substituted or unsubstituted lower alkyl, unsubstituted heteroalkyl, cyano or alkoxy;

R2'represents H, substituted or unsubstituted lower alkyl, or the unsubstituted heteroalkyl,

R3indicates a component selected from the group comprising SR11, Other11and OR11where R11indicates a component selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, the unsubstituted heteroalkyl, diphosphate, triphosphates, acyl, C(O)R12R13C(O)OR12C(O)NR12R13, P(O)(OR12) 2C(O)CHR12R13, SR12and SiR12R13R14in which R12, R13and R14represent components, is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, or R12and R13together with the nitrogen atom or carbon to which they are attached, combine to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms;

R6indicates a simple relationship that is present or absent and, if present, R6and R7United, forming cyclopropyl ring; and

R7denotes CH2-X1or-CH2-combined in the specified cyclopropane ring with R6where X1denotes a leaving group,

R4, R4', R5and R5' components are independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, SR15, OR15, CR15=NR16IO(CH 2)nNR24R25where n is an integer from 1 to 20, preferably n is an integer from 2 to 6;

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted geterotsiklicheskie and substituted or unsubstituted of peptidyl, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms;

and R24and R25independently selected from unsubstituted alkyl and

where at least one of R4, R4', R5and R5' denotes O(CH2)nNR24R25.

As discussed above, X1can be a leaving group. Suitable leaving groups include, but are not limited to, halides, azides, sulfonic esters (for example, alkylsulfonyl, arylsulfonyl), hydronium ions, alkylphenolate, ammoniumchloride esters, alkylphosphonate and fluorinated compounds (e.g., triflate, nonflat, cresylate) and the like. Specific halogen-free, suitable as leaving group is, represents F, Cl and Br. Data selection and other leaving groups suitable for a specific set of reaction conditions is in the range of abilities of the person skilled in the art (see, for example, J. March, Advanced Organic Chemistry, 2nd Edition, John Wiley and Sons, 1992; S.R. Sandler, W. Karo, Organic Functional Group Preparations, 2nd Edition, Academic Press, Inc., 1983; and L.G. Wade, Compendium of Organic Synthetic Methods, John Wiley and Sons, 1980).

In some embodiments, R4, R4', R5and R5' are components that independently selected from H, halogen, NH2, OMe, O(CH2)2N(Me)2and NO2. In some embodiments, at least one of R4, R4', R5and R5' denotes O(CH2)2N(Me)2. In some embodiments, one of R4, R4', R5and R5' denotes O(CH2)2N(Me)2and the other of R4, R4', R5and R5' are H. In other embodiments, R4denotes O(CH2)2N(Me)2and R4', R5and R5' are H.

In some embodiments, R7represents CH2-X1where X1denotes F, Cl or Br and R6no. In some embodiments, the drug is selected so that the leaving group X1indicates a component selected from the group comprising halogen, alkylsulfonyl, arylsulfonyl and azide. In some embodiments, X1denotes Cl or Br

In some embodiments, Z represents O. In some embodiments, X and Z are O.

In some embodiments, R2denotes H, methyl or cyano and R1, R1' and R2' are H. In some embodiments, R1, R1', R2and R2' denote H. In some embodiments, R1, R1' and R2' denote H.

In some embodiments, R3represents a reactive group, which is described below.

A preferred formula for the compounds of formula (14) is the following:

Another preferred option for the compounds of formula (14) is the following:

Additional preferred option for the compounds of formula (14) are the following:

Preferred conjugates duocarmycin and CBI

Peptide, or hydrazine powered disulfide linkers according to this invention can be used in the conjugates, containing as cytotoxic agents analogues duocarmycin or CBI. Preferred conjugates of the present invention is described below with additional details. While nothing else is stated, the substituents are defined as above in the sections on cytotoxins, peptide linkers, hydrazine powered of linkers and disulfide linkers./p>

A. Conjugates with peptide linkers

In a preferred embodiment, the invention relates to a conjugate with a peptide linker having the structure

where X1denotes halogen;

X denotes a component selected from O, S and NR23;

R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl; and

R4, R4', R5and R5' are components that independently selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, OR15and O(CH2)nN(CH3)2where n is an integer from 1 to 20; and

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geterotsiklicheskie, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted, heterotic olkalou cyclic system, having from 4 to 6 components, optionally containing two or more heteroatoms.

Non-restrictive examples of such conjugates include the following structures:

where X1denotes Cl or Br, and

where Ab is an antibody or its fragment.

In another preferred embodiment, the invention relates to a conjugate having the structure

or

where X1denotes a leaving group;

Z and X are the components independently selected from O, S and NR23,

where R23indicates a component selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and acyl; and

R3selected from the group comprising H, substituted alkyl, unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, the unsubstituted heteroaryl, substituted heteroseksualci, the unsubstituted heteroseksualci, halogen, NO2, NR15R16, NC(O)R15, OC(O)NR15R16, OC(O)OR15C(O)R15, OR15and O(CH2)nN(CH3)2,

where n is an integer from 1 to 20;

R15and R16independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, Samusenko is about or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted geterotsiklicheskie, where R15and R16together with the nitrogen atom to which they are attached optionally join to form a substituted or unsubstituted geterotsyklicescoe cyclic system having from 4 to 6 components, optionally containing two or more heteroatoms.

Non-restrictive examples of such conjugates include the following structures:

where each b independently is an integer from 0 to 20, and Ab represents the antibody or fragment.

In other preferred embodiments, the invention relates to a conjugate with a peptide linker selected from the following structures:

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

In other embodiments, the invention relates to a conjugate with a peptide linker selected from the following structures:

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

In other embodiments, the invention relates to a conjugate with a peptide linker having the following structure:

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

B. Compared with the Hydra is nowymi the linkers

In a preferred embodiment, the invention relates to a conjugate with hydrazine powered by a linker having the structure

In another preferred embodiment, the invention relates to a conjugate with hydrazine powered by a linker having the structure

In other preferred embodiments, the invention relates to a conjugate with hydrazine powered by a linker having the structure selected from the

and

where PEG denotes polietilenglikolya fragment and X1denotes Cl or Br.

In other preferred embodiments, the invention relates to a conjugate with hydrazine powered by a linker selected from the following structures:

and

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

In another preferred variant, conjugate with hydrazine powered by a linker selected from the following structures:

and

C. by a disulfide Conjugates with linkers

In a preferred embodiment, the invention relates to a conjugate with a disulfide linker having the structure

and

Non-restrictive examples of such structures include the following:

and

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

LIGANDS

The ligands of the present invention is depicted as "X4". In this invention, X4is an element selected from the group including protected reactive functional groups, unprotected reactive functional groups, detected labels and guides the agents. Preferred ligands are directing agents, such as antibodies and their fragments.

In a preferred embodiment, the group X4can be described as element selected from R29, COOR29C(O)NR29and C(O)NNR29where R29is a component selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted heteroaryl. In another typical embodiment, R29is a component selected from H, OH, NHNH2,

where R30represents a substituted or unsubstituted alkyl having a terminal reactive functional group, substituted or nezamedin the th heteroaryl, having a terminal functional group. The above structures act as reactive protective groups, which can interact, for example, with the side chain of the amino acids of the sending agent, such as antibody, thus connecting the smart agent with a fragment of the linker-drug.

Directing agents

The linker group and the cytotoxins of the present invention may be associated with guiding agents that selectively deliver the payload into the cell, organ or region of the body. Typical directing agents, such as antibodies (e.g., chimeric, humanized antibody and a human antibody), ligands for receptors, lectins, sugars, antibodies and the like, are recognized in this area and applicable without restrictions in the practice of the present invention. Other directing agents include the class of compounds that do not include specific molecular recognition motifs include macromolecules such as poly(ethylene glycol), polysaccharide, polyaminoamide and the like, which add to the cytotoxin molecular weight. Additional molecular weight affect the pharmacokinetics of cytotoxin, such as half-life in serum.

In a typical embodiment, the invention concerns a cytotoxin, linker or cytotoxin conjugate-linker guide agent is, which is biomolecule, for example an antibody, a receptor, a peptide, a lectin, a sugar, a nucleic acid or a combination of both. Technological routes of obtaining typical conjugates according to this invention described in the above schemes.

Biomolecules suitable for use in practice of the present invention, can be obtained from any source. Biomolecules can be isolated from natural sources or can be obtained by synthetic methods. Proteins can be natural or mutated proteins. Mutations can be performed by chemical mutagenesis, site-specific mutagenesis or other methods of inducing mutations, known to specialists in this field. Proteins suitable for use with the present invention, include, for example, enzymes, antigens, antibodies and receptors. Antibodies may be either polyclonal or monoclonal, but the most preferred monoclonal antibodies. The peptides and nucleic acids can be isolated from natural sources or can be partially or completely synthetic in nature.

In a preferred embodiment, the smart agent is an antibody or antibody fragment that is selected on the basis of its specificity relative to the antigen expressed in the target cell or explores nom interesting place. Identified a wide variety of antigens, tumor-specific or specific in relation to other diseases, and antibodies to these antigens are used or proposed for use in the treatment of such tumors or other diseases. Antibodies, which are known in this field can be used in the conjugates according to this invention, in particular, for the treatment of a disease associated antigen target. Non-restrictive examples of antigen targets (and associated diseases), which may be targeted conjugate antibody-linker-drug according to this invention include the following: Her2 (breast cancer), CD20 (lymphoma), EGFR (solid tumor), CD22 (lymphoma, including nahodkinskuju lymphoma), CD52 (chronic lymphocytic leukemia), CD33 (acute myelogenous leukemia), CD4 (lymphomas, autoimmune diseases, including rheumatoid arthritis), CD30 (lymphoma, including nahodkinskuju lymphoma), Mucl8 (melanoma), integrins (solid tumors), PSMA (prostate cancer, adenocarcinoma of prostate cancer), CEA (colorectal cancer), CDlla 5 (psoriasis), CD80 (psoriasis), CD23 (asthma), CD40L (immune thrombocytopenic purpura), CTLA4 (T-cell lymphoma) and BLys (autoimmune diseases, including systemic lupus erythematosus).

In those embodiments where a recognizable fragment is a protein or antibody, the protein can be ... on the EN to the surface or component coassociation the monolayer (SAM) or linked via a spacer elements group with any available reactive peptide residue on the surface of the protein. In preferred embodiments, the reactive groups are amines or carboxylates. In particularly preferred embodiments, the reactive groups are ε-amino groups of lysine residues. In addition, these molecules can adsorb on the surface of the substrate or SAM through non-specific interactions (e.g., chemisorption and physical adsorption).

Identifiable fragments, which are antibodies, can be used for recognition of the analyzed substances, which are proteins, peptides, nucleic acids, saccharides or such small molecules as drugs, herbicides, pesticides, industrial chemicals and military agents. Methods of obtaining antibodies to specific molecules known to experts in this field. See U.S. patent No. 5/147786 (Feng and others) from September 15, 1992; No. 5/334528 (Stanker and others) from August 2, 1994; No. 5/686237 (Al-Bayati, M.A.S.) dated 11 November 1997 and No. 5/573922 (Hoess and others) dated 12 November 1996. Methods of attaching antibodies to the surface is also known in this field. See the work Delamarche and others, Langmuir 12:1944-1946 (1996).

Directing agents can be attached to the linkers of the present invention via the reactive groups. For example, peptides can be attached through an amine, carboxyl, sulfhydryl or hydroxyl group. T the Kai group can be at the end of the peptide, or at an internal site of the peptide chain. Nucleic acids can be attached by means of a reactive group-based (e.g., ekzoticeski Amin) or the hydroxyl group on the fragment of a sugar (for example, 3'- or 5'-hydroxyl). Peptide chain or chains of nucleic acids can be further processed to obtain the derived one or more sites, allowing connection to a circuit suitable reactive groups. See the work Chrisey and others, Nucleic Acids Res. 24: 3031-3039 (1996).

If the peptide or nucleic acid is a fully or partially synthetic molecule, reactive group or a masked reactive group can be included in the synthesis process. Specialists in this field known to many derivative monomers suitable for inclusion reactive groups in peptides and nucleic acids. See, for example, The Peptides: Analysis, Synthesis, Biology, Vol. 2: Special Methods in Peptide Synthesis" Gross, E. and Melenhofer, J., Eds., Academic Press, New York (1980). Many suitable monomers commercially available (Bachem, Sigma and others). After synthesis, the protection can be removed from the masked group, then it becomes available for interaction with the component compounds of the present invention.

Typical guiding agents based on nucleic acids include aptamers, antisense compounds and nucleic acids that form the t triple helix. As a necessary chemical functionality for communication of the sending agent on the basis of the nucleotide with cytotoxin usually use the hydroxyl group of the sugar residue, the amino group from the main balance or phosphate oxygen from the nucleotide. However, the person skilled in the art will easily understand that the nucleic acid can be hung other "unnatural" reactive functionality, using well-known techniques. For example, the hydroxyl group of the sugar residue can be converted into a mercapto - or amino group, using methods well known in the field.

Aptamers or antibodies to nucleic acids) are molecules of single - or double-strand DNA or single-stranded RNA that bind to specific molecular targets. Typically, aptamers act by inhibiting the action of molecular targets, such as proteins, by binding together of targets, circulating in the blood. Aptamers have a chemical functionality and, thus, can covalently contact cytotoxins, which are described here.

Although very diverse molecular targets capable of forming non-covalent, but specific associates with aptamers, including drugs in the form of small molecules, metabolites, cofactors, toxins, drugs based sugars, medicines OS is ove nucleotides, glycoproteins and the like, usually the molecular target contains protein or peptide, including serum proteins, kinny, eicosanoids, cell surface molecules and the like. Examples of aptamers include antithrombine inhibitor from Gilead GS 522 and its derivatives (Gilead Science, Foster City, Calif.). See also work Macaya and others, Proc. Natl. Acad. Sci. USA 90:3745-9 (1993); Bock and others, Nature (London) 355:564-566 (1992) and Wang and others, Biochem. 32:1899-904 (1993).

Aptamers specific about this biomolecules, can be identified using known in the field of methodology. See, for example, 'toole and others (1992) PCT publication no WO 92/14843; Tuerk and Gold (1991) PCT publication no WO 91/19813; Weintraub and Hutchinson (1992) PCT publication No. 92/05285 and Ellington and Szostak, Nature 346: 818 (1990). In short, these techniques usually involve the complexation of molecular targets with a random mixture of oligonucleotides. Complex aptamers as molecular target is separated from uncomplexed oligonucleotides. Aptamers restore from the separated complex and amplified. This cycle is repeated to identify sequences of aptamers with the highest affinity data regarding molecular targets.

For diseases resulting from inappropriate gene expression, the ideal therapy is a specific prevention or reduction of expression of these genes. In principle, it is possible to inhibit, reduce sludge is to disable the production of a specific gene product by hybridization of single-stranded deoxynucleotide or ibadethanelerde, complementary to available sequences in mRNA, or sequence in the transcript, which is essential for processing of pre-mRNA, or sequence in the gene. This is an example of genetic regulation is often called antimuslim, or antigenic, inhibition. Additional efficiency attach via conjugation with nucleic acid alkylating agent such as the agent of the present invention.

Antisense compounds are nucleic acids designed to bind and deprivation of the possibility or prevent the receipt of mRNA that is responsible for generating a specific protein. Antisense compounds include antisense RNA or DNA, single or double-strand, oligonucleotides or their analogs, which can gibridizatsiya specifically regarding individual mRNA species and prevent transcription and/or RNA processing this type of mRNA and/or translation of the encoded polypeptide and thereby reduce the number of the corresponding encoded polypeptide. Ching and others, Proc. Natl. Acad. Sd. U.S.A. 86: 10006-10010 (1989); Broder and others, Ann. Int. Med. 113: 604-618 (1990); Loreau and others, FEBS Letters 274: 53-56 (1990); Holcenberg and others, WO 91/11535; WO 91/09865; WO 91/04753; WO 90/13641; WO 91/13080, WO 91/06629 and EP 386563. Due to its best target sensitivity and selectivity of the antisense oligonucleotides suitable for delivery to terapevticheskii agents, such as cytotoxins of the present invention, the desired molecular target.

Others report that the nucleic acid can be contacted with dual DNA by triple helix formation and to inhibit transcription and/or DNA synthesis. Connection with the triple helices (also referred to as Trenitalia drugs) are oligonucleotides that bind to sequences of double-strand DNA and, as expected, selectively inhibit the transcription of genes that cause disease, such as viral genes, such as HIV and herpes simplex virus, oncogenes, and they stop the production of the protein in the cell nucleus. These medicines are attached directly to double-stranded DNA in the cellular genome, forming a triple helix and preventing the production by the cell of the target protein. See, e.g., PCT publication nos WO 92/10590, WO 92/09705, WO 91/06626 and U.S. patent No. 5176996. Thus, the cytotoxins of the present invention also kongugiruut with sequences of nucleic acids, which form a triple helix.

The site-specificity of nucleic acids (e.g., antisense compounds and transpiring drugs) insignificant effect modification of the phosphodiester bond or a chemical modification of the oligonucleotide end. Consequently, these nucleic acids can be Henichesk the modify, improving the overall stability of binding, increasing the stability in the sense of chemical decomposition, increasing the speed with which the oligonucleotides are transported into cells, and giving the molecules of chemical reactivity. The General approach to the design of different nucleic acids, suitable for antisense therapy, is considered in the works of van der Krol and others, Biotechniques 6: 958-976 (1988) and Stein and others, Cancer Res. 48: 2659-2668 (1988). Therefore, in a typical embodiment, the cytotoxins of the present invention kongugiruut with nucleic acid modifying the phosphodiester bond.

In addition, aptamers, antisense compounds and transferline drugs, bearing the cytotoxins of the present invention can also include nucleotide substitution, addition, deletion or move, until, for as long as specific hybridization or Association with a suitable target sequence as a functional property of the oligonucleotide. For example, some variants use phosphorothioate counterparts, which are more resistant to degradation by nucleases than their natural phosphate diepiriye copies, and therefore is expected to have a higher stability in vivo and greater efficiency (see, for example, Campbell and others, J. Biochem. Biophys. Methods 20: 259-267 (1990)). It is also known that phosphoroamidite derivatives of oligonucleotides binding is carried out with complementary polynucleotide and have additional capacity to accommodate covalently attached type of ligand and will conform to the methods of the present invention. See, e.g., Froehler and others, Nucleic Acids Res. 16(11):4831 (1988).

In some embodiments, aptamers, antisense compounds and transferline medicines contain O-methylribonucleotide (EP publication No. 360609). You can also use chimeric oligonucleotides (Dagle and others, Nucleic Acids Res. 18: 4751 (1990)). For some applications, the antisense oligonucleotides and triple helix can contain polyamidoamine acid (Nielsen and others, Science 254: 1497 (1991) and PCT publication no WO 90/15065) or other cationic derivatives (Letsinger and others, J. Am. Chem. Soc. 110: 4470-4471 (1988)). Other applications may use oligonucleotides, where one or more of fosfolipidnyh relations are substituted isothermic group, such as magnoliopsida link length 2-4 atom, which is described in PCT publication nos WO 92/05186 and 91/06556, or formatline group (Matteucci and others, J. Am. Chem. Soc. 113: 7767-7768 (1991)), or amide group (Nielsen and others, Science 254: 1497-1500 (1991)).

In addition, in the present invention can be used nucleotide analogs, for example, those in which the sugar or base of chemically modified. "Similar" forms of purines and pyrimidines are of the form commonly known in this area, many of which are used as chemotherapeutic agents. Typical, but not exhaustive list includes 4-acetylcytosine, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromure the sludge, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouridine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosidosis, 5'-methoxycarbonylmethyl, 5-methoxymethyl, 2-methylthio-N6-isopentenyladenine, methyl ester uracil-5-exucuse acid, uracil-5-oxiana acid (v), wybutosine, pseudorutile, koozin, 2-tocitizen, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester N-uracil-5-exucuse acid and 2,6-diaminopurine. In addition to well-known reasons to use halogenated base. In addition, the position of the 2'-furanose based can be uncharged substitution volume group. Examples of uncharged volume groups include branched alkali, sugar and branched sugar.

Terminal modification also provides a suitable methodology for conjugation of cytotoxins with nucleic acid modification specificity of cell type, pharmacokinetics, nuclear permeability and absolute rate of cellular uptake for oligonucleotide pharmaceutical agents. For example, known then the iPod substitutions at the 5' and 3' ends to include reactive groups, which allows covalent joining of cytotoxins. See, for example, Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, (1989) Cohen, Ed., CRC Press; Prospects for Antisense Nucleic Acid Therapeutic for Cancer and AIDS (1991), Wickstrom, Ed., Wiley-Liss; Gene Regulation: Biology of Antisense RNA and DNA (1992) Erickson and Izant, Eds., Raven Press and Antisense RNA and DNA (1992), Murray, Ed., Wiley-Liss. General methods relating to antisense compounds, see in the work of Antisense RNA and DNA, (1988), D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Detected Tag

Special label or detected group, used in conjunction with the compounds and methods according to this invention, is usually not a critical aspect of the invention, as long as she does not significantly affect the activity or the applicability of the compounds of this invention. Detectable group may be any material having detektivami physical or chemical property. Such detected label thoroughly developed in the field of immunostimulant, and, in General, the majority of labels suitable in such methods, can be used in the present invention. Thus, a label is any composition, detektiruya spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels in the present invention include magnetic granules (e.g., DYNABEADSTM), Fluor is santie dyes (for example, fluoresceinisothiocyanate, Texas red, rhodamine, and the like), RFID tags (e.g.3H,125I35S14C or32P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic pellets (e.g., polystyrene, polypropylene, latex, and others).

The label can be linked directly or not directly with the compound of the present invention in accordance with methods well known in the field. As stated above, you can use a wide variety of labels, choosing the label depending on the required sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, and ensure its removal.

If the connection of the present invention is a conjugate with a detectable label, the label preferably is an element selected from the group including radioactive isotopes, fluorescent agents, predecessors fluorescent agents, chromophores, enzymes, and combinations thereof. Methods of conjugation of different groups of antibodies are well known in this field. For example, detected by a label, which is often kongugiruut with antibody is an enzyme, such as horseradish peroxidase, alkaline FOS is atasa, β-galactosidase and glucose oxidase.

Non-radioactive labels are often added indirect ways. Typically, a ligand molecule (e.g., Biotin) is covalently associated with the component of the conjugate. Then attach the ligand to another molecule (e.g., streptavidin), which is either essentially is detectable or covalently bound to a signal system, for example with detektivami enzyme, a fluorescent compound or a chemiluminescent compound.

The components of the conjugates of this invention can also be directly conjugated with compounds, which generates a signal, for example, by conjugation with an enzyme or fluorophore. Enzymes of interest as labels, represent, mainly, hydrolases, particularly phosphatases, esterase and glycosidase, or oxidase, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone and other Chemiluminescent compounds include luciferin, and 2,3-dihydropteridine, such as luminal. An overview of the different labels or signal producing systems that can be used, see U.S. patent No. 4391904.

Methods of detecting labels are well known to specialists in this field. Thus, if the label represents, for example the EP, radioactive label, a means of detection include a scintillation counter or photographic film as in autoradiography. If the label represents a fluorescent label, it can be detected by activating fluorochrome in the appropriate wavelength of light and detektywa the resulting fluorescence. It is possible to detect fluorescence visually, using photographic film, using electronic detectors such as charge-coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels can be detected, providing appropriate substrates for the enzyme and detektywa the resulting reaction product. In conclusion, a simple colorimetric labels can be detected simply by observing the color associated with that label. Thus, in various studies on indicators conjugated gold often looks pink, while various conjugated granules are color data granules.

Currently, the preferred fluorescent labels, as they have the advantage of not requiring great caution when working and being suitable for high-performance visual techniques (optical analysis, including digitized images for analysis in an integrated system, including the computer). Preferred IU the key is usually characterized by one or more of the following factors: high sensitivity, high stability, low background, low sensitivity to the environment and high specificity with the introduction of the label. Many fluorescent labels are commercially available from SIGMA chemical company (Saint Louis, MO), Molecular Probes (Eugene, OR), R&D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem. Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known in the art. In addition, the specialist in this field will determine how to choose a suitable fluorophore for a particular application, and, if it is not readily available commercially, the expert is able to synthesize the desired fluorophore again or synthetically modify commercially available fluorescent compounds to obtain the desired fluorescent tag.

In addition, in the present invention are suitable fluorophores in the form of small molecules, natural fluorescent proteins and engineered analogues of such proteins. Such proteins include, for example, green fluorescent proteins of cnidarians (Ward and others, Photochem. Photobiol. 35:803-808 (1982); Levine and others, Comp. Biochem, Physiol, 72B:77-85 (1982)), yellow fluorescent protein from strain Vibrio fischeri (Baldwin and others, Biochemistry 29: 5509-15 (1990)), peridinin-chlorophyll from the dinoflagellate Symbiodinium sp. (Morris and others, Plant Molecular Biology 24: 673:77 (1994)), Phi is biliprotein of the marine blue-green algae, such as Synechococcus, e.g phycoerythrin and phycocyanin (Wilbanks and others, J. Biol Chem. 268: 1226-35 (1993)) and the like.

Usually before bond formation between the cytotoxin and guides (or other) agent and optional spacer elements group activate at least one of chemical functionalities. The person skilled in the art understands that a variety of chemical functionality, including hydroxy, amino and carboxy groups can be activated using a variety of standard methods and conditions. For example, a hydroxyl group cytotoxin or directing agent can be activated by treatment with phosgene, receiving the appropriate chloroformate, or para-nitrophenylphosphate, receiving the corresponding carbonate.

In a typical embodiment, the invention uses a smart agent, which includes the carboxyl functionality. The carboxyl group can be activated, for example by conversion into the corresponding allalone or active ester. This reaction can be carried out in different conditions, as shown in the above work, March, pp. 388-89. In a typical embodiment, allalone get through interaction carboxyl-containing group oxalylamino. Spend the interaction of activated agent with a cytotoxin, or a combination of the cytotoxin-linker the group, receiving the conjugate according to this invention. The person skilled in the art will appreciate that the use of carboxyl-containing directing agents is only illustrative, and that the agents have many other functional groups can be konjugierte with linkers according to this invention.

Reactive functional groups

For simplicity of illustration, the subsequent discussion focuses on the conjugation of the cytotoxin of the present invention with the directing agent. The focus of discussion is an example of a variant according to this invention, from which the person skilled in the art will easily draw conclusions about other options. Focusing the discussion on one variant, do not imply any limitations of the present invention.

Typical compounds of this invention are reactive functional group, which is usually located on the substituted or unsubstituted alkyl or heteroalkyl chain, facilitating its connection to another type. The usual location for reactive group is a terminal position on a chain.

Reactive groups and classes of reactions suitable for use in practice of the present invention are generally those that are well known in the field of chemistry bioconjugate. Reactive functional groupabout to be protected or unprotected, and protected nature of the group can be changed by methods known in the field of organic synthesis. Currently, the predominant classes of reactions with reactive analogues of cytotoxins are classes of reactions, which proceed under relatively mild conditions. They include, but are not limited to, nucleophilic substitution (for example, interaction of amines and alcohols with acylhomoserine, active esters), electrophilic substitution (for example, reactions of enamines) and joining multiple bonds carbon-carbon and carbon-heteroatom (e.g., Michael reaction, joining the Diels-Alder reaction). Data and other suitable reactions are discussed, for example, March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996, and Feeney and others, Modification of Proteins; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982.

Typical types of reactions include the interaction of carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, illimitably, complex thioethers, para-nitroaniline esters, alkyl, alkeline, alkyline and aromatic esters. Hydroxyl groups can be converted into esters, ethers, aldehydes and other Halogenoalkane group is transformed into a new type in the interaction, for example, amine, carboxylate-anion, thiol anion, carbanion, or an alkoxide-ion. Dienophile (for example, maleimide) groups involved in the Diels-alder reaction. Aldehyde or ketone group can be converted into imine, hydrazones, semicarbazones or oximes, or using methods such as attaching the Grignard or accession alkylate. Sulphonylchloride easily interact with amines, forming, for example, sulfonamides. For example, amine or sulfhydryl groups acelerou, alkylate or oxidize. Alkenes can be converted into a number of new species, using cycloaddition reactions, acylation, joining Michael and other Epoxides easily interact with amines and hydroxyl compounds.

The person skilled in the art will readily understand that many of these relations can be obtained in a variety of ways and using a variety of conditions. Production of esters, see, for example, in the above work, March, page 1157; complex thioesters in the above work, March, pp. 362-363, 491, 720-722, 829, 941 and 1172; carbonates - in the above work, March, pp. 346-347; carbamates in the above work, March, page 1156-57; amides in the above work, March, page 1152; urea and thiocarbamide - in the above work, March, page 1174; the floor is giving acetals and ketals can see in the above work Green and others, pp. 178-210 and above the work of the March, page 1146; obtaining aryloxyalkyl derivatives, see Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; the production of esters of enols see in the above work, March, page 1160; N-sulfonylamides see in the work Bundgaard, etc., J. Med. Chem., 31: 2066 (1988); anhydrides in the above work, March, pp. 355-56, 636-37, 990-91 and 1154; N-acylamino - in the above work, March, page 379; N-grounds manniche - in the above work, March, page 800-02 and 828; production of esters of hydroxymethylamino see in the work Petracek and others, Annals NY Acad. Set, 507:353-54 (1987); disulfides in the above work, March, page 1160 and phosphonate esters and phosphoramidates.

Reactive functional groups can be unprotected and are so chosen that are not involved in interactions or affect them. Different reactive functional groups may be protected from participating in the interaction due to the presence of the protective group. The person skilled in the art understands how to protect a specific functional group from the influence of a selected set of reaction conditions. Examples of suitable protective groups, see the work of Greene and others, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

Usually the smart agent is covalently attached to the cytotoxin through their respective helices is their functionalities, using standard chemical techniques. Optionally, the linker or the agent connect to the agent through one or more spacer elements groups. Spacer elements groups can be equivalent or different when used in combination.

Usually before bond formation between the cytotoxin and a reactive functional group and optional spacer elements group, at least one of the chemical functionalities activate. The person skilled in the art will appreciate that a variety of chemical functionality, including hydroxy, amino and carboxy groups can be activated using a variety of standard methods and conditions. In a typical embodiment, the invention contains carboxyl functionality as reactive functional groups. The carboxyl group can be activated, as described here above.

PHARMACEUTICAL PREPARATIONS AND routes of administration

In another preferred embodiment, the present invention relates to a pharmaceutical preparation comprising a compound of this invention and a pharmaceutically acceptable carrier. These compounds, including pharmaceutically acceptable carriers, such as additive salts or their hydrates, can be delivered to the patient using a variety of methods or techniques. Suitable routes of administration include, but is f restricted to this, inhalation, transdermal, oral, rectal administration, the introduction through the mucosa, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections. Preferably, the conjugates of the present invention comprising an antibody or antibody fragment as a guide fragment, parenterally, preferably intravenously.

It is assumed that when used herein, the terms "receive" or "introduction" cover all the ways the direct and indirect delivery of compounds to the designated place of action.

These compounds or their pharmaceutically acceptable salts and/or hydrates you can enter by themselves, in combination with other compounds of this invention and/or in the form of a cocktail, combining with other therapeutic agents. Of course, the choice of therapeutic agents that can be shared to enter with the compounds of this invention, partly depends on the treatment conditions.

For example, if the compound is administered to patients suffering from painful conditions of the body, which depends on autostimulatory (autoinducer), the compounds of this invention can be introduced in the form of cocktails containing agents used to treat pain, infection and other symptoms and side effects commonly associated with this ill is to be neglected. Such agents include, for example, analgesics, antibiotics and other

When administered to a patient undergoing cancer treatment, the compounds can be introduced in the form of a cocktail containing anti-cancer agents and/or additional agents that increase efficiency. Compounds can also be introduced in the form of cocktails containing agents that suppress the side effects of radiation therapy, as antiemetic agents, protective agents against radiation and other

Additional agents that increase efficiency, which can be entered together with the compounds of this invention include, for example, tricyclic antidepressant medications (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); noticecookie and antidepressant medications (e.g., sertraline, trazodone and citalopram); Ca+2-antagonists (e.g. verapamil, nifedipine, nitrendipin and caroverine); amphotericin b; analogues triparanol (e.g., tamoxifen); medicines for arrhythmia (e.g., guanidin); antihypertensive drugs (e.g., reserpine); thiol-output agents (for example, buthionine and sulfoximine) and calcium leucovorin.

The active compounds of this invention administered by themselves or in pharmaceutical compositions where the active link is (connections) is mixed with one or more pharmaceutically acceptable carriers, fillers or diluents. Pharmaceutical compositions for use according to the present invention are usually well-known manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds to drugs that can be used pharmaceutically. Suitable drug depends on the selected method of administration.

For insertion through the mucous membrane in drug use penetrating agents suitable for overcoming the barrier. These penetrating agents commonly known in this field.

For oral administration the compounds can easily be prepared by combining the active compound (compounds) with pharmaceutically acceptable carriers well known in this field. Such media provide an opportunity to prepare the compounds of this invention in the form of tablets, pills, coated tablets, capsules, liquids, gels, syrups, pastes, suspensions and the like, for oral ingestion by a patient to be treated. To obtain pharmaceutical preparations for oral use can solid filler is not necessary to grind in the resulting mixture and, if necessary, to process this mixture of granules, after adding suitable auxiliary agents, getting I have RA tablets or pills. Suitable fillers are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hypromellose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If you want, you can add leavening agents, such as crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt, such as sodium alginate.

The dragee cores provide an acceptable coating. For this purpose you can use concentrated sugar solutions, which may not necessarily contain the Arabian gum, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, solutions glaze and suitable organic solvents or solvent mixtures. You can add to the coating of tablets or pills colorants or pigments to identify or characterize the different combinations of doses of active compounds.

Pharmaceutical preparations which can be used orally include filled capsules made of gelatin, and also soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Filled capsules can contain the active ingredients is mixed with a filler, such as lactose, binders, such as starches, lubricants such as talc or magnesium stearate, and optionally stabilisers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, you can add stabilizers. All preparations for oral administration should be in a unit dosage form suitable for such an introduction.

For buccal injection composition can be in the form of tablets or pellets, prepared by a commonly known method.

For administration by inhalation, the compounds for use in accordance with the present invention are usually delivered in the form of an aerosol spray from the package under pressure or nebulizer with the use of a suitable propellant, for example DICHLORODIFLUOROMETHANE, trichloromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined, providing a valve to deliver a measured amount. It is possible to prepare capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

Connections can be prigot is to provide for parenteral administration by injection, for example bolus injection or continuous infusion. Injection is the preferred method of introduction for the compositions of the present invention. Preparations for injection can be presented, e.g., in ampoules or in mnogochasovykh containers with the addition of preservatives. The composition can be in the form of suspensions, solutions or emulsions in oily or aqueous carriers can contain agents that promote a drug, such as suspendida, stabilizing and/or dispersing agents, can be added such agents, as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salts, such as sodium alginate.

Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In addition, suspensions of the active compounds can be in the form of suitable oil suspensions for injection. Suitable lipophilic solvents or carriers include fatty oils like sesame oil, or synthetic fatty acid esters, as etiloleat or triglycerides, or liposomes. Aqueous suspension for injection may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or Agay is you, increase the solubility of compounds, allowing for the preparation of highly concentrated solutions. For injection, the agents of this invention can be prepared in aqueous solutions, preferably in such physiologically compatible buffers, as Hanks solution, ringer's solution, or physiological salt buffer.

Differently, the active ingredient may be in powder form for recovery before use with a suitable carrier, such as sterile water, not containing pyrogens.

Compounds can also be prepared in the form of compositions for rectal administration, such as suppositories or retention enemas, e.g. containing well-known bases for suppositories, such as cocoa butter or other glycerides.

In addition to the previously described preparations can also be prepared using compounds in the medication for long periods. These drugs for long periods, you can enter through implantation or delivery through the skin (for example, subcutaneously or intramuscularly), by intramuscular injection, or transdermal patch. Thus, it is possible to formulate connections, for example, with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or in the form of slightly soluble derivatives, for example labors worimi salts.

Pharmaceutical compositions may also contain suitable solid or gel-like carriers or fillers. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

The preferred pharmaceutical composition is a composition prepared for injection, such as intravenous injection, and includes from about 0.01 to 100 wt%. conjugate the drug-ligand, taking as 100% the mass of the whole pharmaceutical composition. Conjugate the drug-ligand may be a conjugate of the antibody-cytotoxin, where the antibody is selected in such a way as to be targeted to specific cancer.

LIBRARY

In the scope of the present invention also includes the library of conjugates of cytotoxins cytotoxin-linker and agent-linker for cytotoxins and linkers of the present invention. Typical library include at least 10 compounds, more preferably at least 100 compounds, more preferably at least 1000 compounds and even more preferably at least 100,000 connections.

Libraries exist in this form, it is easy to make a request for a specific property, such as cytotoxicity, degradable linker farm is one way or another reagent decomposition. Typical forms include formats chips, microemissive and the like.

Parallel or combinatorial synthesis has as its main goal the formation of a library of different molecules, all of which have common distinguishing feature identified in this description as the frame. When replacing the various components in each of the changing parts of the skeleton of the molecule increases the amount of study space in the library. Theory and modern medicinal chemistry support the concept of occupied space as a key factor in determining the effectiveness of this compound against a given biological target. When creating a diverse library of molecules, which considers a large share of the target space, it significantly increases the chance of development of high initial connection.

Parallel synthesis is usually carried out on a solid medium such as polymer resin. The frame or other suitable intermediate product binds to the resin by a chemical linker with the possibility of detachment. While the frame is attached to the particle, conduct the reaction for modification.

Variations of the reactants and/or reaction conditions provide structural diversity, which is the criterion for each library.

Attempts to conduct parallel synthesis of small molecules (polygalov with molecular the th weight of 200-1000) were rare before 1990. See, for example, work Camps and other, Annaks de Quimica, 70: 848 (1990). Recently Ellmann revealed parallel synthesis on solid phase carrier (also called "combinatorial") eleven analogues benzodiazepine along with some prostaglandins and beta-turn mimetics. These disclosures are given as examples in U.S. patent No. 5288514. Other appropriate disclosure of the parallel synthesis of small molecules can be found in U.S. patent No. 5324483. This patent discloses a parallel synthesis 4-40 compounds in each of the sixteen different frames. Chen et al also used the strategy of organic synthesis to develop ones libraries synthesized using multistage processes in polymeric media (Chen et al, J. Am. Chem. Soc, 116: 2661-2662 (1994)).

Having a library of unique connections, you can get the library immunoconjugates or antibodies, using as starting point the library autostimulation and methods described here.

SETS

Another aspect of the present invention relates to kits containing one or more compounds or compositions according to this invention and instructions for using the compounds or compositions. In a typical embodiment, the invention concerns a kit for the conjugation of the linker group of the present invention with another molecule. The kit includes linker and instructions for accession is July of this linker to a specific functional group. The kit may also include one or more of the cytotoxic drugs, guides, agents, detectable labels, pharmaceutical salts or buffers. The kit can also include a container, and optionally one or more vials, test tubes, flasks, bottles or syringes. Other formats for sets clear to experts in this field and included in the scope of the present invention.

CLEAN

In another typical embodiment, the present invention relates to a method of selection of molecular targets for compounds ligand-cytotoxin according to this invention, which binds to the ligand X4. This method preferably includes the interaction of cellular drug that contains the target to the immobilized connection with the formation of a complex between the immobilized receptor and connection.

The cytotoxin of the present invention can be mobilitat on affine any media recognized in this field means. Differently, the cytotoxin can be mobilitat using one or more linkers of the present invention.

In another typical embodiment, the invention concerns a matrix for affinity purification, which includes the linker of the present invention.

The method of this invention for separation of a target typically uses one or more techniques of affinity chromatography. Affine who cromatografia enables efficient allocation of view, for example, biological molecules or biopolymers using their recognition sites for certain chemical structures on the media with a high degree of selectivity. The literature is filled with articles, monographs and books by affinity chromatography, including such topics as media for affinity chromatography, custom made components, ligands and their preparation and use. A sample of such links include: Ostrove, Methods Enzymol. 182: 357-71 (1990); Ferment, Bioeng. 70: 199-209 (1990). Huang and others, J. Chromatogr. 492: 431-69 (1989); "Purification of enzymes by heparin-Sepharose affinity Chromatography," J. Chromatogr., 184: 335-45 (1980); Farooqi, Enzyme Eng., 4: 441-2 (1978); Nishikawa, Chem. Technol, 5(9): 564-71 (1975); Guilford and others, in, Pract. High Perform. Liq. Chromatogr., Simpson (ed.), 193-206 (1976); Nishikawa, Proc. Int. Workshop Technoi. Protein Sep. Improv. Blood Plasma Fractionation, Sandberg (ed.), 422-35; (1977) " Affinity Chromatography of enzymes,Affinity Chromatogr., Proc. Int. Symp. 25-38, (1977) (Pub. 1978) and Affinity Chromatography: A Practical Approach, Dean and others (ed.), IRL Press Limited, Oxford, England (1985). Specialists in this field have enough guides the development of specific methods, affinity chromatography, using the materials of this invention.

In the present method can be used as a carrier medium for affinity chromatography of various chemical structures. For example, as materials of media suitable agarose gels and cross-linked agarose gels, as the hydrophilicity makes them relatively free from non-specific binding. Other suitable wear which include spruce, for example, glass granules with controlled pores (CPG), cellulose particles, granules polyacrylamide gel and granules of gel Sephadex™, obtained from dextran and epichlorohydrin.

METHODS of using the CONJUGATES of the DRUG-LIGAND

In addition to the above-described compositions and structures of the present invention also relates to several methods that can be applied in practice, using the compounds and conjugates of this invention. Ways of using conjugate the drug-ligand of the present invention include: destruction or inhibition of growth or replication of tumor cells or cancer cells, cancer treatment, treatment of precancerous lesions, destruction or inhibition of growth or replication of a cell that expresses an autoimmune antibody treatment of autoimmune diseases, the treatment of infectious diseases, prevention of the multiplication of tumor cells or cancer cells, cancer prevention, prevention of reproduction of cells, which expresses an autoimmune antibody prophylaxis of autoimmune diseases and prevention of infectious diseases. These methods of application include introduction to the needy in this animal such as a mammal or a human, an effective amount of the conjugate of the drug-ligand. Preferred ligands for many of the methods described here note the indicators include antibodies and fragments of antibodies, which target specific tumor cell, cancer cell or other target area.

Complex drug-ligand of the present invention is suitable for the treatment of cancer, autoimmune diseases and infectious diseases at the animal. Provided compositions and methods of treatment of tumors, delivering discussing the composition of a pharmaceutically acceptable manner with pharmaceutically effective amount of the composition of the present invention.

The present invention is particularly suitable for treating cancer and inhibiting the multiplication of tumor cells or cancer cells in the animal. Cancer or a precancerous condition, including, but not limited to, tumor metastases, or any disease or disorder characterized by uncontrolled cell growth, can be treated or to produce its prevention through the introduction of complex drug-ligand of the present invention. The complex delivers the drug to the cancer cell or a cancer cell. In one embodiment, the ligand specifically binds to or is associated with the antigen associated with a cancer cell or neoplastic cell. Due to its close proximity with the ligand drug may be absorbed by a tumor cell or cancer cell, for example, by receptor-mediated endocytosis. The antigen can be attached comwholesale cell or a cancer cell or may be an extracellular matrix protein, associated with a tumor cell or cancer cell. Being inside the cell, the linker hydrolytically cleaved by a protease associated with a tumor cell or cancer cell, releasing this medicine. The released drug then becomes free for diffusion and induction of cytotoxic activities. In an alternative embodiment, the drug is cleaved from the complex drug-ligand outside the tumor cell or cancer cell, and then the drug enters the cell.

The ligand may be associated, for example, a tumor cell or cancer cell antigen of a tumor cell or cancer cell, which is located on the surface of tumor cells or cancer cells, or antigen of a tumor cell or cancer cell, which is an extracellular matrix protein associated with tumor cell or cancer cell. The ligand can be designed specifically for a particular type of tumor cells or cancer cells. Therefore, choosing the ligand, it is possible to change the type of tumors or cancers that can be treated effectively.

Typical examples of precancerous lesions, which can be targeted to conjugate the drug-ligand include, but are not limited to: metaplasia, hyperplasia, dysplasia, colorectal polyps, the actinic cedatos, report of the technical cheilitis, the human papilloma virus, leukoplakia, painless ulcers and damage to the skin and Bowen's disease.

Typical examples of cancers or tumors that can present purpose to conjugate the drug-ligand include, but are not limited to: cancer, lung cancer, colon cancer, prostate cancer, lymphoma, melanoma, breast cancer, ovarian cancer, testicular cancer, CNS cancer, renal cancer, kidney cancer, pancreatic cancer, stomach cancer, oral cancer, nasal cancer, cervical cancer, and leukemia. Regular specialist will easily understand that the particular guide ligand used in the conjugate, can be chosen in such a way that he purposefully gave the drug to the tumor tissue to be processed by this drug (namely to choose a smart agent that is specific regarding the tumor-specific antigen). Examples of such guides ligands are well known in this field, their non-restrictive examples include anti-Her2 for the treatment of breast cancer, anti-CD20 for the treatment of lymphoma, anti-PSMA for the treatment of prostate cancer and anti-CD30 for the treatment of lymphomas, including nahodkinskuju lymphoma.

In one embodiment, the present invention relates to a method of destroying cells. This method includes introducing into the cell a certain number of compounds of this invention, dostatochno is to do the specified cells. In a typical embodiment, the compound is administered to a subject bearing this cell. In another typical embodiment, the introduction helps to slow or stop tumor growth, which includes this cell (for example, the cell can be a tumor cell). To introduce with the aim of slowing the growth rate of the cells should be at least 10% less than the growth rate before the introduction. Preferably, the speed of growth has slowed, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or growth completely stopped.

Effective dosing

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient is contained in a therapeutically effective amount, namely in amounts effective to achieve the intended purpose. The actual amount effective for a particular application depends, among other things, on the conditions of treatment. The person skilled in the art can easily perform the determination of an effective amount, especially in light of the here detailed disclosure.

For any compounds described herein therapeutically effective amount can be initially determined from studies of cell culture. Target plasma concentration equal to the concentration of active compounds (compounds which deposits), which are able to inhibit the growth or cell division. In preferred embodiments, the cellular activity is suppressed by at least 25%. Currently, the preferred target plasma concentrations of the active compounds (compounds)that are capable of causing at least about 50%, 75% or even 90% or more in a strong inhibition of cellular activity. The percentage of inhibition of cell activity in a patient can be monitored to assess the correctness of the achieved concentrations of drugs in plasma, and can adjust the dose, increasing it or decreasing, to achieve a desired level of inhibition.

As is well known in this field, a therapeutically effective amount for use in humans can also be determined in animal models. For example, a dose for humans can be formulated thus, in order to achieve a circulating concentration, which is detected, is effective in animals. The dosage in humans can be adjusted by controlling cell inhibition and raising or lowering the dose, as described above.

A therapeutically effective dose can also be determined from data on the application of human connections, for which it is known that they exhibit similar pharmacological activity. The applied dose can be adjusted on the basis of attributes is through biological availability and effectiveness of the introduced compound compared to the known compound.

The person skilled in the art can easily determine the adjustable dose to achieve maximal efficacy in humans, based on the above methods and other methods that are well known in this field.

In the case of local application system circulating in the blood concentration of the introduced compound are not particularly important. In such cases, the compound is administered in such a way as to ensure in a localized area concentration effective to achieve the intended results.

For use in the prevention and/or treatment of diseases related to abnormal cell proliferation, it is preferable circulating concentration of the introduced compound from about 0.001 to 20 μm, preferably from about 0.01 to 5 microns.

Doses for oral administration to a patient compounds described herein typically comprise from about 1 to 10,000 mg/day, more often from about 10 to 1000 mg/day, and most typically from about 50 to 500 mg/day. Typical doses are set by the weight of the patient, constitute from about 0.01 to 150 mg/kg/day, more often from about 0.1 to 15 mg/kg/day, and most typically from about 1 to 10 mg/kg/day, such as 5 mg/kg/day or 3 mg/kg/day.

For other ways you can individually adjust the number of doses and interval between meals, to provide the new levels of the introduced compound in plasma, effective for specific clinical indications to be treated. For example, in one variant, you can enter the connection according to this invention at relatively high concentrations several times a day.

Differently, it may be more desirable to introduce the compound of this invention with a minimum effective concentrations and to apply the regimen with less frequent administration. This will provide a therapeutic mode that corresponds to the severity of the disease of the individual.

Using the description given here, you can plan an effective mode of treatment, which has no significant toxicity and is quite effective for the treatment of clinical symptoms exhibited by a particular patient. This planning should include careful selection of active compounds when considering factors such as efficiency connections, relative bioavailability, the body weight of the patient, the presence and severity of undesirable side effects, the preferred route of administration and toxicity profile of a selected agent.

Compounds, compositions and methods of the present invention is additionally illustrated by the examples below. These examples are provided to illustrate and not to limit the claimed invention.

EXAMPLES

Materialia ways

In the examples below, if not specified differently, temperatures are given in degrees Celsius (°C); the operation is carried out at room temperature or ambient temperature (typically in the range of about 18-25°C); evaporation of the solvent is carried out using a rotary evaporator under reduced pressure (typically 4.5 to 30 mm RT. Art.) with a bath temperature of 60°C, the reactions are usually monitored by way of TLC and reaction times lead for illustration only; melting points are given without amendments; products demonstrate satisfactory1H-NMR and/or microanalytical data; outputs are given for illustration only; and also use the following well-known abbreviations: TPL (melting point), l (liter(s)), ml (milliliters), mmol (mmol), g (grams)mg (milligrams), min (minutes), LC-MS (liquid chromatography-mass spectrometry) and h (hours).1H-NMR spectra were measured on spectrometer Varian Mercury 300 MHz and are consistent with the desired structures. Chemical shifts are given in ppm (ppm) in the low field relative to tetramethylsilane. Mass spectra with elektrorazpredelenie register on the mass spectrometer Perkin Elmer Sciex API 365. Elemental analysis is performed using Robertson Microlit Laboratories, Madison, NJ. For flash chromatography using silicagel E. Merck (230-400 mesh mesh). Analytical HPLC with about ashenai phase is performed on installing HP 1100 or Varian ProStar 210 with column Phenomenex Luna 5 μm C-18(2) 150 mm x 4.6 mm or column Varian Microsorb-MV 0.1 ám C-18, 150 mm x 4,6 mm The flow rate is 1 ml/min with a gradient from 0 to 50% buffer B for 15 minutes, or from 10 to 100% buffer B for 10 min with UV detection at 254 nm. Buffer A: 20 mm ammonium formate + 20% acetonitrile or 0.1% triperoxonane acid in acetonitrile; buffer B: 20 mm ammonium formate + 80% acetonitrile or 0.1% aqueous triperoxonane acid. Preparative HPLC with reversed phase is carried out at the installation Varian ProStar 215 with column Waters Delta Pak 15 μm C-18 300 mm x 7.8 mm

EXAMPLE 1: Synthesis of conjugates with peptide linkers

1.1a Methodology synthesis

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

1.1b Synthesis of compound 1: tert-butyl ether N-[2'-(N'-tert-butoxycarbonylamino)ethyl]valine. To a solution of 2-(N-tert-butoxycarbonylamino)ethylbromide (1 g, 4.5 mmol) and tert-butyl methyl ether valine (0,936 g, 4.5 mmol) in DMF (10 ml) is added potassium carbonate (1.85 g, 13.5 mmol). Thus obtained mixture was stirred at 100°C during the night. The reaction mixture was concentrate and purify the residue by the method of flash chromatography on silica gel, using as eluent a mixture of ethyl acetate/hexane (3/7) and getting listed in the title the connection information in the form of an oil (0.16 g, 12%).1H NMR (CDCl3) δ: 0,94 (fuzzy t, 6H), of 1.44 (s, 9H), and 1,473 1,475 (2s, 9H), of 1.88 (m, 1H), of 2.51 (m, 1H), 2,78 (m, 2H), 3,11 (m, 1H), up 3.22 (m, 1H), 3,39 and 4.13 (2 S.T., 1H), 5,00 (sh, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 205 (M+H+-112), 261 (M+H+-Bu), 317 (M+H+).

1.1c Synthesis of compound 2: N-(2-amino-ethyl)valine. Connection 1 (137 mg, 0.43 mmol) is dissolved in a solution of TN/dichloromethane (2 ml, 1/1) at room temperature. Thus obtained mixture was stirred at room temperature for 30 min, the Reaction mixture was concentrated to dryness, obtaining mentioned in the title compound as an oil (0.18 g, 95%).1H NMR (CD3OD) δ: 1.07 and of 1.16 (2D, 6H), to 2.35 (m, 1H), 3,2 (m, 1H), 3,38 (m, 4H) ppm; LC-MS (ionization elektrorazpredelenie) 217 (M+H+).

1.1d Synthesis of compound 3. To a solution of maleimide-dPEG4-NHS-ester (61 mg, 0.16 and 15 mmol) in dichloromethane (2 ml) is added drop by drop connection 2 (80,7 mg, 0.16 mmol) and diisopropylethylamine (55,5 μl, 0.32 mmol) in dichloromethane (1 ml). Thus obtained mixture is stirred over night. The solvent is removed on a rotary evaporator and the residue purified by the method of flash chromatography on silica gel, using as eluent dichloromethane, then 5% methanol in dichloromethane and finally 100% methanol and getting mentioned in the title compound as a colourless oil (87 mg, 97%).1H NMR (CDCl3) δ: 1,08 (DD, 6H), of 2.25 (m, 1H), 2.49 USD (t, 2H), 2,52 (t, 2H), 3,10-with 3.79 (m, 25H), 6,82 with, 2H) ppm; LC-MS (ionization elektrorazpredelenie) 559 (M+H+).

1.1e Synthesis of compound 4: Fmoc-Cit-PABOH. To a solution of Fmoc-Cit-OH (1.0 g, 2,52 mmol) and 4-aminobenzamide alcohol (341 mg, 2.77 mmol) in dichloromethane (10 ml) and methanol (5 ml) is added 2-ethoxy-1-etoxycarbonyl-1,2-dihydroquinoline [EEDQ] (1.24 g, 5,04 mmol) in one portion. The mixture is stirred in the dark for 16 hours the Solvent is removed on a rotary evaporator and the solid white triturated in ether (100 ml). The resulting suspension is treated with ultrasound for 5 min and then allowed to stand for 30 minutes, the Solid white is collected by filtration, washed with ether and dried in vacuum (1,23 g, 97%).1H NMR (DMSO) δ: (1,32-of 1.52 (m, 2H), 1,52-1,74 (DM, 2H), 2,86-3,06 (DM, 2H), 4,1 (m, 1H), 4,42 (d, 2H), 5,07 (t, 1H), 5.40 to (sh, 2H), 5,97 (t, 1H), 7,19-to 7.95 (m, 12H), 8,10 (d, 1H), becomes 9.97 (s, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 503,1 (M+H+).

1.1f Synthesis of compound 5: Fmoc-Cit-PABC-PNP. To a solution of compound 4 (309 mg, of 0.62 mmol) and para-nitrophenylphosphate (372 mg, of 1.85 mmol) in tetrahydrofuran (30 ml) and 1-methyl-2-pyrrolidine (1 ml) is added pyridine (100 μl, of 1.23 mmol) in one portion. Thus obtained mixture was stirred at room temperature for 30 minutes the Solvent is removed on a rotary evaporator and the residue purified by the method of flash chromatography on silica gel, using as eluent dichloromethane, then 3% methanol in dichloromethane is at the end of the 10% methanol in dichloromethane and getting mentioned in the title compound in the form of a solid white color (97.9 mg, 70%). LC-MS (ionization elektrorazpredelenie) 668 (M+H+).

1.1g Synthesis of compound 6: Fmoc-Lys(Boc)-PABOH. Connection 6 receives, as described above for compound 4, with 98% yield.1H NMR (DMSO) δ: of 1.40 (s, 9H), to 1.38 (m, 2H), 1,50 - 1,74 (DM, 2H), 3.04 from (t, 2H), 3,30 (kV, 3H), 4,19 - or 4.31 (m, 2H), to 4.41 (d, 2H), 4,55 (s, 2H), 7,28 - to 7.68 (m, 12H), of 8.00 (d, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 574 (M+H+).

1.1h Synthesis of compound 7: Fmoc-Lys(Boc)-PABC-PNP. Connection 7 receives, as described above for compound 5, with 70% yield.1H NMR (CDCl3) δ: the 1.44 (s, 9H), 1,49 is 1.60 (m, 6H 20), at 1.73 (m, 1H), 2,00 (m, 1H), 3,11 (m, 1H), 3,20 (sh, 1H), 4,23 (m, 2H), 4,46 (sh, 2H), 4,67 (sh, 1H), 5.56mm (sh, 1H), 7,28 (m, 2H), was 7.36-7,41 (m, 6H), to 7.59 (m, 4H), 7,76 (d, 2H), compared to 8.26 (DD, 2H), 8,45 (sh, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 639 (M+H+-Boc), 684 (M+H+-Bu), 739 (M+H+), 778.

1.1i Synthesis of compound 8: Boc-Val-Cit-OH. To a solution of citrulline (2,54 g, 14,50 mmol) and sodium bicarbonate (1.28 g) in water (40 ml) was added Boc-Val-OSu (4,34 g, 13,81 mmol)dissolved in dimethoxyethane (DME). To increase the solubility of the mixture is added tetrahydrofuran (10 ml). Thus obtained, the mixture is left to mix overnight at room temperature. Add aqueous citric acid (15%, 75 ml) and the mixture is extracted with a mixture of 10% 2-propanol/ethyl acetate (2 x 100 ml). The organic layer was washed with saturated salt solution (2 x 150 ml) and the solvents removed on a rotary evaporator. The obtained solid substance blogosfera dried in vacuum for 5 h and then treated with ether (100 ml). After a short ultrasonic treatment and rubbing collect solid white product by filtration (1.39 g, 27%).1H NMR (CD3OD) δ: 0,91 (DD, 3H), and 0.98 (DD, 3H), of 1.44 (s, 9H), to 1.70 (m, 2H), to 1.87 (m, 2H), 2,02 (m, 2H), 3,11 (t, 2H), with 3.89 (t, 1H), 4,39 5 (kV, 1H), they were 8.22 (d, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 375 (M+H+).

1.1j Synthesis of compound (9): Boc-Val-Cit-PABOH. Connection 9 receives, as described above for compound 4, with 71% yield.1H NMR (CD3OD) δ: of 0.93 and 0.97 (2D, 6H), of 1.44 (s, 9H), was 1.58 (m, 2H), of 1.75 (m, 1H), 1,90 (m, 1H), 2.05 is (m, 1H), 3,10 (m, 1H), 3,19 (m, 1H), 3,91 (d, 1H), to 4.52 (m, 1H), 5.25 in (s, 2H), 7,40 (d, 2H), 7,45 (DD, 2H), to 7.64 (d, 4H), 8,29 (DD, 2H) ppm; LC-MS (ionization elektrorazpredelenie) 480 (M+H+).

1.1k Synthesis of compound 10: Boc-Val-Cit-PABC-PNP. A solution of Boc-Val-Cit-PABOH (178 mg, 0,370 mmol) in THF (8 ml) in CH2Cl2(4 ml) was stirred at room temperature with PNP-chloroformiate (160 mg, 0.80 mmol) and pyridine (65 μl, 0.80 mmol) for 3 hours Added to the reaction mixture in ethyl acetate (100 ml) and 10% aqueous citric acid (50 ml) and the organic layer was washed with saturated salt solution, dried and concentrated and the residue purified by the method of flash chromatography on silica gel, using as eluent 5% methanol and getting mentioned in the title compound in the form of a solid white color (165 mg, 70%).1H NMR (CD3OD) δ: 0,93 (DD, 3H), 0,97 (DD, 3H), of 1.44 (s, 9H), was 1.58 (m, 2H), of 1.75 (m, 1H), 1,89 (m, 1H), 2.05 is (m, 1H), 3,10 (m, 1H), 3,20 (m, 1H), 3,90 (d, 1H), 4,51 (m, 1H), 4,55 (s, 2H), 7,29 (d, 2H) of 7.55 (d, 2H) ppm; LC-MS (ionization elektrorazpredelenie) 545 (M+H+-Boc), 645 (M+H+), 667 (M + Na+), 683 (M+K+).

1.1l Synthesis of compounds 12a. Through a suspension of compound 11 (20 mg, 0,078 mmol) in ethyl acetate (5 ml) was bubbled gaseous HCl for 20 min (until the suspension becomes a transparent solution). The reaction mixture is stirred for another 5 min, then the mixture is concentrated to dryness, obtaining mentioned in the title compound in the form of a solid yellow (26 mg, 100%), which is used in the next stage without additional purification. LC-MS (ionization elektrorazpredelenie) 260 (M+H+-Cl), 295 (M+H+).

1.1m Synthesis of compound 12b. Through a suspension of compound 11 (20 mg, 0,078 mmol) in ethyl acetate (5 ml) was bubbled gaseous HBr for 20 min (until the suspension becomes a transparent solution). The reaction mixture is stirred for another 5 min , then the mixture is concentrated to dryness, obtaining mentioned in the title compound in the form of a solid yellow (33 mg, 100%) which was used in the next stage without additional purification. LC-MS (ionization elektrorazpredelenie) 260 (M+H+-Br), 339 (M+H+), 341(M+H++2).

1.1n Synthesis of compound 13b. To a solution of compound 12a (26 mg, 0,078 mmol) in DMF (2 ml) is added 5-(2-dimethylaminoethoxy)benzofuran-2-carboxylic acid (44 mg, 0,155 mmol) and EDC (30 mg, 0,155 mmol). Thus obtained mixture is displaced is more at room temperature for 2 hours The mixture is concentrated and the residue dissolved in a mixture of H2O/CH3CN/TN (4/1,5/0,5; 6 ml) and placed in the freezer for 3 hours, the Solid yellow is collected by filtration (35 mg, 85%).1H NMR (CD3OD) δ: to 2.67 (s, 3H), 3,01 (s, 6H), to 3.34 (m, 2H), 3,63 (fuzzy t, 1H), with 3.89 (s, 3H), 3,91 (m, 1H), to 4.41 (m, 3H), of 4.54 (m, 1H)and 4.65 (m, 1H), 7,20 (DD, 1H), was 7.36 (d, 1H), 7,54 (s, 1H), to 7.59 (d, 1H), 7,73 (sh, 1H), 11,75 (s, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 490 (M+H+-Cl), 526 (M+H+).

O Synthesis of compound 13c. To a solution of compound 12b (19 mg, 0,0387) in DMF (2 ml) salt 5-(2-dimethylaminoethoxy)benzofuran-2-carboxylic acid, HBr (25 mg, 0,0775 mmol) and PS-carbodiimide (82 mg, mmol/g: 0,94, 0,0775 mmol). The reaction mixture was stirred at room temperature for 24 hours After filtration the filtrate is concentrated and the residue dissolved in a mixture of H2O/CH3CN/TN (2/0,75/0,25; 3 ml) and placed in the freezer for 3 hours, the Solid yellow is collected by filtration and dried, obtaining mentioned in the title compound (18 mg, 82%). LC-MS (ionization elektrorazpredelenie) 490 (M+H+-Br), 570 (M+H+), 572 (M+H++2).

1.1p Synthesis of compound 14a. To a suspension of compound 13a (48 mg, 0.10 mmol) in dichloromethane (4 ml) was added para-nitrophenyl chloroformate (80 mg, 0.40 mmol) and triethylamine (56 μl, 0.40 mmol) at -78°C. the Mixture is slowly warmed to room temperature and stirring is continued for further 30 minutes To the reaction sm is si add the compound N-Boc-N,N'-dimethylethylenediamine (166 mg, 0.80 mmol) and stirred over night. The mixture is concentrated and the residue purified by the method of flash chromatography on silica gel, using as eluent a 1.25% methanol in dichloromethane and receiving specified in the title compound in the form of solid white (71 mg, 100%)1H NMR δ: a 1.45 to 1.47 (m, 9H), 2,69 (s, 3H), of 2.97 (s, 3H), 3,14-to 3.34 (m, 4H), 3,81-to 3.92 (m, 8H), to 4.38-4,47 (m, 3H), 4,70 (d, 1H), 7,05 (DD, 1H), 7,11 (d, 1H), 7,45 (s, 1H), of 7.48 (d, 1H), to 7.99 (s, 1H), 10. 43 (s, 1H) ppm LC-MS (ionization elektrorazpredelenie) 710 (M-H+).

1.1q Synthesis of compound 14b. To a suspension of compound 13b (48 mg, of 0.075 mmol) in dichloromethane (2 ml) is added 4-nitrophenyl chloroformate (80 mg, 0.4 mmol) and triethylamine (40 mg, 0.4 mmol, 56 μl) at 0°C. the Mixture is heated to room temperature and stirring is continued for another 6 hours the Solvent is evaporated and the residue is washed with ether, obtaining an intermediate product. This intermediate product is dissolved in dichloromethane (2 ml) and the reaction solution was added N-Boc-N,N'-dimethylethylenediamine (44 mg, 0.2 mmol) and triethylamine (20 mg, 0.2 mmol, 28 ml). Thus obtained mixture was stirred at room temperature overnight. The mixture is concentrated and the residue purified by HPLC on a column of C-18, using as eluent monitorial (20 mm, pH 7.0) and acetonitrile and receiving specified in the title compound in the form of solid white (31 mg, 54%). LC-MS (ionization with electron what arapalayam) 755 (M+H +).

1.1r Synthesis of compound 14c. To a suspension of compound 13c (24 mg, 0.04 mmol) in CH2Cl2(2 ml) was added para-nitrophenylphosphate (64 mg, 0.32 mmol) and triethylamine (22 μl, 0.16 mmol) at 0°C. the thus Obtained reaction mixture is stirred at room temperature for 18 hours To the reaction mixture is added N-Boc-N,N'-dimethylethylenediamine (94 mg, 0.50 mmol) and stirring is continued for another 50 min, the Reaction mixture was concentrated and the residue purified by the method of flash chromatography on silica gel, using as eluent 5% methanol in dichloromethane and receiving specified in the title compound in the form of solids white (28 mg, 83%). LC-MS (ionization elektrorazpredelenie) 490, 570, 684 (M+H+-Boc), 784 (M+H+), 805 (M+Na+), 722 (M+K+).

1.1s Synthesis of compound 15a. Compound 14a (70 mg, 0.10 mmol) dissolved in triperoxonane acid (5 ml) and the mixture is stirred at room temperature for 30 min and concentrated to dryness, the product (72 mg, 100%) used in the next stage without additional purification. HPLC shows that he has a purity >95%.1H NMR δ: of 2.64 (s, 3H), of 2.93 (s, 3H), 3,19 (s, 3H), 3,30 (t, 1H), 3,79 (s, 3H), 3,85 (s, 3H), 3,81-of 3.85 (m, 1H), 4,27-of 4.49 (m, 3H), 4,59 (d, 1H), and 4.68 (d, 1H), 6,97 (DD, 1H), 7,03 (d, 1H), 7,38 (s, 1H), 7,41 (d, 1H), 8,00 (sh, 1H), 10,61 (sh, 1H) ppm LC-MS (ionization elektrorazpredelenie) 612 (M+H+), 634 (M+Na+).

1.1t Synthesis of compound 15b. 15b get as described above for compounds 15a, with 100% yield.1H NMR (CD3OD) δ: 2,69 (s, 3H), was 2.76 (s, 3H), 2,83 (sh, 1H), 3,01 (s, 6H), is 3.08 (sh, 1H), 3,24 (sh, 2H), 3,42 (m, 2H), 3,63 (sh, 3H), 3,74 (sh, 1H), 3,91 (s, 3H), 3,92 (m, 1H), 4,40 (sh, 2H), 4,57 (sh, 2H), 4,71 (sh, 1H), 7,22 (SD, 1H), was 7.36 (s, 1H), 7,56 (s, 1H), to 7.59 (d, 1H), 8,04 (sh, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 490, 526, 640 (M+H+), 678 (M+K+).

1.1u Synthesis of compound 15c. Compound 15c receive, as described above for compounds 15a, with 100% yield. LC-MS (ionization elektrorazpredelenie) 490, 570, 684 (M+H+), 722 (M+K+).

1.1v Synthesis of compound 16a. To a solution of compound 5 (12.5 mg, 0.019 mmol) and compound 15a (10 mg, of 0.014) in dimethylformamide (200 μl) is added triethylamine (6 μl, 0,044 mmol). Thus obtained mixture was stirred at room temperature overnight. Add to a mixture of ether (5 ml), and solution precipitation solid white. The solid substance is filtered off and purified by the method of flash chromatography on silica gel, using as eluent dichloromethane, then 1% methanol in dichloromethane, 2% methanol in dichloromethane, 3% methanol in dichloromethane and finally 4% methanol in dichloromethane and receiving the connection specified in the form of a solid white color (8,7 mg, 56%). LC-MS (ionization elektrorazpredelenie) 470, 1112 (M+H+), 1134 (M+Na+), 1150 (M+K+).

1.1w Synthesis of compound 16b. To a solution of compound 15b (5 mg, 0,0056 m is ol) in DMF (0.35 ml) is added compound 5 (3.8 mg, 0,0056 mmol) and DIEA (2 μl, to 0.011 mmol). Thus obtained mixture was stirred at room temperature for 5 hours the Mixture is concentrated and the residue purified by the method of flash chromatography on silica gel, using as eluent 10% methanol in dichloromethane and getting mentioned in the title compound as a solid (3 mg, 45%). LC-MS (ionization elektrorazpredelenie) 490, 526, 1169 (M+H+), 1208 (M+K+).

1.1x Synthesis of compound 16c. Compound 16c receive, as described above for compound 16b, with a 50% output. LC-MS (ionization elektrorazpredelenie) 490, 570, 1212 (M+H+), 1250 (M+K+).

1.1y Synthesis of compound 17a. To a solution of compound 16a (8,7 mg, 0,008 mmol) in dimethylformamide (500 μl) is added piperidine (100 μl) in one portion. Thus obtained mixture is stirred for 20 min at room temperature. The solvent is removed on a rotary evaporator and placed the mixture in a high vacuum for 1.5 hours, the Residue is extracted minimum amount of dichloromethane (100 μl) and added to a solution of hexane (3 ml), the solution appears solid white color, which is filtered off and dried (6,7 mg, 96,7%). MS (elektrorazpredelenie) 470, 890,1 (M+H+), 912 (M+Na+), 928 (M+K+).

1.1z Synthesis of compound 17b. Compound 17b receive, as described above for compounds 17a, with 95% yield. LC-MS (ionization elektrorazpredelenie) 947 (M+H+).

1.1aa inches connection 17c. Compound 17c receive, as described above for compounds 17a, with 95% yield. LC-MS (ionization elektrorazpredelenie) 1015 (M+N+).

l.1bb Synthesis of compound 18a. To a solution of compound 17a (4,2 mg of 0.005 mmol) and compound 3 (2,64 mg of 0.005 mmol) in dichloromethane (1 ml) is added in one portion PyBOP (3.7 mg, to 0.007 mmol), and then diisopropylethylamine (1 µl). Thus obtained mixture is stirred over night at room temperature. The solvents are removed on a rotary evaporator. The residue is purified by the method of preparative HPLC, receiving a beige solid (2.6 mg, 38.7 per cent). MS (elektrorazpredelenie) 470, 1431 (M+H+), 1453 (M+Na+), 1469 (M+K+).

1.1cc Synthesis of compound 18b. To a solution of compound 17b (2.2 mg, 0,0025 mmol) and compound 3 in 5% methanol in dichloromethane (400 ml) is added HBTU (9 mg, 0,0046 mmol) and DIEA (1,4 μl, 0,0046 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method prepreparation HPLC, using as eluent 10 mm monitorial and acetonitrile, and receiving the connection specified in the form of oil (1.1 mg, 30%). LC-MS (ionization elektrorazpredelenie) 490, 526, 1488 (M+H+), 1527 (M+K+).

1.1dd Synthesis of compound 18c. To a solution of compound 17c (6.5 mg, 0,0065 mmol) and compound 3 (5.5 mg, 0,0097 mmol) in 5% methanol in dichloromethane (0.5 ml) is added HBTU (3.7 mg, 0,0097 mmol) and DIEA (3,4 MK is, 0,0194 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and purified the residue by the method of flash chromatography on silica gel, using as eluent 30% methanol in dichloromethane and getting mentioned in the title compound as oil (4 mg, 30%). LC-MS (ionization elektrorazpredelenie) 1532 (M+H+), 1554 (M+Na+), 1570 (M+K+).

1.2 methodology for the synthesis of duocarmycin-containing peptide linker without self-destructive spacer

1.2a Reaction A: Through the suspension alkylating kernel (7 mg) in 2 ml of ethyl acetate to pass a slow stream of dry gaseous HBr to obtain a clear solution, which takes approximately 15 min, the Reaction mixture was concentrated and dried overnight under high vacuum.

1.2b Reaction B: To a suspension of brometalia-compound obtained in stage A, in DMF added EDC (10 mg, 0,054 mmol) and 5-nitrobenzeneboronic acid (12 mg, 0,054 mmol) and left to mix for 6 hours Then to this reaction mixture are added ethyl acetate and a saturated solution of salt. The combined organic layers are concentrated after three extraction with ethyl acetate and filtered through silica gel, using a mixture of MeOH/DHM with increasing amounts of MeOH. The product is confirmed by the method of mass when ectromelia, M+1 = 530.

1.2c Reaction C: Give protection to the 4'-OH using methylpiperidine (11 mg, 0,054 mmol) in 2 ml of DHM, 200 μl of allyl alcohol and pyridine (21 μl) for 2 hours the Product was then purified by the method of column chromatography on silica gel and identified by the method of mass spectrometry, MS+1= 654.

1.2d Reaction D: Recovery nitro performed by hydrogenation over Pd/C in a mixture DHM/MeOH (2:1) at a pressure of 40 psi for 45 min, the Product was filtered and the filtrate concentrated and dried in high vacuum. The product is confirmed by the method of mass-spectral analysis MS+1and transferred to the next stage without additional purification.

1.2e Reaction E: To a solution of the above compound (18 mg, 0,024 mmol) in a mixture of MeOH/DHM (2:1, 3 ml) was added Fmoc-Val-citrullin (29 mg, 0.06 mmol), the mixture was stirred for 10 min to dissolve the acid. Add 15 mg, 0.06 mol EEDQ and the reaction mixture is stirred in the dark overnight. Then the reaction mixture was concentrated, rinsed with diethyl ether and the residue purified preparative HPLC with reversed phase, receiving product, which is identified by the method of mass spectrometry M+1=1103.

1.2f Reaction F: Remove the protective Fmoc group performed using 5% piperidine in 1 ml DMF for 10 min After concentrating the reaction mixture, the solid residue TNA is askival diethyl ether. The product is confirmed by the method of mass-spectral analysis, MS+1=880 and M+K = 919.

1.2g Reaction G: To a solution of the free amine in DMF (1.5 ml), obtained in stage F, add Mal-(PEG)4-NHS-ester (20 mg) and the reaction mixture stirred for 1 h Concentration followed by purification by the method of preparative HPLC with reversed phase give 2.8 mg of the compound (overall yield of 11%, based on the alkylating kernel), which is confirmed by the method of mass-spectral analysis, MS+1=2178, M+Na=1300 and M+K=1316.

1.3 Synthesis of peptide linker conjugated to tubulysin A

The ligand can be linked to PEG and peptide linker shown by synthesis.

Synthesis of intermediate products and conjugate the ligand-drug having a peptide linker, where the medication is tubulysin A, shown here above. This basic method can be used with other medication.

1.4a synthesis of a conjugate of the peptide-linker 111

1.4b synthesis of a conjugate of the peptide-linker 112

1.4c synthesis of a conjugate of the peptide-linker 113

EXAMPLE 2: Synthesis of 6-membered conjugates with hydrazine powered linker

2.1 Synthesis of 6-membered gem-dimethyl hydrazine powered linker, anywhereman with duocarmycin derived cytotoxin

2.1a Scheme of the synthesis of compounds 109

2.1b Synthesis of compound 110

To a suspension of Cbz-dimethylalanine (1 g, 3,98 mmol) in 30 ml DHM when the bath temperature with ice added HOAT (catalytic, 0.25 equiv.) DIPEA (2.8 ml, 16 mmol)and then 2-chloro-1,3-dimethylimidazolidine hexaflurophosphate (CIP) (1.2 g, 4.4 mmol). Then to this reaction mixture are added Boc-NN(Me) (643 mol, 4.4 mmol). The reaction mixture was allowed to mix overnight at room temperature. To the reaction mixture are added 10% citric acid solution (100 ml) and extracted with DHM. The organic phase is washed with water and then saturated sodium bicarbonate solution and then with water again. Then the organic phase is concentrated and purified on a column of silica gel with increasing polarity of ethyl acetate in hexane, getting 860 mg, 57% yield of compound 107, which is identified by the method of mass spectrometry, M+1=380 and M+NH4+=397.

Protective Cbz group is removed by catalytic hydrogenation using Pd/C in MeOH and receiving connection 108, which is confirmed by the MS method.

To a solution of PNPC-1918 (10 mg, 0.1 mmol) in 2 ml of DHM add drop by drop a solution of compound 108 (60 mg, 0.25 mmol) in 8 ml DHM and leave the reaction mixture was mixed for 2 days until complete disappearance of the original substance. The reaction mixture is shown that the comfort through a small padding of silica gel and then concentrated and purified by the method of preparative HPLC with reversed phase, getting 4,2 mg of compound 109. The product identified by the method of mass spectrometry, M+1=740. The removal of the Boc-protection connection 109 is carried out by means of the net TN for 20 min, receiving the connection 110. The product identified by the method of mass spectrometry, M+1=640.

111

2.1c Synthesis of compound 111

Mal-PEG4-acetophenone and connection 110 (3 mg, of 0.005 mmol) are combined, concentrated and dried overnight under high vacuum. To this mixture was added 1 ml of 5% solution of acetic acid, prepared the day before, and dried over molecular sieves. The hydrazone formation is completed in less than hours After which the reaction mixture is concentrated and purified by the method of preparative HPLC with reversed phase (monitorial pH = 7)to give 2.8 mg of compound 111 (60% yield). The product identified by the method of mass spectrometry, M+1= 1129, M+NH4= 1146 and M+K = 1168.

2.2 Synthesis of gem-dimethyl 6-membered hydrazine powered linker conjugated to tubulysins cytotoxin

Methodology similar to the one shown in example 2.1 can be applied to the synthesis of seminale-dimethyl 6-membered hydrazine powered linker, forming a complex with a drug, such as tubulysin A.

2.3 Synthesis of hydrazine powered linker conjugated to similar duocarmycin

To a solution of brometalia connection (0,074 mmol) in 3 ml DMF add 5-actional-2-carboxylate (30 mg, 0.15 mmol) and EDC (28 mg, 0.15 mmol) and the resulting mixture is stirred over night. The reaction mixture was concentrated and purified chromatographically on silica gel using 5% MeOH in DHM and receiving 29 mg (74% yield) of product, which is confirmed by the method of mass spectrometry, M+1=523.

To a solution of the compound synthesized in stage C, in 5 ml DHM and 300 μl of allyl alcohol add methylpiperidine (22 mg, 0.11 mmol) and pyridine (44 ml). The reaction mixture was stirred at room temperature for 5 hours Concentration followed by purification by chromatography on silica gel using as eluent a mixture of 5% MeOH/DHM give 48 mg of the desired product (73% yield). The product is confirmed by the method of mass spectrometry, M+1= 650.

A solution of the above compound (8.2 mg, 0.012 mmol) and Mal-PEG4-hydrazine in 5% acetic acid in anhydrous DHM stirred at room temperature for 20 min, and then the solvent is evaporated and spend preparative HPLC with reversed phase using acetonitrile and aqueous phase with humanitarianism buffer, receiving 2.5 mg of the desired final product, which is confirmed by the method of mass spectrometry, M+1= 1063.

Duocarmycins similar conjugated with dimethyl 6-membered hydrazine powered by the linker, incubated in buffer at pH 7.4 for 24 h and examined in time education cyklinowanie product, the resulting cyclization hydrazine powered linker, releasing this duocarmycins similar.

Minimum quantity cyklinowanie product detected after 24 h at pH=7,4, it shows that this type of 6-membered hydrazine powered linker shows a relatively low rate of cyclization.

2.4b Speed cyclization gem-dimethyl 6-membered hydrazine powered linker

Duocarmycins similar conjugated with gem-dimethyl 6-membered hydrazine powered by the linker, incubated in buffer at pH 7.4 and explore time education cyklinowanie product, the resulting cyclization hydrazine powered linker, releasing this duocarmycins similar.

In the presence of 6-membered gem-dimethyl linker cyclization reaction is quite rapid, occurring prior to completion within a few minutes. Thus, the rate of cyclization for gem-dimethyl 6-membered hydrazine powered linker much more than speed to 6-membered linker that does not contain gem-dimethyl fragment.

EXAMPLE 3: Synthesis conjug the tov 5-membered hydrazine powered linker

3.1 methodology for the synthesis of compounds 4

Cbz-DMDA-2,2-diethylmalonate acid (1)

To a solution of 2,2-dimethylmaleic acid (2.0 mg, 0,0151 mol) is added thionyl chloride (1.35 ml, 0,0182 mol) in THF (15 ml) in a flask of 25 ml, equipped with a stirrer, temperature probe and reflux condenser, add a drop of DMF and the reaction mixture is heated at the boil under reflux for 2 h, then cooled to room temperature. This reaction mixture is transferred drop by drop to a solution of Cbz-DMDA (4 mg, 0,0182 mol) and triethylamine (4 ml, 0,0287 mol) in THF (5 ml) at 0oC and stirred for 30 min at this temperature. The solvent is removed in vacuum and the residue is dissolved in 1 N. HCl (50 ml) and extracted with DHM (2 x 25 ml). The combined organic layers extracted with 1 N. NaOH (2 x 25 ml) and the combined aqueous layer was acidified (pH<1) with concentrated HCl and extracted with EtOAc (2 x 25 ml), dried over MgSO4, filtered and concentrated in vacuo to a sticky solid off-white color, 3,44 mg, 68% yield. Connection 1 confirmed method MS: m/z 337,0 [M+1]+.

HPLC retention time: of 3.77 min (mass spectrometry).

Cbz-DMDA-2,2-dimethylmethoxy-Boc-N'-methylhydrazino (2)

To a solution of compound 1 (3.0 mg, 0,0089 mol) and thionyl chloride (0,78 ml, 0,0107 mol) in THF (25 ml) in a three-neck RBF 50 ml equipped with a stirrer, a temperature sensor is s and reflux condenser, add a drop of DMF and the reaction mixture refluxed for 2 h, then cooled to room temperature. Then add this to the reaction mixture drop by drop to a solution of Boc-N-methylhydrazine (1,33 mg, 0,091 mol) and triethylamine (3 ml, 0,0215 mol) in THF (25 ml) at 0oC and stirred for 30 min the Solvent is removed in vacuum and the residue is dissolved in EtOAc(50 ml), dried over MgSO4, filtered and concentrated in vacuo to an oil brown. This oil is dissolved in EtOAc and purified by the method of column chromatography (100% EtOAc)to give to 3.45 mg, 83% yield of a clear oil. Connection 2 confirm method MS: m/z 465,2 [M+1].

HPLC retention time: 3,97 min (mass spectroscopy).

DMDA-2,2-dimethylmethoxy-Boc-N'-methylhydrazino (3)

To a solution of compound 2 (0.5 mg, 0,0011 mol) in MeOH (30 ml) is added 10% Pd/C (15 mg) and the reaction mixture is left to hydrogenation in the device Pair for 30 min, the Catalyst is filtered off and the filtrate was concentrated in vacuo to a clear oil receiving connection 3 (0,38 mg). The product is confirmed by NMR (1H, CDCl3) δ: 1,45 (s, 15H) of 2.45 (s, 3H) 2,85 (s, 6H), and 3.16 (s, 3H) with 4.64 (m, 1H) 10,6 (sh, 1H); NMR (13C, CDCl3) δ: 24,1, 28,57, 35,15, 35,58, 36,66, 47,01, 48,51, 81,11, 155,17, 173,56, 176,24.

The synthesis of compounds 4

In RBF 15 ml, equipped with a mixer, combine the compound 3 (50 mg, 0,1513 mmol), PNPC-1918 (20 mg, 0,0315 mmol) and DHM (5 ml) and the Solution stirred for 30 min, then add triethylamine (25 μl, 0,1794 mmol) and stirred solution light yellow within 1 h the solution was concentrated in vacuo until a yellow oil and purified by the method of column chromatography (100% DHM to a mixture of 1:1 EtOAc/DHM)to give compound 4 in the form of a solid off-white color (22 mg, 84% yield). The product is confirmed by the method of mass spectrometry: m/z 825,7 [M+1]+.

HPLC retention time: 7,65 min (mass spectrometry)

3.2 synthesis of a conjugate of the antibody-drug having a 5-membered hydrazine powered linker

This diagram shows the conjugation of antibodies with complex linker-drug. Such methodologies are well known in the pharmaceutical field. Examples of other reactive sites include maleimide, halogenated that interact with thiols on the ligand, thiols, which interact with disulfides on the ligand, hydrazides, which interact with aldehydes and ketones to the ligand, and hydroxysuccinimide, isocyanates, isothiocyanates and anhydrides, which interact with the amino group on the ligand.

EXAMPLE 4: Synthesis of conjugates with by a disulfide linkers

4.1a Synthesis of compound 1. Into the flask containing PEG4(3.88 g, 20 mmol) is added Triton B (40% solution in methanol, 1.08 ml, 0.25 mmol) and after 15 min, tert-butyl acrylate (3,62 ml, 24 mmol). The mixture is stirred at room temperature overnight. The mixture was concentrated in vacuo and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and getting mentioned in the title compound as a colourless oil (2.35 g, 36%).1H NMR δ: 1,45 (s, 9H), 2,5 (t, 2H), 3,65 (m, 18H).

4.1b Synthesis of compound 2. To a solution of compound 1 (1,17 g, 3.6 mmol) in dichloromethane (10 ml), add triethylamine (532 μl, 4 mmol) and methanesulfonamide (309 μl, 4 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and getting mentioned in the title compound as yellow oil (1.3 g, 89%).1H NMR δ: USD 1.43 (s, 9H), 2,48 (t, 2H), of 3.07 (s, 3H), 3,62-3,70 (m, 14H), 3,76 (m, 2H), 4,37 (m, 2H).

4.1c the Synthesis of compound 3. To a solution of compound 2 (1.3 g, 3.25 mmol) in ethanol (10 ml) is added sodium azide (423 mg, 6.5 mmol). Thus obtained mixture is refluxed over night. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and receiving specified in the header connection in widebase yellow (1.01 g, 90%).1H NMR δ: 1,45 (s, 9H), of 2.50 (t, 2H), 3,40 (t, 2H), 3,62-to 3.73 (m, 16H).

4.1d Synthesis of compound 4. To a solution of compound 3 (470 mg, 1.35 mmol) in ether (5 ml)containing H2O (25 ml), add triphenylphosphine (391 mg, 1.48 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and getting mentioned in the title compound as yellow oil (325 mg, 75%).1H NMR δ: 1,45 (s, 9H), 2,24 (sh, 2H), of 2.51 (t, 2H), 2.91 in (t, 2H), of 3.56 (m, 2H), 3,63-3,66 (m, 12H), and 3.72 (m, 2H 30).

4.1e Synthesis of compound 5. To a solution of 3-mercaptopropionic acid (1.22 g, 11.5 mmol) in methanol (10 ml) add altricial-2 (of 3.78 g, 17,25 mmol). Thus obtained mixture was stirred at room temperature for 3 hours the Solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 30% ethyl acetate in hexane and getting mentioned in the title compound as oil (2,44 g, 98%).1H NMR δ: a 2.8 (t, 2H), 3,05 (t, 2H), 7,14 (m, 1H), to 7.67 (m, 2H), 8,48 (m, 1H).

Compound 5b:1H NMR δ: USD 1.43 (d, 3H), 2,61 (m, 1H), was 2.76 (m, 1H), 3,40 (m, 1H), 7,17 (m, 1H), 7,66 (m, 2H), 8,45 (m, 1H).

4.1f Synthesis of compound 6. 3 Methylbenzothiazolium iodide (1 g, 3.6 mmol) dissolved in 2 N. aqueous sodium hydroxide solution (10 ml) and the mixture is stirred tip is of 6 h at 100°C, then acidified with 6 N. aqueous solution of hydrochloric acid to pH 4 and extracted with diethyl ether. The organic layer is dried over Na2SO4that is evaporated on a rotary evaporator under vacuum, the residue is dissolved in methanol (10 ml) and add compound 5a (776 mg, 3.6 mmol). The mixture is stirred at room temperature for 1 h the Mixture is concentrated to dryness and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and getting mentioned in the title compound as yellow oil (482 mg, 55%).1H NMR δ: 2,85 (m, 2H), 2.95 and (m, 5H), only 6.64 (m, 2H), and 7.3 (m, 1H), and 7.4 (DD, 1H); MS (elektrorazpredelenie) 244 (M+H+), 487 (2M+H+).

Compound 6b:1H NMR δ: of 1.35 (d, 3H), 2,48 (m, 1H), 2,92 (s, 3H), to 3.02 (m, 1H), 3,34 (m, 1H), 6,62 (m, 2H), 7,28 (m, 1H), 7,44 (m, 1H) ; MS (elektrorazpredelenie) 258 (M+H+).

Compound 6c:1H NMR δ: 1,45 (s, 6H), 2,70 (s, 2H), 2,93 (s, 3H), 6,62 (m, 2H), 7,24 (m, 1H), 7,51 (m, 1H); MS (elektrorazpredelenie) 272 (M+H+), 294 (M+Na+), 310 (M+K+).

4.1g Synthesis of compound 7. To a solution of compound 6a (28 mg, 0,115 mmol) in anhydrous methanol (1 ml) add acetylchloride (13 μl, 0,173 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 10% ethyl acetate in hexane and getting listed is in the title compound as an oil (24 mg, 83%).1H NMR δ: 2,08 (m, 2H), 2,93 (s, 3H), 2.95 and (m, 2H), 3,70 (s, 3H), 6,63 (m, 2H), 7,28 (m, 2H), 7,40 (m, 2H) ; MS (elektrorazpredelenie) 258 (M+H+), 280 (M+Na+), 296 (M+K+).

Compound 7b:1H NMR δ: of 1.32 (d, 3H), of 2.45 (m, 1H), 2,92 (s, 3H), of 2.93 (m, 1H), 3,35 (m, 1H), to 3.67 (s, 3H), 6,62 (m, 2H), 7,26 (m, 1H), 7,44 (m, 1H) ; MS (elektrorazpredelenie) 272 (M+H+).

Compound 7c:1H NMR δ: of 1.42 (s, 6H), to 2.66 (s, 2H), 2,93 (s, 3H), 3,62 (s, 3H), 6,62 (m, 2H), 7,24 (m, 1H), 7,51 (m, 1H); MS (elektrorazpredelenie) 286 (M+H+), 308 (M+Na+), 324 (M+K+).

4.1h Synthesis of compound 8. To a solution of compound 7a (24 mg, 0,093 mmol) in dichloromethane (1 ml) add triphosgene (28 mg, 0,093 mmol) and triethylamine (37 μl, 0.28 mmol) at 0°C. the Mixture is stirred for 1 h the Mixture is concentrated to dryness and the residue used in the next stage without additional purification.

The crude material was dissolved in dichloromethane (1 ml) and add compound 8a (35 mg, 0,074 mmol) and DMAP (23 mg, 0,190 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and getting mentioned in the title compound as yellow oil (53 mg, 76%).1H NMR δ: 2,70 (s, 3H), 2,74 (m, 2H), 3,06 (m, 2H), 3,34 (m, 1H), 3,35, and to 3.36 (2s, 3H), 3,63, and to 3.64 (2s, 3H), 3,86 (m, 1H), 3,88 (s, 3H), 3,93, and of 3.94 (2s, 3H), 4,48 (m, 1H), 4,55 (m, 1H), 4,79 (m, 1H), 7,05 (m, 1H), 7,11 (m, 1H), 7,26-7,52 (m, 5H), the 7.85 (d, 1H), 8,1(sh, 1H), 8,98 and 9.08 (2s, 1H) ; MS (elektrorazpredelenie) 753 (M+H+).

Compound 8b:1H NMR δ: 1,38 (m, 3H), 2,52 (m, 1H), 2,69 (m, 3H), and 2.79 (m, 1H), 3.33 and (m, 1H), 3,37 (2s, 3H), of 3.64 (m, 3H), 3,88 (s, 3H), 3,84-3,90 (m, 1H), 3,93 (2s, 3H), 4,48 (m, 1H), 4,57 (m, 1H), 4,78 (m, 1H), 7,06 (m, 1H), 7,12 (m, 1H), 7,26-the 7.43 (m, 3H), 7,50 (m, 2H), 7,86 (m, 1H), 8,1 (sh, 1H), 8,99, the remaining 9.08, 9,13 and which 9.22 (4S, 1H) ; MS (elektrorazpredelenie) 767 (M+H+).

Compound 8c:1H NMR δ: the 1.44 (m, 6H), 2.63 in (d, 2H), 2,70 (s, 3H), at 3.35 (m, 1H), 3,38 and 3,39 (2s, 3H), 3,63, and to 3.64 (2s, 3H), a 3.87 (m, 1H), 3,88 (s, 3H), 3,93, and of 3.94 (2s, 3H), 4,48 (m, 1H), 4,55 (m, 1H), 4,79 (m, 1H), 7,05 (m, 1H), for 7.12 (m, 1H), 7,31-7,39 (m, 3H), 7,49 (m, 2H), 7,89 (d, 1H), 8,1 (sh, 1H), 9,12 and 9,23 (2s, 1H) ; MS (elektrorazpredelenie) 781 (M+H+).

4.1i the Synthesis of compounds 9 and 10. To a solution of compound 8a (0.1 mg) in a mixture of PBS buffer solution (pH 7,2)/methanol (300 μl, 2:1) add 20 mm solution of DTT (100 ál, 15 EQ.) and monitor the reaction by HPLC. The interaction proceeds too rapidly for detection, after a few seconds, the reaction is already completed, quantitatively yielding the product connection 10. The intermediate reaction product connection 9 will not be detected.

4.1j Synthesis of compound (11). To a solution of compound 6a (66 mg, 0.2 mmol) in dichloromethane (1 ml) was added DCC (47 mg, 0.22 mmol), HOBt (31 mg, 0.22 mmol) and compound 4 (50 mg, 0.2 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and getting mentioned in the title compound as yellow oil (70 mg, 62%).1H NMR δ: the 1.44 (s, 9H), of 2.51 (t, 1H), 2.63 in (t, 2H), 2,93 (d, 3H), 3,01 (t, 2H), of 3.45 (m, 2H), 3,55 (m, 2H), to 3.64 (m, 12H), 3,71 (t, 2H), 5,01 (sh, 1H), 6,38 (S.T., 1H), 6,62 (m, 2H), 7,27 (m, 1H), 7,43 (DD, 1H). MS (elektrorazpredelenie) 491 (M-56+H+), 513 (M-56+Na+), 547 (M+H+), 569 (M+Na+).

Compound 11b:1H NMR δ: of 1.34 (d, 3H), 1,45 (s, 9H), is 2.30 (m, 1H), 2,5 (t, 2H), 2,69 (m, 1H), 2,93 (d, 3H), 3,37-3,55 (m, 5H), 3,63 (m, 12H), 3,71 (t, 2H), 4,99 (sh, 1H), 6,13 (S.T., 1H), 6,62 (m, 2H), 7,25 (m, 1H), of 7.48 (DD, 1H). MS (elektrorazpredelenie) 505 (M-56+H+), 527 (M-56+Na+), 543 (M-56+K+), 561 (M+H+), 583 (M+Na+).

Compound 11C: USD 1.43 (s, 3H), 1,45 (s, 9H), 2,46 (s, 2H), 2,5 (t, 2H), of 2.92 and 2.94 (2s, 3H), of 3.33 (m, 2H), 3,47 (t, 2H), 3,63 (m, 12H), 3,70 (t, 2H), 6,06 (S.T., 1H), 6,63 (m, 2H), 7,25 (m, 1H), 7,54 (d, 1H) ; MS (elektrorazpredelenie) 519 (M-56+H+), 541 (M-56+Na+), 575 (M+H+), 597 (M+Na+).

4.1k Synthesis of compound 12: To a suspension of compound 11a (20 mg, 0,037 mmol) in dichloromethane (1 ml), add triethylamine (15 μl, 0.11 mmol) and 2 n solution of phosgene in toluene (55 μl, 0.11 mmol) at 0°C. the Mixture is stirred at room temperature for 1 h the Mixture is concentrated and the residue is dissolved in dichloromethane (1 ml) and added compound 10 (14 mg, 0,030 mmol) and DMAP (9 mg, 0,076 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method of flash chromatography on silica gel, using as eluent 1% methanol in dichloromethane and receiving specified in the header with the Association in the form of a yellow oil (23 mg, 74%).1H NMR δ: the 1.44 (s, 9H), 2.49 USD (t, 2H), to 2.67 (m, 2H), 2.65 and to 2.67 (2s, 3H), of 3.07 (m, 2H), 3.33 and (s, 3H), 3,40 (m, 3H), 3,51 (m, 2H), 3,60 (m, 12H), of 3.69 (m, 2H), a 3.87 (s, 3H), 3,92 (s, 3H), 3,93 5 (m, 1H), to 4.52 (m, 2H), 4,78 (m, 1H), 6,65, 6,74 and 6,97 (3 broad t, 1H), 7,06 (d, 1H), 7,12 (s, 1H), 7,29-7,42 (m, 3H), 7,50 (m, 2H), 7,87 (d, 1H), 8,10 and 8,15 (SS, 1H), 9,79 and 9.58 (2s, 1H) ; MS (elektrorazpredelenie) 986 (M+H+-56), 1042 (M+H+).

Compound 12b:1H NMR δ: 1,32 (m, 3H), of 1.44 (s, 9H), 2,39 (m, 1H), 2,48 (m, 2H), 2,60 (m, 1H), 2,67, and 2,69 (2s, 3H), 3,32, and the 3.35 (2s, 3H), 3,38-and 3.72 (m, 20H), 3,88 (C, 10 3H), 3,93 (s, 3H), of 3.94 (m, 1H), to 4.52 (m, 2H), 4,77 (m, 1H), 6,53, 6,67 and 6,72 (St, 1H), 7,06 (d, 1H), 7,12 (s, 1H), 7,29-7,39 (m, 3H), 7,49 (m, 2H), 7,88 (d, 1H), 8,12 and of 8.25 (SS, 1H), 9,13, 9,36, 10,08 of 10.21 and (4S, 1H) ; MS (elektrorazpredelenie) 1000 (M+H+-56), 1056 (M+H+), 1078 (M+Na+), 1084 (M+K+).

Compound 12c:1H NMR δ: 1,30-of 1.42 (m, 3H), of 1.44 (s, 9H), 2,45-2,52 (m, 4H), 2.69 and of 2.72 (2s, 3H), 3,34 and the 3.35 (2s, 3H), 3,39-and 3.72 (m, 19H), 3,88 (s, 3H), 3,925 and 3,93 (2s, 3H), of 3.94 (m, 1H), 4.53-in (m, 2H), 4,80 (m, 1H), 6,63 (m, 1H), 7,06 (DD, 1H), 7,13 (d, 1H), 7,25-7,39 (m, 3H), 7,50 (m, 2H), 7,89 (d, 1H), 8,10 and 8,27 (SS, 1H), 9,99 and 10,191 (2s, 1H); MS (elektrorazpredelenie) 1014 (M+H+-56), 1070 (M+H+), 1108 (M+K+).

4.1l Synthesis of compound 13. Compound 12a (23 mg, of 0.022 mmol) is dissolved in the solution triperoxonane acid and dichloromethane (1 ml, 1/1) and the mixture is stirred at room temperature for 30 min and concentrated, obtaining the product (21 mg, 100%).1H NMR δ: 2,60 (t, 2H), to 2.67 and $ 2.68 (2s, 3H), of 2.75 (m, 2H), of 3.07 (m, 2H), 3,34 (s, 3H), 3,38-to 3.64 (m, 21H), 3,76 (t, 2H), 3,88 (s, 3H), 3,92 (s, 3H), 3,93 (m, 1H), 4.53-in (m, 2H), 4,78 (m, 1H), 7,06 (d, 1H), 7,13 (s, 1H), 7,31-the 7.43 (m, 3H), 7,49 m, 2H), 7,87 (d, 1H), 8,10 and 8,15 (SS, 1H), 9,44 and 9,65 (2s, 1H); MS (elektrorazpredelenie) 986 (M+H+), 1008 (M+Na+), 1024 (M+K+).

Compound 13b:1H NMR δ: of 1.34 (m, 3H), of 2.56 (m, 1H), 2,62 (m, 2H), 2,68 (m, 3H), 2,8 (m, 1H), 3,35-to 3.36 (2s, 3H), 3,40-3,70 (m, 18H), of 3.77 (t, 2H), 3,88 (s, 3H), 3,93, and 3.95 (2s, 3H), of 3.94 (m, 1H), 4,54 (m, 2H), 4,79 (m, 1H), 7,07 (d, 2H), 7,13 (s, 1H), 7,30-7,42 (m, 3H), 7,49 (m, 2H), 7,88 (d, 1H), 8,11 and 8,25 (2 broad s, 1H), which 9.22, 9,37, 9,80 and 9,92 (4S, 1H) ; MS (elektrorazpredelenie) 1000 (M+H+), 1022 (M+Na+), 1038 (M+K+).

Compound 13c:1H NMR δ: 1,30-1,45 (m, 6H), of 2.54 (m, 2H), 2,61 (m, 2H), 2,68, and 2,69 (2s, 3H), 3,35-to 3.36 (2s, 3H), 3,40-3,70 (m, 17H), of 3.77 (t, 2H), 3,88 (s, 3H), 3,92 3,93 and (2s, 3H), of 3.94 (m, 1H), 4,50 (m, 2H), 4,80 (m, 1H), 7,08 (m, 2H), 7,12 (d, 1H), 7,29-7,39 (m, 3H), 7,49 (m, 2H), 7,89 (m, 1H), 8,10 and of 8.25 (sh 1H), 9,88 and 10,04 (2s, 1H) ; MS (elektrorazpredelenie) 1014 (M+H+), 1036 (M+Na+), 1054 (M+K+).

4.1m Synthesis of compound 14a. To a solution of compound 13a (5.4 mg, 0,0054 mmol) in dichloromethane (1 ml) was added PS-carbodiimide (11,5 mg of 0.94 mmol/g, 0,0108 mmol) and PS-DMAP (7.2 mg, 1,49 mmol/g, 0,0108 mmol). Thus obtained mixture was stirred at room temperature over night, filtered and concentrated, obtaining the product. MS (elektrorazpredelenie) 1082 (M+H+).

4.2 Synthesis of disulfide linker conjugated to tubulysin A

Medicine tubulysin konjugierte with a disulfide linker of the present invention, shown here using the above mechanism. Using similar reaction is ionic schemes, it is possible to synthesize other medications and other linkers of the present invention.

4.3 the Rate of cyclization, disulfide linker

To a solution of compound 8a (0.1 mg) in a mixture of PBS buffer solution (pH 7,2)/methanol (300 μl, 2/1) add 20 mm solution of DTT (100 ál, 15 EQ.) and monitor the reaction by HPLC. In the reaction mixture, a rapid cyclization, the reaction is finished after a few seconds, quantitatively yielding the product 10. The intermediate reaction product 9 will not be detected.

Example 5

Synthesis of compound 32. Through a solution of compound 30 (120 mg, 0.28 mmol) in ethyl acetate (10 ml) was bubbled gaseous HCl for 5 min, the Reaction mixture was stirred at room temperature for another 30 min and then concentrated. Add to the reaction mixture, the ether and collect the white precipitate on the filter funnel. The solid is dried overnight in a vacuum, receiving 100 mg of the desired product, which was confirmed by LC-MS (ionization elektrorazpredelenie) 324 (M+H+) and used in the next stage without additional purification. To a solution of this compound (100 mg, 0.24 mmol) in DMF (5 ml) is added compound 31 (65 mg, 0.26 mmol), HATU (100 mg, 0.26 mmol) and TEA (91 μl, 0.52 mmol). The obtained t is thus the mixture is stirred at room temperature for 3 hours The solvent is evaporated and the residue purified by the method prepreparation HPLC, using as eluent of 0.1% of TN in water and acetonitrile, and receiving a connection 32 in the form of an oil (110 mg, 80%). The desired product is confirmed by LC-MS (ionization elektrorazpredelenie) 555 (M+H+).

Synthesis of compound 33. Mix a solution of compound 32 (110 mg, 0.2 mmol), palladium on coal (20 mg) in DHM (10 ml) and methanol (5 ml) in an atmosphere of hydrogen at atmospheric pressure and room temperature for 12 hours Palladium is filtered off, the reaction mixture was concentrated and the residue purified by the method prepreparation HPLC, using as eluent of 0.1% of TN in water and acetonitrile, and receiving the desired compound in the form of oil (80 mg, 78%). LC-MS (ionization elektrorazpredelenie) 465 (M+H+). To a solution of the residue (80 mg, 0,17 mmol) in dichloromethane (10 ml) and THF (5 ml) is added PNPCl (4-nitrophenylphosphate) (137 mg, of 0.68 mmol) and triethylamine (144 μl, of 1.02 mmol) at 0°C. the resulting mixture is stirred for 30 min at 0°C and then at room temperature for 12 hours This reaction mixture was concentrated in vacuo and precipitated residue using ethyl ether (100 ml), get a connection 33 in the form of a solid yellow (90 mg, 82%), which is dried in vacuum and confirmed by LC-MS (ionization elektrorazpredelenie) 631 (M+H+).

Synthesis soy is inane 46: To a solution of compound 33 (60 mg, 0.1 mmol) in dichloromethane (10 ml) was added Boc-N,N-dimethylethylamine (84 mg, 0.38 mmol) and triethylamine (26 μl, 0.1 mmol) at room temperature. Thus obtained mixture was stirred at room temperature for 12 hours, the Reaction mixture was concentrated in vacuo and the residue precipitated using ethyl ether (100 ml)receive Boc-protected compound 34, which is used in the next stage without additional purification. Boc-protected compound 34 was dissolved in 10 ml of TN and the reaction mixture was stirred at room temperature for 60 min, This reaction mixture was concentrated in vacuo and the residue precipitated using ethyl ether (100 ml), get a connection 46 in the form of a solid yellow color, which is dried in vacuum and confirmed by LC-MS (ionization elektrorazpredelenie) 631 (M+H+).

Synthesis of compound 34: To a solution of 2-bromidrosiphobia (5 g, 24.4 mmol) in DMF (50 ml) add diisopropylethylamine (8.5 ml, 48.8 mmol) and benzylchloride (3,48 ml, 24.4 mmol). Thus obtained mixture was stirred at room temperature for 2 hours, the Reaction mixture was concentrated and the residue purified by the method of flash chromatography on silica gel, using as eluent a mixture of ethyl acetate/hexane (3/7), and receiving the requested connection 34 in the form of an oil (4 g, 64%).1H NMR (CDCl3) δ: 3,54 (sh, 2H), 3,61 (sh, 2H), 5,1 (, 2H), was 7.36 (m, 5H).

Synthesis of compound 35: To a solution of compound 34 (3,34 g 12,99 mmol) and tert-butyl methyl ether valine (3,27 g, 15,59 mmol) in DMF (50 ml) is added potassium carbonate (5,39 g, 38,97 mmol) and potassium iodide (2,59 g, 15,59 mmol). Thus obtained mixture was stirred at 100°C during the night. The reaction mixture was concentrated and the residue purified by the method of flash chromatography on silica gel, using as eluent a mixture of ethyl acetate/hexane (2/8), and receiving the requested connection 35 in the form of oil (3.12 g, 69%).1H NMR (CDCl3) δ: 0,92 (m, 6H), of 1.46 (s, 9H), to 1.86 (m, 1H), 2,53 (m, 1H), 2,80 (m, 2H), 3,18 (m, 1H), and 3.31 (m, 1H), 5,10 (s, 2H), 5.25-inch (sh, 1H), was 7.36 (m, 5H); LC-MS (ionization elektrorazpredelenie) 296 (M+H-tert-butyl+), 352 (M+H+).

Synthesis of compound 36. A solution of compound 35 (3.4 g, 9,72 mmol) and palladium on coal (200 mg) in methanol (30 ml) is placed in an atmosphere of hydrogen at atmospheric pressure and room temperature. Thus obtained mixture was stirred at room temperature for 2 hours Palladium is filtered off and the reaction mixture is concentrated to dryness, obtaining the desired compound 36 in the form of an oil (2.1 g, 98%)

Synthesis of compound 37. To a solution of compound 36 (2.1 g, 9,72 mmol) in dichloromethane (30 ml) is added FmocOSu (ester 9-fluorenylmethoxycarbonyl-N-hydroxysuccinimide) (3.28 g, 9,72 mmol) at 0°C. the resulting mixture is stirred for 2 h at 0°C. Dissolve Itel removed on a rotary evaporator and the residue purified by the method of flash chromatography on silica gel, using as eluent dichloromethane, then with 0.5% methanol in dichloromethane and finally 1% methanol in dichloromethane and getting the desired compound 37 as a colorless oil (2.55 g, 60%).1H NMR (CDCl3) δ: 0,95 (fuzzy t, 6H), to 1.48 (s, 9H), 1,90 (m, 1H), to 2.55 (m, 1H), 2,82 (m, 2H), 3,18 (m, 1H), 3,32 (m, 1H), 4,24 (m, 1H), 4,37 (m, 2H), 5.40 to (sh, 1H), 7,30 (m, 2H), 7,39 (m, 2H), 7,60 (d, 2H), of 7.75 (d, 2H) ppm; LC-MS (ionization elektrorazpredelenie) 383 (M+H-tert-butyl+), 440 (M+H+), 462 (M+Na+), 478 (M+K+).

Synthesis of compound 38. Through a solution of compound 37 (177 mg, 0.4 mmol) in a mixture of tetrahydrofuran-water (3/1,8 ml) was bubbled gaseous HCl for 5 min, the Reaction mixture was stirred at 37°C overnight, then concentrated to dryness, obtaining the desired compound 38 in the form of a solid (168 mg, 98%), which is confirmed by LC-MS (ionization elektrorazpredelenie) 383 (M+H+), 405 (M+Na+), and used in the next stage without additional purification. LC-MS (ionization elektrorazpredelenie) 383 (M+H+), 405 (M+Na+).

Synthesis of compound 39. To a solution of compound 5 (525 mg, of 0.79 mmol) in DMF (5 ml) is added N-Boc-N,N'-dimethylethylenediamine (177 mg, of 0.94 mmol). Thus obtained mixture was stirred at room temperature for 30 min the Solvent is removed and the residue purified by the method of flash chromatography on silica gel, using as eluent dichloromethane, then 2 methanol in dichloromethane and finally 5% methanol in dichloromethane and getting the desired compound 39 as a colourless oil (364 mg, 65%).1H NMR (CD3OD) δ: of 1.39 (s, 9H), and 1.56 (m, 2H), 1.70 to (m, 1H), equal to 1.82 (m, 1H), 2,70 and 2.82 (2s, 3H), 2,90 (s, 3H), to 3.09 (m, 1H), 3,17 (m, 1H), 3,30-3,37 (m, 4H), to 4.16 (t, 1H), 4,27 (m, 1H), 4,33 (d, 2H), 5,02 (sh, 2H), 7.24 to and 7.36 (m, 6H), 7,51-the 7.65 (m, 4H), 7,74 (d, 2H) ppm; LC-MS (ionization elektrorazpredelenie) 618 (M+H-Boc+), 662 (M+H-tert-butyl+), 718 (M+H+), 740 (M+Na+), 1435 (2M+H+).

Synthesis of compound 40. The connection 40 receives, as described above for compounds 17a, with 98% yield. LC-MS (ionization elektrorazpredelenie) 396 (M+H-Boc+), 496 (M+H+), 517 (M+Na+), 533 (M+K+), 992 (2M+H+).

Synthesis of compound 41. To a solution of compound 40 (138 mg, 0.28 mmol) in DMF (4 ml) is added compound 38 (110 mg, 0.28 mmol), HOBt (36 mg, 0.28 mmol) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (50 mg, 0.28 mmol). Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method prepreparation HPLC, using as eluent of 0.1% of TN in water and acetonitrile, and receiving the requested connection 41 in the form of oil (178 mg, 70%).1H NMR (CD3OD) δ: 1,04 and 1,11 (2D, 6H), 40 (s, 9H), was 1.58 (m, 2H), 1.77 in (m, 1H), of 1.88 (m, 1H), 2,24 (m, 1H), 2,72, and 2,84 (2s, 3H), of 2.92 (s, 3H), 3,10-3,18 (m, 4H), 3,35-of 3.46 (m, 6H), 3,82 (d, 1H), 4,22 (t, 1H), to 4.41 (m, 2H), 4,59 (m, 1H), 5,04 (sh, 2H), 7,28-7,40 (m, 6H), 7,55 (m, 2H), 7,63 (m, 2H), 7,78 (d, 2H) ppm; LC-MS (ionization elektrorazpredelenie) 760 (M+H-Boc+), 804 (M+H-tert-butyl+), 860 (M+H+), 882 (M+Na+), 899 (M+K+)

Synthesis of compound 42. The connection 42 receives, as described above for compounds 17a, with 98% yield. LC-MS (ionization elektrorazpredelenie) 538 (M+H-Boc+), 582 (M+H-tert-butyl+), 638 (M+H+), 660 (M+Na+).

Synthesis of compound 43. To a solution of compound 42 (23 mg, being 0.036 mmol) in dichloromethane (1 ml) add GMBS (ester N-(maleimidomethyl)succinimide) (14 mg, 0.05 mmol) and diisopropylethylamine (8,4 μl, 0.05 mmol) at 0°C. the Mixture is slowly warmed to room temperature and continue stirring for further 30 minutes the Solvent is evaporated and the residue purified by the method prepreparation HPLC, using as eluent of 0.1% of TN in water and acetonitrile, and receiving the requested connection 43 in the form of an oil (26 mg, 79%).1H NMR (CD3OD) δ: of 1.06 and 1.12 (2D, 6H), of 1.41 (s, 9H), to 1.59 (m, 2H), 1,78 (m, 1H), 1,86-of 1.93 (m, 3H), 2,24 (m, 3H), 2,74, and 2,84 (2s, 3H), 2,93 (sh, 3H), 3,13-up 3.22 (m, 4H), 3,40-of 3.60 (m, 8H), 3,82 (d, 1H), 4,60 (m, 1H), of 5.05 (sh, 2H), 6,80 (s, 2H), 7,32 (m, 2H), EUR 7.57 (d, 2H), 8,78 (d, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 703 (M+H-Boc+), 747 (M+H-tert-butyl+), 803 (M+H+), 825 (M+Na+), 841 (M+K+).

Synthesis of compound 44. The connection 44 receive, as described above for compounds 15a, with 98% yield. LC-MS (ionization elektrorazpredelenie) 703 (M+H+), 725 (M+Na+).

Synthesis of compound 45. To a solution of compound 44 (15 mg, to 0.016 mmol) and compound 33 (10 mg, to 0.016 mmol) in DMF (0.8 ml) add diisopropylethylamine (5,5 μl, 0,032 IMO the b) at room temperature. Thus obtained mixture was stirred at room temperature overnight. The solvent is evaporated and the residue purified by the method prepreparation HPLC, using as eluent of 0.1% of TN in water and acetonitrile, and receiving the requested connection 45 in the form of oil (10 mg, 45%).1H NMR (CD3OD) δ: 1,02-of 1.13 (m, 6H), of 1.55 (m, 2H), 1,74 (m, 1H), 1,84-of 1.92 (m, 3H), 2,20-of 2.27 (m, 3H), 2.95 and-3,14 (m, 16H), 3,47-a-3.84 (m, 12H), 3,98 (m, 1H), 4,2-4,34 (m, 3H), of 4.57 (m, 1H), 4,69 (m, 2H), 5,07-5,17 (m, 2H,), 6,78 (s, 2H), 7,16-of 7.23 (m, 3H), 7,30 (m, 1H), 7,38-7,47 (m, 3H), 7,52-7,58 (m, 3H), 7,81-a 7.92 (m, 2H), 8,25 (sh, 1H) ppm; LC-MS (ionization elektrorazpredelenie) 1194 (M+H+), 1215 (M+Na+), 1233 (M+K+).

Example 6

Synthesis of compound (2). A solution of compound 1 (100 mg, 0.24 mmol) and 10% Pd-C (35 mg) in a mixture of MeOH/CH2Cl2(1/2, 10 ml) Tegaserod in vacuum at 40 C. the resulting mixture was placed in an atmosphere of hydrogen and stirred at 25°C for 7 o'clock This reaction mixture is filtered through celite (washed with CH2Cl2). The solvent is removed in vacuum. Chromatography on silica gel with elution with mixture of EtOAc/hexane (2/8) gives compound 2 (77 mg, 98%).1H NMR (DMSO-d6) δ: 10,36 (s, 1H), 8,04 (d, 1H, J=8,2 Hz), 7,72 (d, 1H, J=8,2 Hz), to 7.61 (sh, 1H), 7,45 (t, 1H, J=8,4 Hz)7,261 (t, 1H, J=8,4 Hz)4,06 (m, 4H), to 3.73 (m, 1H), of 1.52 (s, 9H).

Synthesis of compound (4). A solution of compound 2 (35 mg, 0.1 mmol) in 4M HCl-EtOAc (5 ml) was stirred at 25°C in Ar atmosphere for 30 min Rast is oritel removed in vacuum. To the residue add 5-acetylindole-2-carboxylic acid (24,4 mg, 0.12 mmol). Add a solution of EDC (22.9 mg, 0.12 mmol) in DMF (3 ml) and the reaction mixture was stirred at 25°C for 5 hours the Solvent is removed. The crude product chromatographic on silica gel, elwira 10% MeOH in CH2CCl2and receiving connection 4 (40,7 mg, 93%).1H NMR (DMSO-d6) δ: 12,13 (s, 1H), 10,47 (s, 1H); to 8.45 (s, 1H), 8,10 (d, 1H, J=8,4 Hz), of 7.96 (sh, 1H), a 7.85 (d, 2H, J=8,4 Hz), 7,54 (d, 1H, J=8,4 Hz), 7,51 (t, 1H, J=8,2 Hz), was 7.36 (t, 1H, J=7,6), 7,35 (s, 1H), 4,81 (t, 1H, 11.2 Hz), of 4.54 (DD, 1H, 8,8 Hz)to 4.23 (m, 1H), 4,01 (DD, 1H, J=10,2 Hz), 3,86 (DD, 1H, J=10,7 Hz), 2,61 (s, 3H).

Synthesis of compound (5). 4-Methyl-1-piperazinecarboxamide hydrochloride (19.9 mg, 0.1 mmol) are added to a solution of compound 4 (20 mg, 0.05 mmol) and anhydrous pyridine (25 μl, 0.3 mmol) in 3% allyl alcohol in dry methylene chloride (4 ml) and the mixture is stirred for 16 hours Purification of the crude product on silica gel gives compound 5 (23,6 mg, 91%).1H NMR (DMSO-d6) δ : a 12.03 (s, 1H), to 8.41 (s, 1H), 8,21 (s, 1H), 8,01 (d, 1H, J=8,4 Hz), 7,88 (d, 1H, J=8,4 Hz), 7,82 (DD, 1H, J=8,4 Hz), 7,58 (t, 1H, J=8.1 Hz), 7,51 (d, 1H, J=8,4 Hz), 7,46 (t, 1H, J=7,6 Hz), 7,37 (s, 1H), a 4.86 (t, 1H, J=10,8 Hz), of 4.57 (DD, 1H, J=10,8 Hz), to 4.38 (m, 1H), 4,06 (DD, 1H, J=10,8 Hz), 3,86 (DD, 1H, J=ll Hz), 3,41 (broad, 4H), 3,29 (broad, 4H), 2,82 (s, 3H), 2.57 m (s, 3H).

Synthesis of compound (7). A solution of compound 5 (13 mg, 24 µmol) and linker 6 (of 16.9 mg, 31 μmol) in 5% acetic acid in dry methylene chloride (1 ml) is stirred for 30 min at 25°C. the Solvent is fully removed in vacuum and purified by HPLC (column SymmetryPrep C 18, 7 μm, 19 x 150 mm)to give compound 7 (18.5 mg, 81%). MS: calculated for C48H57ClN8O11(M+H) m/z 958,38 found 958,10.

EXAMPLE 7: studies of the proliferation

The biological activity of cytotoxic compounds of the present invention can be studied using well-known study of proliferation3H-thymidine. This is a convenient way to quantify cell proliferation, which measures DNA synthesis by determining the incorporation of exogenous radiochango3H-thymidine. This study is vysokoproizvoditel and may use a large number of connections.

To study cell promyelocytic leukemia HL-60 were cultured in RPMI medium containing 10% fetal calf serum (FCS), deactivated by heating. The day of the test cells are harvested, washed and re-suspended at a concentration of 0.5 x 106 cells/ml in RPMI containing 10% FCS. 100 μl of cell suspension added to 96-cell-based tablets. Make serial dilutions (3-fold increments) doxorubicin (positive control) or the test compounds and add 100 ál of connections per cell. At the end add 10 ál of 100 mccu/ml3H-thymidine per cell and incubated tablets within 24 hours Collect cells in tablets, using 96-ACE CNY harvester (Packard Instruments), and measure on the counter Packard Top Count. Build chetyrehpaltsevye logistic curves to enable3H-thymidine as a function of polyarnosti drugs using the software Prism to estimate IC50.

The compounds of this invention typically have a value IC50in the above-described study from approximately 1 PM to 100 nm, preferably from about 10 PM to 10 nm.

EXAMPLE 8: the Conjugation of molecules of the drug-linker-antibody

This example describes the conditions of interaction and methodology for conjugation of molecules of the drug-linker of the present invention (optionally including other groups, such as spacers, reactive functional groups and the like) with the antibody as the directing agent X4. These terms and methodology are supposed to be only a typical example, and not limitation. In this area there are other approaches to the conjugation of molecules of the drug-linker to the antibody.

The described method of conjugation is based on the introduction of free tylnej groups in antibody interaction lysine antibody with 2-aminothiophenol with subsequent interaction of the molecules of the drug-linker with active maleimide group. First, the antibody to be conjugated, change the buffer 0,1M phosphate buffer, pH 8.0, containing 50 mm NaCl, 2 mm DTPA, pH 8.0, concentri the comfort to 5-10 mg/ml Getting tialata carried out by adding to the antibody 2-aminosilane. Number 2-aminothieno to be added, determined in preliminary experiments and vary from one antibody to another. In preliminary experiments, titration, adding to the antibody of the increasing number of 2-aminothieno, followed by incubation with the antibody for one hour at room temperature, the antibody vymalivayut in 50 mm HEPES buffer, pH to 6.0 using a column of Sephadex G-25, and quickly determine the number entered tylnej groups through interaction with dithiodipyridine (DTDP). As a result of interaction tylnej groups with DTDP there is a release of dipyridine that control at 324 nm. Using samples with a protein concentration of 0.5-1.0 mg/ml For accurate determination of protein concentration in the samples determine the absorbance at 280 nm and then an aliquot of each sample (0.9 ml) incubated with 0.1 ml of DTDP (5 mm initial solution in ethanol) for 10 min at room temperature. Also incubated blank samples containing only buffer plus DTDP. After 10 min, measure the absorbance at 324 nm and determine the number of present thiols, using the extinction coefficient of dipyridine 19800M-1.

Usually requires a degree of etiolirovaniya three tirinya groups on the antibody. For example, for one specific antibodies is and this reach, adding a 15-fold molar excess of 2-aminothieno followed by incubation at room temperature for 1 h Therefore, the subject conjugation of antibody incubated with 2 aminothiophenol when required molar ratio and then vymalivayut in conjugation buffer (50 mm HEPES-buffer, pH of 6.0, containing 5 mm glycine, 3% glycerol and 2 mm DTPA). Etiolirovannye material kept on ice until determine the amount of the thiol, as described above.

After checking the number of input tylnej groups add molecule drug-linker containing the active maleimido group, with 3-fold molar excess to Tilney group. The conjugation reaction is carried out in the conjugation buffer containing a final concentration of 5% dimethyl ether of ethylene glycol (or a suitable alternative solvent). Usually the original solution of the drug-linker dissolved in a mixture of 90% dimethyl ether of ethylene glycol and 10% dimethyl sulfoxide. To add to the antibody can be added to the original solution directly to etiolirovannuyu the antibody that has a sufficient number of added dimethyl ether of ethylene glycol to bring the final concentration to 5%, or pre-diluted in the conjugation buffer containing a final concentration of 10% dimethyl ether of ethylene glycol, followed by relax is of an equal volume tarirovannogo antibodies.

Subject conjugation reaction mixture is incubated at room temperature for 2 h under stirring. After incubation, the reaction mixture was centrifuged at 14000 rpm./min for 15 min and the pH adjusted to 7.2, if not immediately produce cleaning. Purification of the conjugate produced by the method of chromatography, using a number of ways. The conjugate can be cleared by applying the method of size-exclusive chromatography on a column of Sephacryl S200, pre-equilibrated with 50 mm HEPES-buffer, pH of 7.2, containing 5 mm glycine, 50 mm NaCl, and 3% glycerol. The chromatography is performed with a linear flow velocity of 28 cm/PM Fractions containing the conjugate are collected, combined and concentrated. Differently, cleaning can be carried out by the method of ion-exchange chromatography. Conditions vary from one antibody to another, and each case requires optimization. For example, the reaction mixture is conjugate antibody-drug contribute in a column of SP-Sepharose pre-equilibrated in 50 mm HEPES, 5 mm glycine, 3% glycerol, pH of 6.0. Conjugate antibodies elute using a gradient of 0-1 M NaCl in the equilibrating buffer. The fractions containing the conjugate are pooled, adjusted pH to 7.2 and, if necessary, concentrate the sample.

Each one claimed in the patent applications, patents, publications and other published documents mentioned or to which reference was made in the data is m description included here in its entirety by reference, to the extent as if it were specifically and individually indicated that each individual patent application, patent, publication, and another published document is included as a reference.

While the present invention is described with reference to its specific variants, the person skilled in the art should understand that you can make various changes and substitutions on equivalents, without departing from the true spirit and scope of the present invention and appended claims. In addition, it is possible to make many modifications to adapt a particular situation, material, consider the composition, method, stage or stages of the process to the purpose, spirit and scope of the present invention. As expected, all such modifications are included in the scope of the attached claims.

1. The compound of the formula

where D is a
or
X represents O;
Z is selected from O or NH;
R1represents C(O)2R8where R8represents lower alkyl;
R2represents lower alkyl;
R3is a OR11; where
R11represents C(O)NR12R13in which
R12and R13 form a 6-membered geterotsyklicescoe cyclic system, containing two nitrogen atom, optionally substituted lower alkyl;
R4represents NR15where R4connects D with F, and R15selected from H or lower alkyl;
R7denotes CH2-X1where X1represents halogen,
F denotes a linker comprising the structure:

where AA1indicates one or more components, independently selected from the group comprising natural amino acids and synthetic α-amino acids, and AA1represents enzymatically cleaved peptide sequence;
with represents 2, 3 or 4;
o represents 0 or 1;
where, when equal to 1, -L3-NH has a structure selected from the group consisting of


L4denotes a linker component, in which L4does not contain a carboxylic acyl group directly attached to an M-end (AA1)withand L4contains1-6alkyl or C1-6heteroalkyl with the nitrogen atom as the heteroatoms, optionally substituted by =O; and
X4not necessarily associated with the antibody and is a component selected from
and

2. The compound according to claim 1, where 4contains an acyclic fragment.

3. The compound according to any one of claims 1 and 2, where L4increases the solubility of the compounds in comparison with the connection, not having L4.

4. The compound according to any one of claims 1 and 2, where L4reducing the aggregation of connections compared to the connection that does not have L4.

5. The compound according to any one of claims 1 and 2, where L4contains polietilenglikolya fragment.

6. The compound according to claim 5, where polietilenglikolya fragment contains 3-12 duplicate links.

7. The connection according to claim 6, where polietilenglikolya fragment contains 2-6 duplicate links.

8. The connection according to claim 7, where polietilenglikolya fragment contains 4 duplicate link.

9. The compound according to any one of claims 1 and 2 or 6-8, where (AA1)with
represents a peptide sequence that is cleaved by a protease expressed in tumor tissue.

10. The connection according to claim 9, where the protease is a lysosomal protease.

11. The compound according to any one of claims 1 and 2, 6-8, 10, where C is an integer 2 or 3.

12. The compound according to any one of claims 1 and 2, 6-8, 10, where the amino acid (AA1)withlocated closest to the medicinal fragment selected from the group including: Ala, Asn, Asp, Cit, Cys, Gin, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

13. The compound according to any one of claims 1 and 2, 6-8, 10, if (AA1)cpresented yet a peptide sequence, selected from the group comprising Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO:1), β-Ala-Leu-Ala-Leu (SEQ ID NO:2) and Gly-Phe-Leu-Gly (SEQ ID NO:3).

14. The compound according to any one of claims 1 and 2, 6-8, 10, if (AA1)cis a Val-Cit or Val-Lys.

15. The compound according to claim 1, having the structure:

or

16. A compound selected from the group consisting of:




or

where each b independently is an integer from 0 to 20, and where Ab represents the antibody or fragment.

17. The compound according to claim 1, which is selected from the group including:



and

where X denotes Cl or Br, and Ab represents the antibody or fragment.

18. The compound having the structure

where D is a
or
X represents O;
Z is selected from O or NH;
R1represents C(O)2R8where R8represents lower alkyl;
R 2represents lower alkyl;
R3represents-O - and is linked to N;
R4represents the components that are independently selected from the group consisting of OR15and-O(CH2)2NR15R16,
and R15and R16independently selected from hydrogen or lower alkyl; and
R7denotes CH2-X1where X1represents halogen,
X4not necessarily associated with the antibody, and is a component selected from
and
L4denotes a linker component containing polietilenglikolya fragment comprising 2-6 duplicate links;
p is 0 or 1;
N denotes the in vivo degradable linker comprising the structure:

where n1an integer from 1 to 10;
n2is 0, 1 or 2;
each R24is a component that is independently selected from the group comprising H or lower alkyl; and
I refers to any connection, either:

where n3is 0 or 1 provided that when n3is 0, n2not equal to 0; and n4is 1, 2 or 3,
where, if I denotes a bond, n1equal to 3 and n2is 1, then D cannot be:

or

where R is Me or CH2-CH2-NMe2.

19. Connection p, where the substitution on phenyl cycle represents a para-substitution.

20. The compound according to any one of p or 19, where n1is 2, 3 or 4.

21. Connection claim 20, where n1equal to 3.

22. The compound according to any one of p and 19 or 21, where n2is 1.

23. Connection p.22, where I denotes the connection.

24. The compound according to any one of p and 19, 21 or 23, where N forms a 6-membered auto linker at the cleavage.

25. Connection p.22, where n30 and n4equal to 2.

26. The compound according to any one of p and 19 or 21, where N form two 5-membered auto linker at the cleavage.

27. The compound according to any one of p and 19, 21 or 23, where N forms a 5-membered auto linker, N forms a 7-membered auto linker, or N forms a 5-membered auto linker and 6-membered auto linker at the cleavage.

28. The compound according to any one of p and 19 or 21, where H contains the structure:

29. Connection p, where n1is 2, 3 or 4.

30. Connection p, where n1equal to 3.

31. The compound according to any one of p or 30, where each R24independently selected from CH3and N.

32. The compound according to any one of PP and 30, where each R24denotes N.

33. The compound according to any one of p and 1, 21 or 23, where N has the structure:

34. Connection p, where n1equal to 3.

35. Connection p, where each R24independently selected from CH3and N.

36. Connection p, where N has the structure:

37. Connection p, where H contains genialne dimethyl substitution.

38. The compound according to any one of p and 19, 21, 23, 25, 29 and 30 or 34-37, where D denotes the cytotoxic drug.

39. The compound according to any one of p and 19, 21, 23, 25, 29 and 30 or 34-37, where L4contains an acyclic fragment.

40. The compound according to any one of p and 19, 21, 23, 25, 29 and 30 or 34-37, where L4increases the solubility of the compounds in comparison with the connection, not having L4.

41. The compound according to any one of p and 19, 21, 23, 25, 29 and 30 or 34-37, where L4reducing the aggregation of connections compared to the connection that does not have L4.

42. Connection p where polietilenglikolya fragment contains 4 duplicate link.

43. Connection p having the structure:

or

44. Connection p having the structure:

or

45. Connection p having the structure:

or

where PEG is polie lenghly fragment, and X1denotes Cl or Br.

46. Connection p having the structure:

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

47. Connection p, having a structure selected from the group including:
,
,
and

where X1denotes Cl or Br, and Ab represents the antibody or fragment.

48. The compound of the formula

where D is a
or
X represents O;
Z is selected from O or NH;
R1represents C(O)2R8where R8represents lower alkyl;
R2represents lower alkyl;
R3represents-O - and is linked to J;
R4represent the group consisting of OR15and O(CH2)2NR15R16and R15and R16independently selected from hydrogen or lower alkyl; and
R7denotes CH2-X1where X1represents halogen,
X4not necessarily associated with the antibody, and is a component selected from
and
L4denotes a linker component containing FR is gment of polyethylene glycol, including 2-6 duplicate links;
p is 0 or 1;
J denotes the in vivo degradable linker comprising the structure:

where each R24is a component that is independently selected from the group comprising H or lower alkyl; and d is an integer from 0, 1, 2, 3, 4, 5 and 6.

49. Connection p, where J has the structure:

50. Connection § 49, where d is 1 or 2.

51. Connection p, where J has the structure:

52. The compound according to any one of p-51, where D is a cytotoxic drug.

53. The compound according to any one of p-51, where L4contains an acyclic fragment.

54. The compound according to any one of p-51, where L4increases the solubility of the compounds in comparison with the connection, not having L4.

55. The compound according to any one of p-51, where L4reducing the aggregation of connections compared to the connection that does not have L4.

56. Connection p where polietilenglikolya fragment contains 4 duplicate link.

57. Connection p having the structure:

or

58. Connection p, having a structure selected from the group including:
,
,

,

and

where X denotes Cl or Br, and Ab represents the antibody or fragment.

59. The compound having the structure

where D is a
or
X represents O;
Z is selected from O or NH;
R1represents C(O)2R9where R9represents lower alkyl;
R2represents lower alkyl; and
R3is a OR11; where
R11represents C(O)NR12R13in which
R12and R13form a 6-membered geterotsyklicescoe cyclic system containing two nitrogen atom, optionally substituted lower alkyl;
R4attach D to N; and
R7denotes CH2-X1where X1is a halogen;
X4not necessarily associated with the antibody, and R29is a component selected from
and
L4denotes a linker component that represents an unsubstituted lower alkyl;
p is 0 or 1;
N denotes the in vivo degradable linker comprising the structure:

where q is 0, 1, 2, 3, 4, 5 or 6; and
where each R24

60. Connection p, where N forms a 6-membered auto linker decomposition.

61. Connection p, where N form two 5-membered auto linker decomposition.

62. The compound according to any one of p-61, where D is a cytotoxic drug.

63. Connection p having the structure:

or

where Ab represents the antibody or its fragment.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: present invention relates to compounds of formula

, in which A is a counter ion, a=1-3, b=0-3, X=1-6C alkyl, R1=1-6C alkyl, one or R2 and R3 is 1-6C alkyl and the other is XN+Hb(R1)3-b, or R2 and R3 form a methylenedioxy group, one or R4 and R5 is a halogen and the other is a halogen-substituted 1-6C alkyl, or R4 and R5 are bonded to form a 6-10C aromatic ring or a substituted 6-10C aromatic ring in which the substitute is selected from 1-6C alkoxy, halogen and halogen-substituted 1-6C alkyl. The invention also relates to a method of measuring content of analysed substance capable of ensuring proportional colour change as a result of a reaction in a biological fluid, involving the following steps: ensuring availability of the disclosed tetrazolium salt as an indicator and determination of concentration of the said analysed substance in the biological fluid using the said tetrazolium salt which is used as an indicator.

EFFECT: agents are highly effective.

24 cl, 7 dwg, 1 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula or its pharmaceutically acceptable salt, where R1 and R2 each independently denotes a hydrogen atom, a halogen atom, a lower alkyl, a hydroxyl group, a cyano group or a lower alkoxy; R3 independently denotes a hydrogen atom, a halogen atom, a lower alkyl, a lower alkoxy, a hydroxyalkyl, trifluoromethyl, lower alkenyl or cyano group; R4 independently denotes a hydrogen atom, a lower alkyl, a lower alkoxy, a halogen atom, trifluoromethyl, hydroxyalkyl optionally substituted with a lower alkyl, aminoalkyl optionally substituted with lower alkyl, alkanoyl, carboxyl group, lower alkoxycarbonyl or cyano group; Q denotes a nitrogen atom; R5 and R6 each independently denotes a hydrogen atom, a lower alkyl, a halogen atom, a lower alkylsulfonyl, a lower alkylsulfanyl, alkanoyl, formyl, aryl, mono- or di-(lower) alkylcarbamoyl or mono- or di-(lower) alkylsulfamoyl; and further as indicated in the formula of invention. The invention also relates to a glucokinase activator containing the compound in paragraph 1 and to a therapeutic agent based on said compounds.

EFFECT: novel compounds which can be useful in treating and preventing diabetes and obesity are obtained and described.

29 cl, 227 ex, 6 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I and their pharmaceutically acceptable salts. Disclosed compounds have inhibitory effect on CDK1 kinase and can be used to prepare medicinal agents for treating diseases associated with abnormal cell cycle development. In formula I , R1 is hydrogen, -C(O)OR9 or R2-(X)n-; X is (lower)alkylene, hydroxy(lower)alkylene, cyclised(lower)alkylne or mono- or dihalogen(lower)alkylene; R2 is a group, where denotes a phenyl or a 5-6-member heteroaromatic ring containing 1-2 heteroatoms selected from a group comprising oxygen, sulphur and nitrogen atoms; R5, R6 and R7 are independently selected from a group comprising hydroxy, hydrogen, (lower)alkyl, halogen and (lower)alkoxy; R4 is a halogen, , (O)k(CH2CH2O)y-R10, , -S-R12 or -O-(CH2)tR14, where denotes a phenyl, a cycloalkyl ring containing 3-6 carbon atoms, a 4-6-member heterocycloalkyl containing 3-5 carbon atoms and 1-2 heteroatoms selected from a group comprising oxygen, nitrogen and sulphur atoms; R9, R11, R15 and R16 independently denote (lower)alkyl; R10 and R12 denote (lower)alkyl; R14 denotes perfluoro(lower)alkyl or -NR15R16; R17 and R18 independently denote hydrogen, , F, OCH3 and -C(=O)CH3; n and k are equal to 0 or 1; m, w, y and z are equal an integer from 0 to 3; and t equals an integer from 0 to 6.

EFFECT: invention also relates to a pharmaceutical composition having antiproliferative activity, containing one or more of the disclosed compounds.

65 cl, 1 tbl, 49 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of general formula (I): wherein dashed lines present single or double bonds, and the values of radicals R1, R2, R3, R4 are described in cl. 1 of the patent claim. Besides the invention refers to application and a based pharmaceutical composition for prevention and treatment of neurodegenerative diseases and other diseases wherein cell dystrophy and/or cell loss (apoptosis) caused by free radicals act the main part.

EFFECT: production of new compounds and the based pharmaceutical composition which can find application in medicine for prevention and treatment of neurodegenerative diseases.

6 cl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of general formula I and to their pharmaceutically acceptable acid addition salts. The compounds of the present invention exhibit the properties of glycine carrier 1 (GlyT-1) inhibitors. In formula I , R1 represents -OR1', -SR1' or morpholinyl; R1' represents lower alkyl, halogen-substituted lower alkyl, or represents -(CH2)n-lower cycloalkyl; R2 represents -S(O)2-lower alkyl, -S(O)2NH-lower alkyl, NO2 or CN; X1 represents CR3 or N; X2 represents CR3' or N; R3/R3' independently represent hydrogen, halogen, lower alkyl, CN, NO2, -S(O)2-phenyl, -S(O)2-lower alkyl, -S(O)2-pyridine-2, 3 or 4-yl, phenyl optionally substituted with one or two substitutes specified from the group consisting of NO2 or halogen, or represent halogen-substituted lower alkyl, or represent -C(O)-lower alkyl; n has a value of 0, 1 or 2. The invention also concerns a drug containing one or more compounds of the invention and pharmaceutically appropriate excipients.

EFFECT: preparation of the compounds exhibiting the properties of glycine carrier inhibitors.

20 cl, 1 tbl, 133 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I) , where R1 is selected from group, including: phenyl, unsubstituted or mono-, di- or tri-substituted independently with lower alkyl, lower alkoxy group, halogen or lower halogenalkyl; naphtyl; tetrahydronaphtyl; C3-7cycloalkyl; -(CHR3)m-phenyl, where m stands for 1, 2, or 3; and phenyl is unsubstituted or mono-, di- or tri-substituted with lower alkoxy group, and where R3 is independently selected from hydrogen and lower alkyl; -(CH2)n-heteroaryl, where n stands for 1, 2 or 3; term "heteroaryl" relates to aromatic 5- or 6- member ring or bicyclic 9-member aromatic groups, which can include 1, 2 or 3 atoms, selected from nitrogen and/or sulphur; -(CH2)n-heteroaryl, where n stands for 1, 2 or 3; term "heteroaryl" relates to aromatic 5- or 6- member ring or bicyclic 9-member aromatic groups, which can include 1, 2 or 3 atoms, selected from nitrogen and/or sulphur, and heteroaryl is mono-, di- or tri-substituted independently with lower alkoxy group; and R2 is selected from group including: n-butyl; phenyl, unsubstituted or mono-, di- or tri-substituted independently with lower alkyl, halogen or lower alkoxy group; heteroaryl, where term "heteroaryl" relates to aromatic 5-member ring, which can include 1, 2 or 3 atoms, selected from nitrogen and/or sulphur; unsubstituted or mono-, di- or tri-substituted independently with lower alkoxy group; -C(O)-NR4R5; where R4 and R5 stand for lower alkyl or together with nitrogen atom, to which they are bound, form 5-member heterocycle, which can additionally contain heteroatom, selected from N or S, and to their pharmaceutically acceptable salts. Invention also relates to pharmaceutical composition.

EFFECT: obtaining novel biologically active compounds, able to inhibit DPP-IV.

13 cl, 43 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I)

, pharmaceutical compositions based on the said compounds, as well as methods of using said compounds in preparing medicinal agents.

EFFECT: obtaining compounds and a composition which can inhibit phosphatase cdc25, particularly phosphatase cdc25-C and can be particularly used for treating cancer.

12 cl, 56 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula I and their pharmaceutically acceptable salts. The disclosed compounds have inhibitory effect on CDK1 kinase. In formula I , R1 is hydrogen or R2-(X)n-; X is a lower alkylene or cyclic lower alkylene; R2 denotes ; where denotes phenyl; cycloalkyl containing 3-6 carbon atoms; 4-6-member heterocycloalkyl ring having 3-5 carbon atoms and 1-2 oxygen atoms; R5, R6 and R7 are independently selected from a group containing hydrogen or halide; R4 is hydrogen or -(O)k(CH2CH2O)y-R10; R19 is hydrogen; R20 is hydrogen or -C(O)-R11; R10 and R11 is a lower alkyl; n and k are equal to 0 or 1; y is an integer from 0 to 3.

EFFECT: obtaining a pharmaceutical composition with inhibitory effect on CDK1 kinase, containing one or more of the disclosed compounds.

15 cl, 10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to compounds of formula I or to pharmaceutically acceptable salts thereof, where Ar is imidazole or pyrazole, where the said Ar can be substituted with substitute(s) selected from a group consisting of a C1-C6 alkyl group, a phenyl group and a halogen atom, each of Y1, Y2 and Y3 is a carbon ot nitrogen atom, A is an oxygen atom, a sulphur atom or a group of formula -SO2-, R1 is a hydrogen atom, a C1-C6 alkyl group which can be substituted with one phenyl group (where the said phenyl group can be substituted with one substitute selected from a group consisting of a halogen atom and a C1-C6 alkyl group), or a phenyl group, R2 is a C1-C6 alkyl group, R3 is (i) a C1-C18 alkyl group, (ii) C2-C8 alkenyl group, (iii) C2-C8 alkynyl group, (iv) C3-C8 cycloalkyl group, (v) C1-C6 alkyl group substituted with 1-3 substitutes selected from a group given in paragraph 1 of the formula of invention, or (vi) a phenyl group, a naphthyl group, a pyrazolyl group, a pyridyl group, an indolyl group, a quinolinyl group or an isoquinolinyl group, where each of the said groups can be substituted with 1-3 substitutes selected from a group given in paragraph 1, R4 is a hydrogen atom or a C1-C6 alkyl group, and R5 is (i) C1-C10 alkyl group, (ii) C1-C10 alkyl group which is substituted with one or two substitutes selected from a group given in paragraph 1, (iii) C2-C8 alkenyl group which can be substituted with a phenyl group, or (iv) phenyl group, naphthyl group, thienyl group, pyrrolyl group, pyrazolyl group, pyridyl group, furanyl group, benzothienyl group, isoquinolinyl group, isoxazolyl group, thiazolyl group, benzothiadiazolyl group, benzoxadiazolyl group, phenyl group, condensed with a 5-7-member saturated hydrocarbon ring which can contain one or two oxygen atoms as ring members, uracyl group or tetrahydroisoquinolinyl group, where each of the said groups can be substituted with 1-5 substitutes selected from a group given in paragraph 1, provided that when Ar is a group of formula 5, which can be substituted with a C1-C6 alkyl group, R5 is not a C1-C10 alkyl group, and the formula (I) compound is not 5-(3,5-dichlorophenylthio)-4-isopropyl-2-methane-sulfonylaminomethyl-1-methyl-1H-imidazole or 5-(3,5-dichlorophenylthio)-4-isopropyl-1-methyl-2-p-toluene-sulfonylaminomethyl-1H-imidazole. The invention also relates to a pharmaceutical composition based on the formula I compound and to formula II compounds, radicals of which are defined in the formula of invention.

EFFECT: obtaining novel compounds with inhibitory effect on the bond between S1P and its Edg-1 (SIP1) receptor.

32 cl, 43 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel benzene derivatives of general formula (I) or salts thereof: [Chem. 12]

(Symbols in the given formula have the following values X1:-NR12-C(=O)- or -C(=O)-NR12-, X2 : -NR13 -C(=O)-, Ring A is a 6-member ring, if necessary having 1 or 2 double bonds and if necessary having 1-3 heteroatoms selected from N, O, Ring B is a benzene ring or a 6-member heteroaryl ring having 1-3 heteroatoms selected from N, R is a hydrogen atom or a residue of β-D- glucopyranoside uronic acid; R1-R8 are identical or different and each denotes a hydrogen atom, a halogen atom, -O-(lower alkyl), R9-R11 are identical or different and each denotes a hydrogen atom, lower alkyl, -O-(lower alkyl), -(CH2)n-N(lower alkyl)2, -(CH2)n-NH(lower alkyl), -(CH2)n-N(lower alkyl) (if necessary substituted with -C=O; a 6-member heterocycle having 1-3 heteroatoms selected from N, S, O) -(CH2)n-(C=O)-N(lower alkyl)2, -(CH2)n-(C-O)-N(lower alkyl) (if necessary substituted with -C=O, alkyl, a 6-member heterocycle having 1-3 heteroatoms selected from N) -(CH2)n- if necessary substituted with alkyl, -COCH3, -SO2CH3, -COOCH3, -C=O, CF3, -OCH3, OH, halogen; 5-7-member heterocycle having 1-3 heteroatoms selected from N, S, O), -(CH2)n-O- (if necessary substituted with alkyl; 6-member heterocycle having 1-3 heteroatoms selected from N), n is an integer from 0 to 3, R12 and R13 denote a hydrogen atom, provided that in R1-R11, when two lower alkyls are bonded to a nitrogen atom, they can together form a 3-8-member nitrogen-containing heterocycle.) The invention also relates to benzene derivatives of general formula (II), to a pharmaceutical composition, as well as to use of the said compounds.

EFFECT: obtaining novel biologically active compounds which are active as inhibitors of activated blood-coagulation factor X.

16 cl, 365 ex, 42 tbl

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry, and specifically to compounds of general formula I , where A is an oxygen atom, an alkylene, alkenyl or hetero alkylene group, in which the CH2 group is substituted with a NH group, where the said groups can be optionally substituted with OH, =O or CH2OH groups, X1, X2, X3, X4 and X5 independently represent nitrogen atoms or groups of formula CH or CR4, Cy is cycloalkylene or heterocycloalkylene group containing at least one nitrogen atom, R1 is a hydrogen atom, an alkyl or alkyloxy group, R2 is a halogen atom, a hydroxy group, an alkyl or heteroalkyl residue, where the said groups can be optionally substituted with OH, NH2 groups and/or a =O group, R3 is a group of formula -B-Y, in which B denotes an alkylene, alkenyl or heteroalkylene group, where the said groups can be optionally substituted with OH, NH2, COOH groups or a =O group, and Y is an optionally substituted phenyl, optionally substituted heteroaryl group containing 5 or 6 ring atoms, or an optionally substituted bicyclic heterocycle in which one ring is phenyl or pyridyl, and the other is a 5-, 6- or 7-member heteroaryl or heterocycloalkyl group which contains up to 3 heteroatoms selected from nitrogen, oxygen and sulphur atoms, R4 is a halogen atom, n equals 0, 1 or 2 and m equals 0 or 1, or their pharmaceutically acceptable salts, solvates and hydrates. The invention also relates to a pharmaceutical composition based on the formula I compound and use of the compound or the pharmaceutical composition to treat bacterial infections.

EFFECT: obtaining novel compounds possessing useful biological properties.

12 cl, 7 ex

FIELD: chemistry.

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

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

14 cl, 2 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula (I') which have inhibitory effect on ALK kinase: , where n' is selected from 1 and 2; R'2 is selected from halogen; R'3 is selected from -S(O)2NR'5R'6, -S(O)2R'6 and -C(O)NR'5R'6, where R'5 is selected from hydrogen and C1-6alkyl, and R'1 is selected from C1-6alkyl; and R'1 is selected from phenyl which is substituted with 3 radicals independently selected from C2-6alkoxy group, C1-6alkyl, -X'R'4 and -OXR'4, where X' denotes a bond, and R'4 is selected from piperazinyl, piperidinyl, pyrrolidinyl, morpholino, where R'4 can be optionally substituted with 1-3 radicals independently selected from C1-6 alkyl, provided that the following compound is excluded .

EFFECT: design of a method of inhibiting and using compounds for making a medicinal agent for treating diseases which respond to ALK kinase inhibition.

7 cl, 61 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel benzene derivatives of general formula (I) or salts thereof: [Chem. 12]

(Symbols in the given formula have the following values X1:-NR12-C(=O)- or -C(=O)-NR12-, X2 : -NR13 -C(=O)-, Ring A is a 6-member ring, if necessary having 1 or 2 double bonds and if necessary having 1-3 heteroatoms selected from N, O, Ring B is a benzene ring or a 6-member heteroaryl ring having 1-3 heteroatoms selected from N, R is a hydrogen atom or a residue of β-D- glucopyranoside uronic acid; R1-R8 are identical or different and each denotes a hydrogen atom, a halogen atom, -O-(lower alkyl), R9-R11 are identical or different and each denotes a hydrogen atom, lower alkyl, -O-(lower alkyl), -(CH2)n-N(lower alkyl)2, -(CH2)n-NH(lower alkyl), -(CH2)n-N(lower alkyl) (if necessary substituted with -C=O; a 6-member heterocycle having 1-3 heteroatoms selected from N, S, O) -(CH2)n-(C=O)-N(lower alkyl)2, -(CH2)n-(C-O)-N(lower alkyl) (if necessary substituted with -C=O, alkyl, a 6-member heterocycle having 1-3 heteroatoms selected from N) -(CH2)n- if necessary substituted with alkyl, -COCH3, -SO2CH3, -COOCH3, -C=O, CF3, -OCH3, OH, halogen; 5-7-member heterocycle having 1-3 heteroatoms selected from N, S, O), -(CH2)n-O- (if necessary substituted with alkyl; 6-member heterocycle having 1-3 heteroatoms selected from N), n is an integer from 0 to 3, R12 and R13 denote a hydrogen atom, provided that in R1-R11, when two lower alkyls are bonded to a nitrogen atom, they can together form a 3-8-member nitrogen-containing heterocycle.) The invention also relates to benzene derivatives of general formula (II), to a pharmaceutical composition, as well as to use of the said compounds.

EFFECT: obtaining novel biologically active compounds which are active as inhibitors of activated blood-coagulation factor X.

16 cl, 365 ex, 42 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (I) , where D is pyrrolidine, X is a radical selected from a group containing C=O, SO2, (C=O)-NH, CH2, O-(C=O), (C=O)-O, (C=S)-NH, Y is a radical selected from a group containing - (CH2)n-E-(CH2)m-L-(CH2)k, where E is O, k, m and n are separately and independently equal to 0, 1, 2 and 3, Z is an alkyl, where the alkyl is CH2 or CH2CH2, A is a radical selected from a group containing benzyl, substituted benzyl, phenyl, substituted phenyl, alkyl and substituted alkyl, cycloalkyl, heterocyclyl, aryl, alkyloxyalkyl, substituted alkyloxyalkyl, alkyloxyaryl, B is a radical of formula (II) , where R1 is selected from a group containing H, alkyl and substituted alkyl, cycloalkyl, R2 is selected from a group containing H, benzyl, substituted benzyl, phenyl, substituted phenyl, alkyl and substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, cycloalkylalkyl, substituted cycloalkylalkyl, G is an amino group which is substituted by a heteroaryl or substituted heteroaryl, and where Q and L each independently denotes a radical selected from a group containing (C=O)-NH, NH, CH2, NH-(C=O)-NH, NH-(C=O), NH-SO2, NRc, (C=O)-NRc, and where Ra, Rb, Rc and Rd each independently denotes a radical selected from a group containing H, alkyl, cycloalkyl, heterocyclyl, aryl, substituted aryl, as well as to use of these compounds as integrin inhibitor and for making a medicinal and a diagnostic agent, to a pharmaceutical composition based on these compounds and to methods of treating integrin-associated condition.

EFFECT: novel compounds which are integrin inhibitors and can be used to treat diseases where angiogenesis inhibition is necessary are obtained and described.

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula I , where R1 is selected from a group comprising hydrogen, lower alkyl, cycloalkyl or lower cycloalkylalkyl, where the cycloalkyl ring can be substituted with lower alkoxyalkyl, lower alkoxyalkyl, and tetrahydropyranyl and lower heterocyclylalkyl, where the heterocyclic ring is oxetanyl or tetrahydropyranyl, which can be substituted with a halogen; R2 is selected from a group comprising hydrogen, lower alkyl, cycloalkyl or lower cycloalkylalkyl, where the cycloalkyl ring can be substituted with lower alkoxyalkyl, lower alkoxyalkyl, and tetrahydropyranyl or lower heterocyclylalkyl, where the heterocyclic ring is oxetanyl or tetrahydropyranyl which can be substituted with a halogen; or R1 and R2 together with the nitrogen atom to which they are bonded form a 4-, 5- or 6-member saturated or partially unsaturated heterocyclic ring which optionally contains the same heteroatom selected from oxygen or sulphur, where the said saturated or partially heterocyclic ring is unsubstituted or substituted with one or two groups independently selected from a group consisting of lower alkyl, halogen, halogenalkyl, cyano group, hydroxy group, lower hydroxyalkyl, lower alkoxy group, oxo group; A is selected from , and , where m equals 0 or 1; R3 is a lower alkyl; n equals 0; R4 is a lower alkyl; p equals 1; q equals 0, 1 or 2; R5 is hydrogen; and their pharmaceutically acceptable salts. The invention also relates to a pharmaceutical composition based on formula I compounds.

EFFECT: new quinoline derivatives are obtained, which have antagonistic effect on histamine 3 receptors (H3 receptors).

18 cl, 4 tbl, 86 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to benzazepin derivatives of formula (I), where R1 is unsubstituted cyclobutyl, R2 is 3-pyrazinyl, substituted CON(H)(Me) or 2-pyridinyl-M-pyrrolidinyl, where the said pyrrolidinyl group is substituted with a =O group; which is: methylamide 5-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy) pyrazine-2-carboxylic acid

or 1-{6-[(3-cyclbutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-3-pyridinyl}-2-pyrrolidinone

EFFECT: obtaining compounds which have affinity to histamine H3 receptor and pharmaceutical compositons containing said compounds.

11 cl, 288 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula

,

where the carbon atom denoted * is in R- or S-configuration; X is a concentrated bicyclic carbocycle or heterocycle selected from a group consisting of benzofuranyl, benzo[b]thiophenyl, benzoisothiazolyl, indazolyl, indolyl, benzooxazolyl, benzothiazolyl, indenyl, indanyl, dihydrobenzocycloheptenyl, naphthyl, tetrahydronaphthyl, quinolinyl, isoquinolinyl, quinoxalinyl, 2H-chromenyl, imidazo[1.2-a]pyridinyl, pyrazolo[1.5-a]pyridinyl, and condensed bicyclic carbocycle or condensed bicyclic heterocycle, optionally substituted with substitutes (1 to 4) which are defined below for R14; R1 is H, C1-C6-alkyl, C3-C6-cyclalkyl, C1-C3-alkyl, substituted OR11, -NR9R10 or -CN; R2 is H, C1-C6-alkyl, or gem-dimethyl; R3 is H, -OR11, C1-C6-alkyl or halogen; R4 is H, halogen, -OR11, -CN, C1-C6-alkyl, C1-C6-alkyl, substituted -NR9R10, C3-C6-cycloalkyl, substituted -NR9R10, C(O)R12; or R4 is morpholinyl, piperidinyl, pyrimidinyl, pyridazinyl, pyrazinyl, pyrrolyl, isoxazolyl, pyrrolidinyl, piperazinyl, 2-oxo-2H-pyridinyl, [1.2.4]triazolo[4.3-a]pyridinyl, 3-oxo-[1.2.4]triazolo[4.3-a]pyridinyl, quinoxalinyl, which are optionally substituted with substitutes (1 to 4) which are defined below for R14; R5 is H or C1-C6-alkyl; R6 is H, C1-C6-alkyl, or -OR11; R7 is H; R8 is H, -OR9, C1-C6-alkyl, -CN; R9 is H or C1-C4-alkyl; R10 is H or C1-C4-alkyl; or R9 and R10 taken together with the nitrogen atom to which they are bonded form morpholine; R11 is H, C1-C4-alkyl; R12 is C1-C6-alkyl; R14 in each case is independently selected from a substitute selected from a group consisting of halogen, -OR11, -NR11R12, C1-C6-alkyl, which is optionally substituted with 1-3 substitutes, in each case independently selected from a group consisting of C1-C3-alkyl, aryl; or to pharmaceutically acceptable salts thereof. The invention also relates to a pharmaceutical composition, to a method of obtaining formula (I) compounds, as well as to a method of treating disorders.

EFFECT: obtaining new biological active compounds having norepinephrine, dopamine and serotonin reuptake selective inhibitory activity.

90 cl, 162 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula I

where A, B and D each denotes N or CR5, where one of A, B and D denotes N, R1 denotes OR6, R2 denotes halogen, C1-C4alkyl, halogen(C1-C4)alkyl or OR7, R3 denotes a heteroarylalkyl group in which the heteroaryl fragment contains 5-6 atoms in the ring, at least one of which is an N atom, and the alkyl fragment with a branched or straight chain contains 1-5 carbon atoms, R4 denotes C3-C10cyclalkyl, C6-C14aryl, unsubstituted or substituted with one or more substitutes selected from a group comprising halogen, alkoxy, terazol-5-yl, 2-(heterocyclyl)tetrazol-5-yl or a carboxy group; heteroaryl containing 5-6 atoms in the ring; heterocyclic group saturated or partially saturated, containing 5-6 atoms in the ring, at least one of which is an N atom, unsubstituted or substituted with one or more substitutes selected from a group comprising alkoxy, alkoxyalkoxy, oxo, alkoxycarbonyl, alkylsulfanyl, alkylsufonyl or phenylsulfonyl; R5 denotes H; R6 denotes H or C1-C4alkyl with a branched or straight chain, unsubstituted or substituted with one or more halogens, R7 denotes H or C1-C12alkyl with a branched or straight chain, unsubstituted or substituted with one or more substitutes selected from a group which includes halogen; C3-C10cyclalkyl; saturated heterocyclic group containing 5-6 atoms in the ring, at least one of which is an O atom, or a heterocylcylalkyl group in which the heterocyclic fragment is saturated, partially saturated or unsaturated and contains 5-10 atoms in the ring, at least one of which is an O atom; or to a pharmaceutically acceptable salt thereof, as well as to a pharmaceutical composition for inhibiting PDE4 enzyme activity and to use of the said compound to prepare a medicinal agent.

EFFECT: novel compounds which can be used in medicine are obtained and described.

65 cl, 17 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel therapeutically suitable derivatives of pyridazin-3(2H)-one of formula and pharmaceutical compositions containing the said derivatives. These compounds are used for treating, preventing or inhibiting corresponding pathological conditions, diseases or disorders, mainly asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, atopic dermatitis, psoriasis or irritable colon syndrome.

EFFECT: obtaining compounds which are active and selective phosphodiesterase 4 (PDE4) inhibitors.

11 cl, 1 tbl, 182 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel pyrimidine derivatives having FAK inhibitory activity of formula (I), where R0 is hydrogen; R1 is a 5- or 6-member heterocycle containing 1 or 2 nitrogen atoms substituted with (C1-C7)alkyl, hydroxyl group, dialkylamino group or a 6-member heterocycle containing one nitrogen atom; R2 is hydrogen; R3 is carbamoyl substituted once or twice with (C1-C7)alkyl; a 5-member heterocycle containing 4 nitrogen atoms; SO2N(R12)R13, where R12 is hydrogen or (lower)alkyl, and R13 is hydrogen, (C1-C7)alkyl, (C1-C7)alkoxy(C1-C7)alkyl, di(C1-C7)alkylamino(C1-C7)alkyl, hydroxy(C1-C7)alkyl, or R12 and R13 together a nitrogen atom with which they are bonded form a 6-member heterocycle containing two nitrogen atoms, where the said heterocycle is not substituted or substituted with (C1-C7)alkyl; R4 is hydrogen; R5 is a halide; R6 is hydrogen; R7 is hydrogen; (C1-C7)alkoxy; carbamoyl which is not substituted or substituted with (lower)alkyl; a 5- or 6-member heterocycle containing 1 or 2 nitrogen or oxygen atoms, unsubstituted or substituted with di(C1-C7)alkylamino, (C1-C7)alkyl, hydroxy, 6-member heterocycle containing 1 or 2 nitrogen or oxygen ring atoms, unsubstituted or substituted with (C1-C7)alkyl; 6-member heterocycle-oxy containing 1 nitrogen ring atom, unsubstituted or substituted with (C1-C7)alkyl; heterocycle(C1-C7)alkyloxy, where heterocycle denotes a 5- or 6-member heterocycle containing 1 or 2 nitrogen or oxygen ring atoms which is not substituted or substituted with (C1-C7)alkyl; R8 is hydrogen; halide; (C1-C7)alkoxy, carbamoyl unsubstituted or substituted with (C1-C7)alkyl; heterocycle(C1-C7)alkyloxy, where heterocycle denotes a 5-member heterocycle containing 1 nitrogen ring atom, unsubstituted or substituted with (C1-C7)alkyl; 5- or 6-member heterocycle containing 1 or 2 nitrogen or oxygen atoms, unsubstituted or substituted with one or two substitutes independently selected from hydroxy, (C1-C7)alkyl, aminocarbonyl and (C1-C7)alkylamino; 6-member heterocycle-oxy, containing 1 nitrogen ring atom, unsubstituted or substituted 1-5 times with (C1-C7)alkyl or di(C1-C7)alkylamino; or R7 and R8 together with atoms with which they are bonded form a 6-member heterocycle containing two nitrogen or oxygen atoms, unsubstituted or substituted once or twice with (C1-C7)alkyl or oxo group; R9 is hydrogen; R10 is (C1-C7)alkoxy, as well as to their pharmaceutically acceptable salts. The invention also relates to a pharmaceutical composition and synthesis method.

EFFECT: novel compounds have useful biological activity.

4 cl, 167 ex

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