Released polymer conjugates based on aliphatic biologically decomposing agents

FIELD: chemistry.

SUBSTANCE: activated polymer derivatives of bicin are described as well as conjugates obtained with the aid of the derivates. Also modes of obtaining and application of the bicin derivatives are described.

EFFECT: obtaining of bicin derivatives.

24 cl, 11 dwg, 2 tbl, 21 ex

 

The SCOPE TO WHICH the INVENTION RELATES

The present invention relates to branched polymers applicable to increase the circulation time of biologically active substancesin vivo. This invention also relates to conjugates made with these polymers.

PREREQUISITES TO the CREATION of INVENTIONS

Some of the original concept of "attachment" of the peptides or polypeptides to poly(ethylene glycol) PEG and similar water-soluble poly(alkalinized) are disclosed in U.S. patent No. 4179337, the description of which is incorporated herein by reference. Polypeptides, modified these polymers exhibit lower immunogenicity/antigenicity and circulate in the bloodstream for a longer time than unmodified versions.

For conjugation of poly(alkalisation) one of the end hydroxyl groups converted into reactive functional group. This process is often referred to as "activation", and the product is called "activated poly(alkalization)". Other, essentially pantagonia polymers, "activate" or functionalitywith this way.

Activated polymers are subjected to interaction with therapeutic agents having a nucleophilic functional groups, which serve as the area accession. One nucleophilic function the national group, usually used as the area of attachment, are ε-amino group of lysine. Free carboxylic groups, suitably activated carbonyl groups, oxidized carbohydrate part of the molecule and mercaptopropyl can also be used as a scope attachment.

Insulin and hemoglobin are among the first conjugated therapeutic agents. These relatively large polypeptides contain a few free ε-amino areas join. Could be attached sufficient number of polymers to reduce immunogenicity and increase circulation without significant loss of biological activity.

However, it was found that excessive conjugation of polymers and/or conjugation involving therapeutically active part of the molecule, which groups are associated with biological activity, often leads to loss of activity and, consequently, to the loss of therapeutic efficacy. This often happens with peptides of low molecular weight, which have a small number of areas of joining, not related to biological activity. Many ones medicines also lacks a sufficient number of regions joining to obtain a favorable modification of the polymer.

One option to overcome the texts of the above problems is the use of a longer polymers with higher molecular weight. Depending on the desired molecular weight, these substances can be difficult to obtain and expensive to use. In addition, they sometimes give a slight improvement compared to the more readily available polymers.

An alternative proposal is the attachment of two chains of polymer through the triazine ring to the amino groups of the protein. See, for example, Enzyme, 26, 49-53 (1981) Proc. Soc. .. Biol. Med., 188, 364-9 (1988). Triazine, however, is a toxic substance, the contents of which are difficult to reduce to acceptable levels after conjugation. In addition, the triazine is a planar group and can be substituted only with double polymer. A flat structure is rigidly fixed at the two chains of polymer. This limits the advantages of polymer conjugation to approximately the same degree, which is achieved by increasing the length of the polymer chain. Thus, polymers, activated without the presence of triazine, would make a significant contribution to the state of the art.

In the above cases, however, the biologically active polymer conjugates were obtained in the presence of essentially stable to hydrolysis links (bridges) between the polymer and the source of biologically active molecules. Thus, there were obtained durable conjugates, which are rather permanently bound than the prodrugs of the AK ones (where the original molecule is eventually released in vivo.

In public, U.S. patent No. 5643575, 5919455 and 6113906 described additional improvements related to megacephaly PEG having a common connection point to the nucleophile through aliphatic linker. Unlike earlier branched polymer conjugates based on triazine, aliphatic linkers provide an opportunity to avoid the toxic effects of triazine, and also give other benefits. In addition, over the years, have also been proposed some methods of making prodrugs. Prodrugs include chemical derivatives of biologically active parent compound, which, when introduced, will, ultimately, to release the active parent compoundin vivo.The use of prodrugs allows you to change the start and/or duration of action of biologically active compoundsin vivo.Prodrugs are often biologically inert or essentially inactive form of the original or active compounds. The rate of release of the active drug may be affected by several factors, including the rate of hydrolysis of the linker that connects the source of the biologically active compound with a carrier prodrugs.

Have been described, some prodrugs based on the ester or phosphate linkages. In most cases, mainly complex is oevery communication type, used to obtain prodrugs, provides t1/2for hydrolysis up to several days in the aquatic environment. Although one might expect that received a prodrug, the majority of conjugates excreted from the body to achieve a sufficient degree of hydrolysis ofin vivo.Thus, it would be preferable to provide prodrugs that have a connection, enabling more rapid hydrolysis regard, the polymer-drugin vivoorder the original drug connection was formed faster.

Also described prodrugs based on amide or urethane linkages. In General, it is known that the amide bond is highly resistant to hydrolysis. However, recently it was found that C-terminal amides ε-amino acids readily undergo hydrolysis at 25°C and a pH of 7.4 when the N-end N-gidroksietilirovanny one or two hydroxyethylene groups. Bis N-2-hydroxymethylglycinate (bichenovii) type of molecules is the key for such hydrolysis reactions. Such groups bisinuosa type have recently been used in the synthesis of prodrugs, see public application for the grant of a U.S. patent 10/218,167, the contents of which are incorporated herein by reference.

There is still space for improvement in the development of prodrugs. The present invention relates to the AKOM improvement.

SUMMARY of the INVENTION

In one aspect this invention relates to compounds of formula (I):

where R1and R2independently selected from the group consisting of essentially neoantigenic polymer residues, H, C1-6Akilov, C2-6alkenyl, C2-6the quinil, aralkyl and terminal branching groups, provided that R1and R2both are not H;

Z is selected from parts of the molecules are actively transported into the target cell, hydrophobic parts of the molecule bifunctional linking parts of the molecules and their combinations;

Y1-3may be the same or different and selected from O, S or NR11;

L1and L2may be the same or different bifunctional linkers;

R3-R11, R24and R25may be the same or different and selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6the quinil, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic;

L3and L 4may be the same or different and selected from:

-C(O)(CR30R31)Y15(CR32R33)C(O)- or

-C(O)(CR30R31)(CR32R33)C(O)-

where Y15selected from O, S, NR34or CH2and

R30-34may be the same or different and selected from H, alkyl, alkenyl, quinil, heteroalkyl or aryl;

A is selected from the leaving groups, functional groups, biologically active parts of the molecule and OH;

a, b, c, d and e are independently 0 or 1,

m, n, o and p independently represent positive integers

f and g are 0 or 1, provided that

at least one of (f + a) or (g + c) equals 2.

Another aspect of the present invention includes bifunctional compounds formed when at least one of R1) and (R2) represents a polymeric residue, including alpha and the omega end of the binder group. In this aspect of the present invention it is possible to attach two equivalent biologically active component of the medicinal product, the protein, polypeptide, oligonucleotide, etc. to the polymer (preferably PEG) bichenovii system. An example of such a bifunctional polymer conjugate is illustrated below as formula (IIa) and (IIb):

where Z represents an where Y4represents O, S or NR11and L5represents a bifunctional bridge, and all the other options described above.

Also describes how to obtain the compounds of the present invention and methods of treatment using them.

For the purposes of the present invention, it is understood that the term "residue" refers to the connection part, to which it refers, which remains after conducting a substitution reaction in which the carrier is attached part of the polymeric prodrugs.

For the purposes of the present invention it is clear that each term "polymer residue" or "PEG balance" means the portion of the polymer or PEG, which remains after the reaction with a biologically active compound.

For the purposes of the present invention it is clear that the term "alkyl" includes straight, branched, substituted, for example halo-, alkoxy-, nitro-, C1-2alkali, C3-8cycloalkyl or substituted cycloalkyl, etc.

For the purposes of the present invention, it is clear that "substituted" includes the addition or substitution of one or more atoms in the functional group or join one or more different atoms.

For the purposes of the present invention substituted alkali include carboxyacid, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethyl; the replacement of the military of alkenyl, including carboxyaldehyde, aminoalkyl, dialkanolamine, hydroxyalkyl and mercaptoethanol; substituted alkinyl, including carboxykinase, aminoalkyl, dialkanolamine, hydroxyalkyl and mercaptoethanol; substituted cycloalkyl, including those parts of the molecule, as a 4-chlorocyclohexane; arily, including those parts of the molecule, as naphthyl; substituted arily, including those parts of the molecule, such as 3-bromo-phenyl; aralkyl, including such items as toluyl; heteroalkyl, including such parts as ethylthiophene; substituted heteroalkyl, including the number of such parts, such as 3-methoxy-thiophene; alkoxy includes such parts of a molecule as methoxy; and phenoxy includes such parts of a molecule, such as 3-nitrophenoxy. It is clear that " halo " includes fluorine, chlorine, iodine and bromine.

The term "sufficient" for the purposes of the present invention will indicate the amount that provides a therapeutic effect in the sense as understood by the person skilled in the field.

For the purposes of the present invention will be clear that the "essentially neoantigenic" and "essentially neoantigenic" includes all polymeric materials, which in this area means non-toxic and do not cause appreciable immune response in mammals.

For the purposes of the present invention will be clear that the "positively the e integer" will denote a positive integer, preferably about 1 to 6, and more preferably 1 or 2.

One main advantage of the present invention is that bichenovii the linker gives the possibility to control the rate of hydrolysis of the prodrug, preferably releasing components in pure form with different speedsin vivoandin vitro.For example, various bifunctional part of the molecule, including the remains of amino acids or short peptides, can be included as part of any of the L1-3to change the speed of hydrolysis of the prodrugs and/or entry into the cell, and so onin vivoandin vitro.

Another advantage of this invention lies in the fact that these target compounds delivered via polymeric transport system, often show a marked increase in solubility in water and the duration of the circulation in vivo.

BRIEF DESCRIPTION of DRAWINGS

Figure 1-11 schematically presents methods for obtaining the compounds of the present invention, which is described in detail in the description and the examples.

DETAILED description of the INVENTION

A. FORMULA (I)

In one embodiment, this invention relates to compounds of formula (I)

where R1and R2may be the same or different and selected from the group consisting and is essentially neoantigenic polymer residues, H, C1-6Akilov, C2-6alkenyl, C2-6the quinil, aralkyl and terminal branching groups, provided that R1and R2both are not H;

Z is selected from parts of the molecules are actively transported into a target cell, hydrophobic parts of the molecule bifunctional linking parts of the molecules and their combinations;

Y1-3may be the same or different and selected from O, S or NR11;

L1and L2may be the same or different bifunctional linkers; R3-R11, R24and R25may be the same or different and selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6the quinil, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic;

L3and L4may be the same or different and selected from: -C(O)(CR30R31)Y15(CR32R33)C(O)- or-C(O)(CR30R31)(CR32R33)C(O)-

where Y15selected from O, S, NR34or CH2and

R30-34may be the same or different and selected the from H, of alkyl, alkenyl, quinil, heteroalkyl or aryl;

A is selected from the leaving groups, functional groups, biologically active parts of the molecule and OH;

a, b, c, d, and e are independently 0 or 1,

m, n, o and p independently represent positive integers

f and g are 0 or 1, provided that

at least one of (f + a) or (g + c) equals 2.

In some preferred aspects of the present invention one or more of R1and R2includes neoantigenic polymer residue, such as the group of polyethylene glycol (PEG). Optional, R1-2includes cap group, designated here as J. Preferred J groups used to kupirovaniya polymer include those parts of the molecule, as OH, NH2, SH, CO2H, C1-6the alkyl such as CH3and compounds of the formulas (IIIa) and (IIIb):

where all variables are defined previously.

Another embodiment of the invention are compounds of formula IV and V:

wherea, b, c, d, f, and gare positive integers, and all other variables are defined above.

In another aspect of this invention R1and R2together with the atoms to which they are attached, can form a bridge structure having the formula:

where R70-80may be the same or different and selected from hydrogen, C1-6Akilov, C2-6alkenyl, C2-6the quinil, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic;

n' is a positive integer, preferably from about 1 to about 7, and all other variables are defined previously.

In relation to other variables that make up the formula of the present invention, in some aspects of this invention, preferred are the following:

in some aspects, R1and R2represent the remains of polyalkylated, and, more preferably, the residues of ethylene glycol.

In other aspects of R1and R2represent a branched end groups, based bicine, described in more detail below, enabling numerous branching of polymer chains; R3-R10and R24-25each represents a hydrogen; a, b, c, d, f, m, n, o and p are each preferably equal to 1;

e is preferably 0 or 1;

Z not only is em a ,as defined above, or, alternatively, Z contains amino acid residue, a peptide residue, a group that is actively transported into a target cell, hydrophobic or with a combination of such properties, so that when combined with A biologically active groups are formed prodrugs, which are released from bichenovii polymer parts formula (I), (II), etc. see Also public USSN 09/758,993, the content of which is incorporated herein by reference.

B. ESSENTIALLY PANTAGONIA POLYMERS

As indicated above, R1and R2each is preferably a water-soluble polymer residue, which preferably are essentially panthenyl, such as polyalkylene (ICS) and, more preferably, the polyethylene glycols, such as MPEG. To illustrate and not to limit, the residual part R1and R2containing the polyethylene glycol may be selected from:

J-O-(CH2CH2O)x-,

J-O-(CH2CH2O)x-CH2C(O)-O-,

J-O-(CH2CH2O)X-CH2CH2NR12-,

J-O-(CH2CH2O)X-CH2CH2SH-,

-OC(O)CH2-O-(CH2CH20)x-CH2C(O)-O-,

-NR12CH2CH2-O-(CH2CH2O)x-CH2CH2NR12and

-SHCH2CH2-O-(CH2CH2O) -CH2CH2SH-.

where x represents the degree of polymerization;

R12selected from hydrogen, C1-6Akilov, C2-6alkenyl, C2-6alkinyl, C3-12branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic, and J represents the cap-grouping described above with respect to formula II.

In one particularly preferred embodiment, R1-2selected from CH3-O-(CH2CH2O)x-, CH3-O-(CH2CH2O)x-CH2C(O)-O-CH3O-(CH2CH2O)x-CH2CH2NH - and CH3-O-(CH2CH2O)x-CH2CH2SH-, where x is a positive integer, preferably selected so that the average molecular weight is from about 2,000 to about 25,000 Da. In alternative aspects of the present invention, the molecular weight of the polymer is in the range from several hundred up to 40,000 or more, depending on the requirements of a specialist. PEG is mainly represented by the structure: -O-(CH2CH2O)x-,

and R1and R 2preferably contain the remains of this formula.

The degree of polymerization for the polymer (x) can be from about 10 to about 2300. Represents the number of repeating units in the polymer series and depends on the molecular weight of the polymer. Part of the molecule (J) represents nephropidae described above, i.e. a group that is at the end of the polymer, and, in some aspects, can be selected any one of NH2, OH, SH, CO2H, C1-6Akilov or other activating end groups of PEG, known to specialists in this field.

Also suitable polypropylenglycol, branched PEG derivatives described in the public U.S. patent No. 5643575 (patent '575), "star-PEG" and multibeam PEG described in the catalog "Polyethylene Glycol and Derivatives for Biomedical Application", Shearwater Corporation, 2001. Description of any set forth below is included here as a reference. Branching, presented in the patent '575, gives a secondary or tertiary branching from bichenovii group, as a way to increase the loading of the polymer, a biologically active molecule or an enzyme from one attachment point. It will be understood that the water-soluble polymer can be functionally activated to join bifunctional binder groups, if required, without any experimental work.

Although PAO's and PEG can significantly distinguish between the I-average molecular weight, preferably, R1and R2each has an average molecular weight of from about 2000 to about 25,000 Yes in most aspects of this invention.

Included here polymeric substance is preferably soluble in water at room temperature. Non-limiting list of such polymers includes polyalkylbenzene the homopolymers, such as polyethylene glycol (PEG), or polypropylenglycol, polyoxyethylenated polyols, copolymers and block copolymers, provided that there is a water-solubility of these block copolymers.

In another embodiment, and alternatively, the polymers based on PAO, R1and R2each optionally selected from one or more essentially neoantigenic substances such as dextran, polyvinyl alcohols, polymers based on carbohydrates, hydroxypropylmethacrylamide (HPMA), polyalkyleneglycol, and/or their copolymers. See also public U.S. patent No. 6153655, the content of which is incorporated herein by reference. Specialists in this field will be clear that use the same type of activation described here as for PAO, and PEG. Professionals in this field also it is clear that the above list is only to illustrate, and that covers all polymer compounds having opican the mi here properties and that also discusses other derivatives polyalkylated, such as polypropyleneglycol, etc.

The polymers of the present invention can also be copolymerizable with bifunctional compounds such as poly(allenglish)diamines to obtain interpenetrating polymer networks that are suitable for use in permeable contact lenses, wound dressings, devices for drug delivery and the like. Spatial constraints and the solubility of such branching can be easily identified by experts in the field. It is preferable, however, that the molecular weight polymers with multiple branching does not exceed the 80,000 Dalton.

C. BIFUNCTIONAL BRIDGE GROUP: L1L2L3and L4

In many aspects of the present invention and, in particular, in the formula (I), L1, L2, L3and/or L4represent bridging groups, which facilitate the accession bisinuosa derivative to the polymer chains, for example, R1and/or R2. The resulting connection can be either direct, or be carried out via the optional linking group, known to specialists in this field. In the description mentioned other Lxgroup, and it is clear that they can be selected from the same group that Lsub> 1. In this aspect of the present invention, L1and L2may be the same or different, and selected from:

-NR19(CR14R15)tO-

-NR19(CR14R15)t(CR16CR17O)qNR19-

- (CR14R15)tNR19-

- (CR14R15)tO-

-NR19(CR14R15)tNR19-

-NR19(CR14R15)t(CR16CR17O)q-

-NR19(CR16CR17O)t-

-NR19(CR16CR17O)t(CR14R15)qNR19-

-NR19(CR16CR17O)t-

-O(CR14R15)t-NR19-

-O(CR14R15)tNR19-

-O(CR14R15)tO-

-O(CR16CR17O)tNR19-

where R14-R17and R19independently selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6alkinyl, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1 6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic; and

R18select the group, consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6alkinyl, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic, NO2, haloalkyl and halogen; and

t and q each are individually selected positive integers, preferably from about 1 to about 4.

In other aspects of this invention L1and/or L2may include amino acid residue. The amino acid may be selected from any known natural L-amino acids, e.g. alanine, valine, leucine, isoleucine, glycine, serine, threonine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, lysine, arginine, histidine, Proline and/or their combinations, several, without specific instructions. In those cases where L1and/or L2include peptide, the size of this peptide are within a range from about 2 to about 10 amino acid residues. In another preferred embodiment, this peptide is Gly-Phe-Leu-Gly.

Amino acid OST the TCI preferably have the formula

where X' represents O, S or NR26, Y5represents O, S or NR27and R26, R27and R28independently selected from the same group, which determines R3but each preferably represents H or lower alkyl (i.e. C1-6alkyl); and f represents a positive integer from about 1 to about 10, preferably 1.

Derivatives and analogues of natural amino acids, as well as various well-known in this field unnatural amino acid (D or L), hydrophobic or not hydrophobic, also included in the scope of this invention. For example only, the analogues of amino acids and derivatives include 2-amino-adipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidine acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoethanol acid, 3-aminoadamantane acid, 2-aminopimelic acid, 2,4-aminobutyric acid, desmosine, 2,2-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, N-ethylasparagine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, ALLO-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, Norvaline, norleucine, ornithine and others that are too numerous, h is ordinary to mention, listed in 63 Federal register (Fed. Reg.), 29620, 29622, incorporated herein by reference.

For example, short peptides are the peptides in the range of from 2 to about 10 or more amino acid residues mentioned above.

More preferably, L1and L2may be the same or different and selected from:

-NH(CH2CH2)2About-

-NH(CH2CH2)(CH2CH2O)NH-

-O(CH2CH2)NH-

-O(CH2CH2)O-

-NH(CH2CH2)NH-

-NH(CH2CH2)(CH2CH2O)-

-NH(CH2CH2O)-

-NH(CH2CH2O)(CH2)NH-

-NH(CH2CH2O)2-

-O(CH2)3NH-

-O(CH2)3O-

-O(CH2CH2O)2NH-

In another embodiment of this invention L3and L4may be the same or different, and selected from:

-C(O)CR30R31OCR32R33C(O)-;

-C(O)CR30R31NR34CR32R33C(O)-;

-C(O)CR30R31SCR32R33C(O)-, or

-C(O)(CR30R31)nC(O)-;

where R30-34independently selected from H, C1-6of alkyl, C2-6alkenyl, C2-6the quinil, C1-6heteroalkyl or aryl, and

n is a positive integer, preferably from about 2 to 3.

Predpochtitel is but L3and L4selected from the

-C(O)CH2OCH2C(O)-;

-C(O)CH2NHCH2C(O)-;

-C(O)CH2SCH2C(O)-;

-C(O)CH2CH2CH2C(O)-, or

-C(O)CH2CH2C(O)-.

The main advantage of this invention is that the specialist is able to control the speed of hydrolysis or release of biologically active molecules or drugs from polymeric bichenovii platform. Depending on the specific linkers, the person skilled in the art can modify the connection to control the rate of hydrolysis. This preferred aspect of the invention allows the technician to adjust the rate at which the biologically active part of the molecule is delivered to the intended target. In those situations in which it would be desirable for the rapid release of a biological part of the molecule or drug, the incorporation of linkers L3and L4provides a higher rate of hydrolysis. On the contrary, for the former polymer-based platforms Bizina described in the public patent application U.S. 10/218,167, where the release of this part of the molecule or drug from said platform often depends on conditions such as pH or the presence of enzymes, linkers L3and L4thanks nahimana interaction,is able to significantly increase the speed, when the biologically active part of the molecule or drug is released from the polymer platform regardless of the conditions of pH or the presence or absence of enzymes. In the presence of enzymes, however, the rate of hydrolysis will be controlled either negimaru interaction, when possible, or enzymatic reactions, which in any case proceed faster. Thus, the rate of hydrolysis to a high degree depends on the type of linkers used between bichenovii part of the molecule and a PEG-part. Thus, this invention allows the use of both persistent and released linkers on any branch bichenovii part of the molecule, provided that at least one of the branches includes a disposable part of the linkers L1-L3or L2-L4.

D. Z PART of the MOLECULES AND THEIR FUNCTION

In one aspect of the present invention Z is an L5-C(=Y4), where L5is a bifunctional linker selected from the group which characterizes the L1and L2and Y4selected from the same groups that characterize Y1-3. In this aspect of the present invention, the group Z acts as a bridge between group a and the remainder bichenovii transport forms.

In other aspects of this invention, Z represents a part of the olekuly, which is actively transported into a target cell, hydrophobic portion of the molecule, and combinations thereof. Although Z is preferably a monovalent, optionally Z may be bivalent or multivalent, so that gives you the ability to attach more than one And group to the polymer-based bezina. To achieve active transport Z can include an amino acid, a peptide residue or a residue polyamine, for example, any of the above in relation to the L1and L2the remainder of the sugar, the balance of fatty acids, C6-18alkyl, substituted aryl, heteroaryl, -C(=O), -C(=S) - or-C(=NR29), where R29represents H, lower alkyl, etc

This aspect of the present invention in a broad sense based on the principle that biologically active substances that are suitable for inclusion in the conjugates of drugs with polymer-based bicine, can themselves be substances/compounds which are not active after hydrolytic release of the song, coupled with Bizina, but which will become active after undergoing a further chemical process/reaction. In this embodiment, therapeutic or diagnostic agent, peptide, polypeptide, etc. which is delivered to the bloodstream via a polymer-based system bicine, will remain inactive until Pronichev the treatment or to the active transfer of interest to a target cell, where it is activated by chemical processes within the cell, such as an enzyme or enzyme system present in this tissue or cell.

Prodrugs according to this aspect of the invention is manufactured in such a way that the hydrolysis of the polymer conjugate based Bizina flowingin vivoenzyme conjugate with the release of the active biological substance (here denoted by "A") in the extracellular fluid, staying connected with Z-part. Biologically active substances in this aspect of the invention preferably, but not exclusively, represent a therapeutic and/or diagnostic agents with a small molecular weight. For example, one possible combination of Z-A is a leucine-doxorubicin, the other hand is a associated with amino acid camptothecin or paclitaxel, and the fabric being treated is a tumor tissue.

Not bound by any theory or hypothesis, as it may work the invention, it is believed that depending on the optional part of the molecule, is selected as the amplifier transfer, the speed of transport of biologically active substances in tumor cells is represented by the delivery of biologically active substances in the extracellular space of the tissue, for example tissue, demonstrate what she EPR effect, in protected form or in the form of accelerated migration.

In one embodiment, the power transfer (Z) selected from known substances for the transport system of the cell membranes. For example only, it is known that cells actively carry some nutrients and endocrine factors, and the like, and such nutrients, or the like, which are readily used to reinforce the active transport of biologically effective substances into target cells. Examples of such nutrients include amino acid residues, peptides, such as short peptides, the size of which is within the range from about 2 to about 10 residues, or more simple sugars and fatty acids, endocrine factors and the like.

Short peptides, such as peptides in the range of from 2 to about 10, or more amino acid residues, as mentioned above.In this embodiment of the invention it is believed that this peptide accelerators transfer does not need to be hydrophobic, but I believe that they act in other ways to enhance the capture and/or protection of the United agents small molecular weight from premature hydrolysis of the systemic blood. For example, suppose that the peptide accelerators transfer, such as, polyalanine, and other accelerators transfer of similar molecular weight, prostranstvenno prevent the removal of the biologically active agent hydrolytic enzymes plasma, but then split in the target cell under the action of various peptides and/or proteases, such as cathepsins.

In some preferred aspects, Z is a hydrophobic part of the molecule. Not bound by any theory or hypothesis what is the contribution of hydrophobicity in efficiency, it is believed that the hydrophobic portion inhibits extracellular cleavage of the amplifier transfer from biologically active agent, inhibiting the effects of hydrolytic enzymes, etc. that are present in the extracellular tissue space, for example in the plasma. Thus, some preferred the amplifiers transfer include, for example, hydrophobic amino acids such as alanine, valine, leucine, isoleucine, methionine, Proline, phenylalanine, tyrosine and tryptophan, as well as and non-natural derivatives, such as γ-amino acid and its analogues, as mentioned above.

In another embodiment, the accelerator migration represents a hydrophobic organic part of the molecule. For example only organic part represents alkyl of length C6-18or more substituted aryl or heteroaryl or unsubstituted. Also assume that the accelerator migration presents an organic part, covers and includes an organic functional group containing, for example, -C(=S) and/or-C(=O).

E. G UPPY AND FORMULAS (I)

1. Leaving group

In those aspects where A represents a leaving group, suitable part of the molecule include, without limitation, groups such as N-hydroxybenzotriazole, halogen, N-hydroxyphthalimide, p-nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl; thiazolidinedione, O-allodapini or

or

other suitable leaving groups will be apparent to experts in this field.

For the purposes of the present invention under the leaving groups refers to such groups, which are able to interact with the nucleophile, in the desired target, i.e. biologically active molecules, the bifunctional spacer, intermediate substance, etc. Data of the target, thus, contain a group for substitution, as, for example, NH2groups in proteins, peptides, enzymes, natural or chemically synthesized therapeutic molecules, such as doxorubicin, spacers, such as one-deputizing the diamines.

Compounds according to this invention can also include spacer elements group between bichenovii group and the leaving group or, if required, attached to the target substance (drug). Spacer elements of the molecule may be heteroalkyl, alkoxy, alkyl, with the holding of up to 18 carbon atoms or even additional polymer chain. Spacer elements of the molecule can be added using standard methods of synthesis. It is clear that these parts of the molecules selected for (A), can also interact with other parts of the molecule, in addition to the biologically active nucleophiles.

2. Functional group

"A" can also be functional groups. Non-limiting examples of such functional groups include maleimides, vinyl, remains sulfone, hydroxy, amino, carboxy, mercapto, hydrazide, carbazate and the like, which may be attached to bichenovii part through amine-containing spacer. After joining bichenovii part of the functional group (for example, maleimide) can be used to attach bizin-polymer to the target, such as a cysteine residue of the polypeptide, the amino acid or the peptide spacer, etc.

3. Biologically active molecules

In those embodiments of formula (I)where A represents the balance of compounds containing amine - or hydroxyl. Non-limiting list of such suitable compounds include residues of organic compounds, enzymes, proteins, polypeptides, etc. Organic compounds include, without limitation, such items as anthracycline compounds, including daunorubicin, doxorubicin; p-aminoaniline mustard, melphalan, Ara-C (C is tainabilit) and related antimetabolites connection for example, gemcitabine, etc. Alternative, this part of the molecule may be the residue of a cardiovascular agent containing amine or hydroxyl, antineoplastic agents, such as camptothecin and paclitaxel, anti-infective, anti-fungal such as nystatin, fluconazole and amphotericin B, the means for removal of anxiety, gastrointestinal means, means for activation of the Central nervous system analgesic, means for fertility, means for contraception, anti-inflammatory agents, steroidal agents, substances, etc.

In addition to the above biologically active portion of the molecule may also be a residue of an enzyme, protein, polypeptide, oligonucleotide, monoclonal antibodies, single-chain antigen-binding proteins (SCA), such as, CC49, and discusses their fragments. Suitable proteins include, but not limited to, polypeptides, enzymes, peptides and the like, having at least one available group for attachment of a polymer, for example, ε-amino, Costinesti, N-terminal amino group include compounds having a physiological or pharmacological action, as well as substances that are able to catalyze reactions in organic solvents.

Of interest are proteins, polypeptides, Pat the water, include, but are not limited to, hemoglobin, serum proteins such as blood factors including factors VII, VIII and IX; immunoglobulins, cytokines such as interleukins, i.e. with IL-1 through IL-13, etc., α, β and γ interferons, colony stimulating factors including granulocyte colony stimulating factors, platelet-derived growth factors and protein that activates phospholipase (PLAP). Other proteins, mainly representing the biological and therapeutic interest include insulin, plant proteins such as lectins and ricini, factors tumor necrosis and related proteins, growth factors such as transforming growth factors such as TGFα or TGFβ and epidermal growth factors, hormones, somatomedins, erythropoietin, pigmentary open the hormones, hypothalamic releasing factors, antidiuretic hormones, prolactin, Holodny gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, and the like. Interest antibodies include IgG, IgE, IgM, IgA, IgD and fragments thereof.

Some proteins, such as interleukins, interferons and colony stimulating factors also exist in deglycosylation form, usually as a result of the use of recombinant technology. Deglycosylation options are also among the proteins n the standing of the invention.

Enzymes of interest include carbohydrate-specific enzymes, proteolytic enzymes, oxidoreductase, transferases, hydrolases, LiAZ, isomerase, and ligase. Not limited to the specific examples, the examples of enzymes of interest include asparaginase, arginase, argininosuccinate, adenoidectomies, superoxide dismutase, endotoxins, catalase, chymotrypsin, lipase, uricase, adenosinetriphosphatase, tyrosinase and bilirubinometer. Interest carbohydrate-specific enzymes include glucoseoxidase, glucholazy, galactosidase, glucocerebrosidase, glucoronidase etc.

Here also includes any part of a biological polymer, demonstrating the biological activityin vivo. This part includes the amino acid sequence, nucleic acids (DNA, RNA), peptide nucleic acid (NCP), antibody fragments, single-chain binding proteins, see, for example, U.S. patent No. 4946778, the description of which is incorporated herein by reference, binding molecules comprising fused antibodies or fragments, polyclonal antibodies, monoclonal antibodies and catalytic antibodies.

Proteins or parts thereof may be obtained or selected using methods known to experts in this field, such as the culture is Kana, extraction from animal sources, or methods of recombinant DNA. It also examines the sources of transgenic proteins, polypeptides, amino acid sequences and such. Such substances derived from transgenic animals, i.e. mice, pigs, cows, etc. in which these proteins are secreted in milk, blood or tissue. Transgenic insects and baculovirus expression systems are also considered as sources. Moreover, mutant variants of proteins, such as mutant interferon, also included in the scope of this invention.

Other proteins of interest, are allergenic proteins, such as ragweed, antigen E, bee venom, tick-borne allergen, and the like. The foregoing is illustrative of the proteins that are suitable for the present invention. It is clear that these proteins are defined here, is not mentioned specifically, but with an available amino group, also refers to and included in the scope of the present invention.

In a preferred aspect of the present invention the amino - or hydroxyl-containing compound is a biologically active compound which is suitable for medical or diagnostic use in the treatment of animals, for example mammals, including humans, for States that require such treatment. The implies is provided, the above list is illustrating and not limiting for compounds that can be modified. For specialists in this field will be obvious that other such compounds/compositions can be similarly modified without redundant experimental work. It is clear that biologically active substances that are not mentioned separately, but having the appropriate group of accession, is also provided and are included in the scope of the present invention.

The only limitation regarding the type of amino - or hydroxyl-containing molecules that are suitable for inclusion in this invention, is available at least one (primary or secondary) amine - or hydroxyl, which can react and bind with the polymer conjugate, and no significant loss of biological activity after release and regeneration of the parent compound system prodrugs.

F. SYNTHESIS BIZIN-LINKED POLYMERS

Synthesis of specific polymers based Bizina set forth in the Examples. Referring to Figure 1, for the purpose of illustration, one preferred method includes:

1) interaction blocked bifunctional linker with an anhydride, such as, for example, diglycolic anhydride, with the formation of blocked bifunctional JV is Ysera, such as:

2) accession blocked bifunctional spacer to each hydroxyl acid protected molecule Bizina such as:

where tBu represents a protective group, and all other variables are the same as defined previously for formula (I)

3) unlocking the resulting intermediate compound and its interaction with activated polymer, such as PNP-PEG and SC-PEG in the basic environment interaction,

4) removing protection from bichenovii acid and subsequent activation of this acid with a suitable activating group, such as thiazolidinedione, in terms of the binding.

It will be clear that other, known in the field of protective groups can be used instead of the t-Bu. The activated PEG or polymer derived Bizina now able to react and conjugates with drug, a peptide, a spacer, etc.

Non-limiting list of suitable binding agents include 1,3-aminobutiramida carbodiimide (DIPC), any suitable dialkylammonium, 2-halo-1-alkyl-pyridine halide (Mukaiyama reagent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), a cyclic anhydride papapostolou acid (PPACA) and phenyldichlorophosphine, etc. available, for example, from the comm is Ronski sources, such as Sigma-Aldrich Chemical, or synthesized using known technologies.

Preferably, the substituents react in an inert solvent, such as tetrahydrofuran (THF), acetonitrile (CH3CN), methylene chloride (DCM), chloroform (CHCl3), dimethylformamide (DMF) or mixtures thereof. Suitable bases include dimethylaminopyridine (DMAP), diisopropylethylamine, pyridine, triethylamine, KOH, potassium t-piperonyl and NaOH, etc. Usually the reaction is carried out at a temperature of from about 0°C to about 22°C (room temperature).

An alternative method of obtaining derivatives of bicine includes:

1) interaction with one equivalent of an elongated blocked bifunctional linker with one equivalent of acid is protected bichenovii part of the molecule with derivatization, such as:

where tBu represents a protective group, and all other variables are the same as defined previously for formula (I).

2) unlocking the resulting intermediate compound and its interaction with activated polymer, such as PNP-PEG and SC-PEG when the basic conditions of binding,

3) removing protection from bichenovii acid and subsequent activation of this acid with a suitable activating group, such as thiazolidinedione, in terms of St. the statements.

Another method of obtaining derivatives of Betina:

1) first elongated blocked bifunctional linker reacts with acidic protected bichenovii intermediate connection with the formation of:

2) then the second block-bifunctional linker reacts with the intermediate connection stage 1 education:

3) is then obtained above, the resulting intermediate compound unlocks and subjected to interaction with the activated polymer, such as PNP-PEG and SC-PEG in the basic conditions of binding,

4) finally, with bichenovii acid removes the protection, and then activate a suitable activating group, such as thiazolidinedione, in terms of the binding.

Regardless of the option chosen, some of the preferred compounds, which result described here, the methods of synthesis include:

where A1 represents a leaving group, such as:

or other leaving group, such as described above in section E 1.

Interaction bizin-activated polymers with appropriate targeted leads is the result of the transformation of the activated polymer conjugates, turning And1in A2where a2represents the balance of biologically active molecules, spacer, etc.

G. MULTIPLE-BOOT POLYMER

In another aspect this invention relates to mnogochislennym polymeric compounds based on bezina. In particular, the main derived Bizina additionally modify the order to include one or more end groups of branching. Preferably, the end groups of the branching represent a group of the formula:

where Y5represents O, S or NR46;

L6is a bifunctional linker selected from the same group, which determines the L1;

L8is a bifunctional linker selected from the same group, which determines the L3;

R40-R46may be the same or different and selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6the quinil, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6GE is arealake;

j, j', k and k' are each independently 0 or a positive integer;

q is 0 or 1;

u, h, v, and w are independently selected positive integers;

R50selected from the group consisting of essentially neoantigenic polymer residues, C1-6Akilov, C2-6alkenyl, C2-6the quinil, aralkyl, and

where L7is a bifunctional linker selected from the same group, which determines the L1;

L9is a bifunctional linker selected from the same group, which determines the L3;

R60selected from the group consisting of essentially neoantigenic polymer residues, C1-6Akilov, C2-6alkenyl, C2-6the quinil, aralkyl, and all other variables are defined above.

The resulting branched derivatives Bizina have a structure represented by the formula:

where all the variables are already defined above.

As shown below and in the examples, the intermediate compound derived Bizina containing blocked primary amine, reacts with two equivalents of activated bisinuosa polymer with the formation of bichenovii polymer systems containing up to four polymer chains, which connect the die in a single attachment point on a biologically active molecule, the enzyme, targets, etc. This process can be repeated to education vicimization derived by reacting two equivalents of polymer derived Bizina with four chains described above, with one equivalent of blocked primary amine derived bezina.

H. DIAGNOSIS IN VIVO

Another aspect of this invention relates to the conjugates according to the invention need not be received with a diagnostic label that is associated with the above-described power transfer, where the label chosen for the purpose of diagnosis or visualization. Thus, a suitable label is produced by any suitable binding part of the molecule, for example, amino acid balance, with any standardized emitting isotope, a radio-opaque label, magnetic resonance labels or other non-radioactive labels, suitable for magnetic resonance imaging, labels, fluorescent type, labels, manifests visible color and/or is able to fluoresce in ultraviolet, infrared or electrochemical stimulation, for visualization of tumor tissue during surgical interventions, etc. are Optional, diagnostic label include therapeutic conjugate of the molecule, or associated with it, giving you the ability to monitor the distribution of the population therapeutic biologically active substances in the organism of an animal or human.

In another embodiment of the invention labeled conjugates according to the invention can be easily obtained by methods known in this field, with a suitable label, including, for example, radioisotope labels. For example only, they include131Iodine,125Iodine,99mTechnetium and/or111Iodine to obtain radioimmunoscintigraphy agents for the selective capture of tumor cells in vivo. For example, in the field of technology, there are various ways of binding of the peptide with Tc-99m, including, just for example, shown in U.S. patent No. 5328679; 5888474; 5997844 and 5997845, incorporated herein by reference.

Approximately, for anatomical localization of the tumor tissue in a patient's body, conjugate label is injected into the patient or animal suspected of having a tumor. Over a period of time sufficient for the localization of the labeled immunoglobulin in the location (s) the location of the tumor, register the signal label, for example, visual, x-ray radiography, computed tomography, NMR tomography, instrumental detection of fluorescent labels, photoscenery device such as a gamma camera, or any other method or device, in accordance with the nature of the selected tag.

The detected signal is then converted into an image or anatomically and/and and physiologically determine the size of the tumor. The image makes it possible to determine the location of the tumor in vivo and to develop an appropriate treatment plan. In these versions of the invention, the labeled portion of the molecule itself is a therapeutic tool, as defined by the signal provides information about the localization during treatment, providing the basis for subsequent diagnostic and therapeutic interventions.

I. METHODS of TREATMENT

Another aspect of the present invention relates to methods of treatment of various conditions mammals. These methods comprise the administration to a mammal in need of such treatment, an effective amount of a prodrug, such as a conjugate of doxorubicin-bicin associated with PEG, obtained as described here. These compositions are effective, inter alia, for the treatment of neoplastic disease, reducing tumor mass, prevention of metastasis of tumors and prevention of recurrence of tumor growth/tumors in a mammal.

The number of input prodrug will depend on the source of the molecule, for example, peptide, polypeptide, protein, enzyme, etc. enclosed in it. Mainly, the number of prodrugs used in the methods of treatment, is such a number, which effectively achieves the desired therapeutic result, mlekopitayushchie is. Naturally, different doses of the compounds, prodrugs will to some extent be varied depending on the initial connection, the speed of hydrolysis in vivo, the molecular weight of the polymer, etc. Specialist in this field will determine the optimal dose of the prodrug, selected on the basis of clinical experience and therapeutic indications. The specific dose will be obvious to those skilled without redundant experimental work.

The compositions of the present invention can be included in one or more pharmaceutical compositions for the introduction of mammals. The pharmaceutical compositions can be in the form of solution, suspension, tablet, capsule or the like, obtained in accordance with methods well known in the field. It is also expected that such compositions can be administered orally and/or parenterally depending on the desires of a specialist. The solution and/or suspension of this composition can be used, for example, as a carrier for injection or infiltration of this composition by any means known in this field, for example, intravenous, intramuscular, subcutaneous injection and the like.

This introduction can be done by infusion into the space or cavity of the body, as well as inhalation and/or intranasal route. In predpochtitelnei of the present invention, however, the prodrug is administered parenterally to a mammal in need of it.

EXAMPLES

The following examples serve to further understanding of the invention, but not for any limitation of the actual volume of the present invention. The underlined and bold numbers indicated in the Examples correspond to the numbers shown in Fig. 1 through 11.

General methods. All reactions were carried out in an atmosphere of nitrogen or argon. Commercial reagents were used without additional purification. All connections PEG was dried under vacuum or by azeotropic distillation from toluene before use. NMR spectra were obtained using Varian Mercury®300 NMR spectrometer and deuterated chloroform as solvent unless otherwise stated. Chemical shifts (δ) are presented in parts per million (ppm) weak fields from tetramethylsilane (TMS).

Method HPLC. The reaction mixture and the purity of intermediates and final products was controlled by devices Beckman Coulter System Gold® HPLC using a column ZOBAX® 300 SB C-8 reversed-phase (150x4,6 mm) or column Phenomenex Jupiter® 300A C18 reversed-phase (150x4,6 mm) with multi-wave UV detector, using a gradient of 30 to 90% acetonitrile in 0.5% triperoxonane acid (TFA) with a flow rate of 1 ml/min

EXAMPLE 1

SYNTHESIS of COMPOUND (1)

To a solution of di-tert-BUTYLCARBAMATE (15 g, 86 mmol) in 1,4-dioxane (150 ml), cooled to 5°C in an ice bath, was added dropwise a solution of 2,2'-(Ethylenedioxy)bis(ethylamine) (25,85 g of 174.4 mmol) in 1,4-dioxane (100 ml) for 1 h the Reaction mixture was allowed to warm to room temperature and was stirred for another two hours. The solvent was removed under reduced pressure, and the residue was dissolved in methylene chloride (DCM, 150 ml), washed with water (3 x 150 ml), dried (MgSO4), filtered, and the solvent evaporated under reduced pressure obtaining at the output connection1(at 13.84 g of 68.8 mmol, 80%).13C NMR (75.5 MHz, CDCl3) δ, 115,76, 79,03, 73,45, 70,12, 40,31, 28,39.

EXAMPLE 2

SYNTHESIS of COMPOUND (3)

A solution of compound1(3.0 g, 12.1 mmol), diglycolic anhydride (2, 1.26 g, 10.9 mmol), and DMAP (1.4 g, 11.5 mmol) in anhydrous DCM (30 ml) was stirred at room temperature for 18 hours. The mixture was washed using 0.1 G. of HCl (30 ml)and the organic layer was dried (anhydrous sodium sulfate), filtered, and the solvent was removed under reduced pressure obtaining at the output connection3(1.5 g, 4.14 mmol, 38%).13C NMR (75.5 MHz, CDCl3) δ, 173,37, 171,27, 169,94, 169,59, 157,81, 155,96, 81,15, 79,30, 71,78-68,76 (m), 41,59, 40,13, 38,94, 38,73, 28,27.

EXAMPLE 3

SYNTHESIS of COMPOUND (6)

A solution of compound4(24,0 g, 0,228 mol) and5(12.0 g, 0.061 mol) in anhydrous methylene chloride (DCM, 400 ml) premesis is whether at room temperature for 18 hours. The reaction mixture was washed with water (4 x 150 ml)and the organic layer was dried over anhydrous sodium sulfate, followed by filtration and removal of solventin vacuoobtaining at the output connection6(6,1 g, 0,0279 mol, 46%).13C NMR (67,8 MHz, CDCl3) δ, 172,1, 81,4, 59,5, 57,0, 56,3, 27,8.

EXAMPLE 4

SYNTHESIS of COMPOUND (7)

To a solution of compound3(0.5 g, 1.37 mmol),6(0,090 g, 0.41 mmol), DMAP (and 0.46 g, 3.8 mmol), and scandium triflate (0.04 g, is 0.023 mmol) in anhydrous DCM (10 ml), cooled to 0°C, was added EDC (0.35 g, 1.8 mmol). The mixture was left in an ice bath to heat to room temperature over night. This mixture was washed with water, and then 0.1 G. of HCl. The organic layer was dried (anhydrous sodium sulfate), filtered, and the solvent was removed under reduced pressure to obtain compound7(0,37 g, 0.41 mmol, ˜100%).

13C NMR (75.5 MHz, CDCl3) δ, 170,15, 169,38, 168,64, 155,73, 81,23, 79,05, 70,95, 69,86, 69,58, 68,33, 67,55, 63,18, 53,09, 52,85, 40,31, 38,59, 28,37, 28,14.

EXAMPLE 5

SYNTHESIS of COMPOUND (8)

To a solution of compound7(0,38 g, 0.42 mmol) in DCM (8 ml) was added triperoxonane acid (TFA, 2 ml) and the solution was stirred for 15 minutes at room temperature, followed by removal of solvent under reduced pressure to obtain compound8(0,38 g, 0.42 mmol, ˜100 %). The structure of8confirmed with POM is using 13C NMR.

EXAMPLE 6

SYNTHESIS of COMPOUND (10)

Preparing a solution of compound8(0,38 g, 0.42 mmol) and DMAP (0.16 g, 1.3 mmol) in anhydrous DCM (30 ml), 22 ml of which was first added to a solution of compound9(8.0 g, 0.66 mmol) in DCM (30 ml). The resulting mixture was stirred at room temperature for 6 hours followed by the addition of the remaining solution connection8and was stirred for another 12 hours. The solvent was partially removed under reduced pressure and the product was besieged ethyl simple ether, filtered and the residue was vykristallizovyvalas from 2-propanol (IPA, 160 ml) to obtain the output connection10(7,8 g to 0.63 mmol, 95%).13C NMR (75.5 MHz, CDCl3) δ, 169,82, 169,10, 168,32, 155,87, 80,85, 71,51-67,28 (PEG), 63,48, 62,88, 58,61, 55,80, 52,55, 40,44, 38,26, 27,88.

EXAMPLE 7

SYNTHESIS of COMPOUND (11)

A solution of compound10(6.7 g, 0.27 mmol) in DCM (68 ml) and TFA (34 ml) was stirred at room temperature for 15 hours, followed by partial removal of the solvent under reduced pressure. Product besieged ethyl simple ether, filtered, and washed with ethyl simple broadcast receiving the connection11(6.7 g, 0.27 mmol, ˜100%).13C NMR (75.5 MHz, CDCl3) δ, 169,04, 168,67, 168,37, 155,94, 71,51-68,04 (PEG), 63,48, 62,71, 58,59, 55,07, 52,84, 40,43, 38,23.

EXAMPLE 8

SYNTHESIS of COMPOUND (12)

The solution is soedineniya 11(6,9 g, 0.27 mmol), 2-mercaptothiazoline (0.10 g, 0.84 mmol), and DMAP (0,136 g, 1.12 mmol) in DCM (70 ml) was cooled to 0°C, followed by addition of EDC (0.16 g, 0.84 mmol). This mixture was allowed to warm to room temperature and was stirred for 12 hours. The solvent was partially removed under reduced pressure and the product was besieged ethyl simple ether, filtered, and vykristallizovyvalas of IPA (140 ml) to obtain the output connection12(6.0 g, 0.23 mmol, 87%).13C NMR (67,8 MHz, CDCl3) δ, 201,05, 172,52, 169,10, 168,31, 155,85, 71,51-67,11 (PEG), 63,46, 63,08, 60,47, 58,58, 55,33, 52,55, 40,44, 38,28, 28,71.

EXAMPLE 9

SYNTHESIS of COMPOUND (13)

To a solution of compound12(2.0 g, 0,089 mmol) and doxorubicin hydrochloride (0,103 g, 0,179 mmol) in a mixture of DCM/DMF (20 ml/20 ml) was added DMAP (0,043 g, 0.35 mmol). This mixture was stirred in nitrogen atmosphere for 18 hours with subsequent partial removal of solvent under reduced pressure. Derived PEG besieged ethyl simple ether, collected by filtration and twice vykristallizovyvalas from DMF/IPA (8 ml 32 ml) to obtain the output connection13(1.6 g, 0,065 mmol, 73%).13C NMR (67,8 MHz, CDCl3) δ, 313,32, 186,56, 186,18, 169,50, 168,93, 168,55, 160,58, 155,99, 155,85, 155,23, 135,38, 135,05, 133,46, 133,28, 120,47, 119,39, 118,17, 111,14, 110,93, 100,54, 72,0-69,0 (PEG), 68,01, 65,17, 63,67, 62,65, 58,68, 56,41, 54,07, 40,54, 38,40, 35,51, 33,56, 29,73, 16,69.

EXAMPLE 10

SYNTHESIS of COMPOUND (15)

The solution is connected to the I 14(3.0 g, 12.1 mmol),2(1.26 g, 10.9 mmol), and DMAP (1.4 g, 11.5 mmol) in anhydrous DCM (30 ml) was stirred at room temperature for 18 hours. The mixture is washed using 0.1 G. of HCl (30 ml)and the organic layer is dried (anhydrous sodium sulfate), filtered, and the solvent is removed under reduced pressure obtaining at the output connection15. The structure of15confirm with13C NMR.

EXAMPLE 11

SYNTHESIS of COMPOUND (16)

To a solution of compound15(0.5 g, 1.37 mmol),6(0,090 g, 0.41 mmol), DMAP (and 0.46 g, 3.8 mmol), and scandium triflate (0.04 g, is 0.023 mmol) in anhydrous DCM (10 ml), cooled to 0°C, add EDC (0.35 g, 1.8 mmol). The mixture is left in an ice bath to heat to room temperature over night. This mixture is washed with water, and then 0.1 G. of HCl. The organic layer is dried (anhydrous sodium sulfate), filtered, and the solvent is removed under reduced pressure to obtain compound16. The structure of16confirm using13C NMR.

EXAMPLE 12

SYNTHESIS of COMPOUND (17)

Connection17.To a solution of compound16(0,38 g, 0.42 mmol) in DCM (8 ml) is added triperoxonane acid (TFA, 2 ml)and the solution stirred for 15 minutes at room temperature, followed by removal of solvent under reduced pressure to obtain compound17. The structure is ur connection 17confirm using13C NMR.

EXAMPLE 13

SYNTHESIS of COMPOUND (18)

Prepare a solution of the compound17(0,38 g, 0.42 mmol) and DMAP (0.16 g, 1.3 mmol) in anhydrous DCM (30 ml), 22 ml which is first added to a solution of9a(8.0 g, 0.66 mmol) in DCM (30 ml). The resulting mixture was stirred at room temperature for 6 hours followed by the addition of the remaining solution connection18and stirred for another 12 hours. The solvent was partially removed under reduced pressure and the product precipitated with ethyl simple ether, filtered, and the residue vykristallizovyvalas from 2-propanol (IPA, 160 ml) to obtain the output connection18. The structure of18confirm using13C NMR.

EXAMPLE 14

SYNTHESIS of COMPOUND (19)

A solution of compound18(6.7 g, 0.27 mmol) in DCM (68 ml) and TFA (34 ml) was stirred at room temperature for 15 hours, followed by partial removal of the solvent under reduced pressure. The product is precipitated with ethyl simple ether, filtered and washed with ethyl simple broadcast receiving the connection19. The structure of19confirm with13C NMR.

EXAMPLE 15

SYNTHESIS of COMPOUND (20)

A solution of compound19(6,9 g, 0.27 mmol), 2-mercaptothiazoline (0.10 g, 0.84 mmol), and DMAP (0,136 g, 1.12 mmol) in DCM (70 is l) is cooled to 0° C, followed by addition of EDC (0.16 g, 0.84 mmol). Mixture is allowed to warm to room temperature and stirred for 12 hours. The solvent was partially removed under reduced pressure and the product precipitated with ethyl simple ether, filtered and vykristallizovyvalas of IPA (140 ml) to obtain the output connection20. The structure of20confirm with13C NMR.

EXAMPLE 16

SYNTHESIS of COMPOUND (21)

Connection21.To a solution of compound20(2.0 g, 0,089 mmol) and doxorubicin hydrochloride (0,103 g, 0,179 mmol) in a mixture of DCM/DMF (20 ml/20 ml) is added DMAP (0,043 g, 0.35 mmol). This mixture is stirred under nitrogen atmosphere for 18 hours with subsequent partial removal of solvent under reduced pressure. Derived PEG-precipitated with ethyl simple ether, collected by filtration and twice vykristallizovyvalas from DMF/IPA (8 ml 32 ml) to obtain the output connection21. The structure 21 is confirmed using13C NMR.

EXAMPLE 17

SYNTHESIS of COMPOUND 26

Connection26get in conditions similar to the conditions for the connection18.

EXAMPLE 18

SYNTHESIS of COMPOUND 29

Connection29get in conditions similar to the conditions for the connection21.

EXAMPLE 19

SYNTHESIS of COMPOUND 35

Connection35can be obtained in the us is the conditions, similar conditions obtain connection13except that the reaction takes only one equivalent of the compound3and one equivalent of the compound6.

EXAMPLE 20

SYNTHESIS of COMPOUND 43

Connection43can be obtained in conditions similar to the conditions for the connection13,except that one equivalent of the compound3interacts with one equivalent of the compound6with the formation of compound36. Then one equivalent of the compound36interacts with one equivalent of the compound37with the formation of compound38. Other reaction conditions are the same with the connection43.

EXAMPLE 21

ALGAE PROTEIN

Materials and methods

Lysozyme protein chicken eggs (EC 3.2.1.17), lysozyme bacterial substrate(Micrococcus lysodeikticus),andPBS buffer (10 mm phosphate, pH of 7.4, 138 mm NaCl, and 2.7 mm KCl) was acquired by the company Sigma Inc. (St. Louis, MO). Tris-glycine SDS gel electrophoresis of industrial production and the working buffer was received from the company Invitrogen (Carlsbad, CA). The plasma of rats used for the determination of hydrolysis of conjugates ofin vitroprocessed in EDTA and stored frozen. IL-2 was purchased at the company PeproTech (Princeton, NJ), and GFP was acquired by the company Clontech (Palo Alto, CA). All measurementsin vivoconducted three times and the standard deviation of ±5 %was established for the measurement of in vitro.

Obtaining a single of conjugates of PEG-lysozyme

Lysozyme hen egg has a molecular weight of 14500 and 6 lysine residues. With rapid stirring powdered activated PEG, in a molar ratio in the reaction is 1:1 (PEG:lysozyme)was added to a solution of lysozyme (5 mg/ml in 0.1 M phosphate buffer, pH 7.3. After stirring for 45 min at 25°C, the reaction mixture was treated with 0.2 M sodium phosphate (pH 5,1) to a final pH of 6.5. The reaction mixture was dialyzed against 20 mm sodium phosphate, pH 5,1, 4°C, using a membrane filter with border transmission molecular weight 6000-8000. The conductivity of the sample after dialysis should be less than 2 MSM. The selection of a single conjugate PEG-lysozyme was performed on a cation exchange column (Poros HS) using a solvent system of 20 mm of sodium phosphate with a pH of 5.1 with a gradient of NaCl. Peak single conjugate PEG-lysozyme was collected and concentrated using ultrafree" filter centrifuge with a membrane 10k NMWL (Millipore Corp., Bedford, MA). The output unit cleared conjugate PEG-lysozyme was about 20-30%.

Getting multiple conjugates of PEG-lysozyme

With rapid stirring the activated PEG linker with a molar ratio in the reaction of 30:1 (PEG:lysozyme) was added to a solution of lysozyme (5 mg/ml in 0.1 M phosphate buffer, pH 7.3. After paramesh the tion for 45 min at room temperature, the reaction mixture was treated with 0.2 M sodium phosphate (pH 5,1) to a final pH of 6.5. The reaction mixture was diluted with H2O and separated on a column Hiload Superdex 200 with a speed of 1 ml/min Column buffer containing 20 mm sodium phosphate (pH 6,8) and 140 mm NaCl. The peak fractions were collected and concentrated using ultrafree" filter centrifuge with a membrane 30k NMWL (Millipore Corp., Bedford, MA). The yield of purified multiple conjugate PEG-lysozyme was approximately 85%, and the number of the PEG molecule of lysozyme according to the fluorimetric analysis was 5-6.

Determining the concentration

The concentration of conjugate PEG-lysozyme was determined using the UV using the extinction coefficient 2,39 ml/MGSM at 280 nm in 0.1 M sodium phosphate, pH 7.3.

Analysis of the enzymatic activity of lysozyme

The reaction conditions mentioned above, lysozyme activity disappears after conjugation with only a single PEG. On the release of lysozyme indicated the restoration of the activity of lysozyme in different conditions of release and was confirmed by SDS gel electrophoresis. In a typical analysis of the activity of lysozyme, 0.2 ml of 0.02% (wt./about)M. lysodeikticus(substrate) was added to 0,12-0,24 mg of lysozyme in 50 µg 66 mm potassium phosphate, pH of 6.2, containing 0.01% of BSA, in 96-well title the tablet. The absorption at 450 nm was carried out for 5 min. sink Rate of absorption was used as a measure of enzyme activity. One unit of enzymatic AK is Yunosti was amended 0.001 units of optical density/min at 25° C at 450 nm.

The release of lysozyme in the plasma of rats and in the chemical buffer

The conjugates of PEG-lysozyme in phosphate buffer, pH 6.5, was subjected to buffer exchange with PBS, pH 7,4, to monitor the release into the plasma of rats. Measured stability in PBS at 37°C. the Conjugates were also subjected to buffer exchange with H2O for release in Tris-buffer, pH 8.5. Centrifuge tube Method 10 K (Millipore Corp., Bedford, MA) was used for single conjugates of PEG-lysozyme, whereas Method 30K used for multiple conjugates of PEG-lysozyme. The release of lysozyme from single or multiple conjugates of PEG-lysozyme was performed at a concentration of 0.15 mg/ml in an atmosphere of N2. At the specified time took aliquots were neutralized by the action of 0.2 M phosphate (pH 5,1) up to pH 6.5, and stored at -20°C until further analysis.

RESULTS

Table 1
Properties of PEG-bicin-doxorubicin
Connectiont1/2(rp) hMM% activityIC50(nm)
133,2252331,80455

Table 2
Soon the th release Paglinawan of lysozyme in the plasma of rats and in the buffer *
ConnectionPlasmaPBS, 25°CPBS, 37°CpH 8.5, 37°C
13A536121,3
13b648152

*data are presented ast1/2in hours.

Release in plasma was observed within 3 days, the release into the buffer with a pH of 8.5 for 5 days and the release in PBS for 7 days. PBS contains 138 mm NaCl, 2.7 mm KCl and 10 mm phosphate, pH of 7.4. The release of lysozyme was determined to restore the activity of lysozyme was confirmed using gel electrophoresis.

1. The compound containing the formula (I)

where R1and R2independently selected from the group consisting of polyalkyleneglycol, H, C1-6Akilov, C2-6alkenyl,2-6the quinil, aralkyl and terminal branching groups;

Z is selected from parts of the molecules are actively transported into the target cell, hydrophobic parts of the molecule bifunctional linking parts of the molecules and their combinations;

Y1-3may be the same or different and selected from O, S or NR11;

L1and L2may be the same or different bifunctional linkers, in the abusive group, consisting of

-NR19(CR14R15)tO-;

-NR19(CR14R15)t(CR16CR17O)qNR19-;

- (CR14R15)tNR19-;

- (CR14R15)tO-;

-NR19(CR14R15)tNR19-;

-NR19(CR14R15)t(CR16CR17O)q-;

-NR19(CR16CR17O)t-;

-NR19(CR16CR17O)t(CR14R15)qNR19-;

-O(CR14R15)t-NR19-;

-O(CR14R15)tO-;

-O(CR16CR17O)tNR19-

where R14-R17and R19independently selected from the group consisting of hydrogen, C1-6Akilov,2-6alkenyl,2-6alkinyl,3-12branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,2-6substituted alkenyl,2-6substituted alkinyl,3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic; and

p num="359"> R18selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6alkinyl, C3-12branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic, NO2, haloalkyl and halogen; and

t and q are each individually selected positive integers from about 1 to 4;

R3-R11, R24and R25may be the same or different and selected from the group consisting of hydrogen, C1-6Akilov,2-6alkenyl,2-6the quinil,3-19branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,2-6substituted alkenyl,2-6substituted alkinyl,3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy and C1-6heteroatomic;

L3and L4may be the same or different and selected from the

-C(O)CR30R31OCR32R33C(O)-;

-C(O)CR30R31NR34CR32R33C(O)-;

-C()CR 30R31SCR32R33C(O)-; or

-C(O)(CR30R31)nC(O)-,

where R30-34independently selected from H, C1-6of alkyl, C2-6alkenyl,2-6the quinil, C1-6heteroalkyl or aryl, and

n represents a positive integer from about 2 to about 3,

And selected from the aminecontaining biologically active parts of the molecule;

a, b, C, d and e are independently 0 or 1,

m, n, o and p independently represent a positive integer from 1 to 6,

f and g are 0 or 1, provided that

at least one of (f+a) or (g+C) equals 2.

2. The compound according to claim 1, where each R3-R10, R24-25and R30-34represents hydrogen.

3. The compound according to claim 1, where each a, b, C, d, f, g, m, n, o and p is 1 and e is 0 or 1.

4. The compound according to claim 1, where each of C and g is 0.

5. The compound according to claim 1, where each of a and f is equal to 0.

6. The compound according to claim 1, where each of C, g and d is equal to 0.

7. The compound according to claim 1, where each of a, b and f are equal to 0.

8. The compound according to claim 1, where R1includes polyethylene glycol.

9. The compound according to claim 1, where R2includes polyethylene glycol.

10. The compound according to claim 1, where R1or R2also include CEP-group J, selected from the group consisting of HE, NH2, SH, CO2H, C1-6the alkyl parts of the molecule

11. The compound of claim 8 selected from the group consisting of

and (IIb)

12. The compound according to claim 1, where R1selected from the group consisting of

J-O-(CH2CH2O)x-;

J-O-(CH2CH2O)x-CH2C(O)-O-;

J-O-(CH2CH2O)X-CH2CH2NR12-;

J-O-(CH2CH2O)X-CH2CH2SH-;

-OC(O)CH2-O-(CH2CH2O)x-CH2C(O)-O-;

-NR12CH2CH2-O-(CH2CH2O)x-CH2CH2NR12-; and

-SHCH2CH2-O-(CH2CH2O)x-CH2CH2SH-,

where x represents the degree of polymerization;

R12selected of the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6the quinil, C3-12branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6Ala is XI, phenoxy and C1-6heteroatomic, and J represents the cap group.

13. The compound according to claim 1, where R2selected from the group consisting of

J-O-(CH2CH2O)x-;

J-O-(CH2CH2O)x-CH2C(O)-O-;

J-O-(CH2CH2O)X-CH2CH2NR12-;

J-O-(CH2CH2O)X-CH2CH2SH-;

-OC(O)CH2-O-(CH2CH2O)x-CH2C(O)-O-;

-NR13CH2CH2-O-(CH2CH2O)x-CH2CH2NR13-; and

-SHCH2CH2-O-(CH2CH2O)x-CH2CH2SH-,

where x represents the degree of polymerization;

R13selected from the group consisting of hydrogen, C1-6Akilov,2-6alkenyl,2-6the quinil,3-12branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,2-6substituted alkenyl,2-6substituted alkinyl,3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic, and J represents the cap group.

14. The compound according to claim 1, where R1-2individually selected from the group consisting of

CH3-O-(CH2CH2O)x-;

CH3-Is-(CH 2CH2O)x-CH2C(O)-O-;

CH3O-(CH2CH2O)x-CH2CH2NH-; and

CH3-O-(CH2CH2O)x-CH2CH2SH-,

where x represents the degree of polymerization.

15. The compound according to claim 1, where each R1and R2contains polymer residue of the formula-O-(CH2CH2O)x-,

where x represents the degree of polymerization.

16. The connection indicated in paragraph 15, where each of R1and R2has an average molecular weight of from about 2000 Da to about 25,000 Da.

17. The compound according to claim 1, where the specified limit branched group contains the formula

where Y5represents O, S or NR46;

L6is a bifunctional linker selected from the same group, which determines the L1;

L8is a bifunctional linker selected from the same group, which determines the L3;

R40-R46may be the same or different and selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6the quinil, C3-19branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8samisen the x cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic;

each j, j', k and k' are independently 0 or a positive integer;

q is 0 or 1;

g, h, v, and w are independently selected positive integers from 1 to 6;

R50selected from the group consisting of polyalkyleneglycol, C1-6Akilov,2-6alkenyl,2-6the quinil, aralkyl, and

where L7is a bifunctional linker selected from the same group, which determines the L1;

L9is a bifunctional linker selected from the same group, which determines the L3;

R60selected from the group consisting of polyalkyleneglycol, C1-6Akilov,2-6alkenyl,2-6the quinil and Arakelov.

18. The connection 17 containing structure

19. The connection 17 containing structure

20. A method of obtaining a polymer conjugate that includes the interaction of the compounds of formula

where A1represents a leaving group;

R1and R2independently selected from the group consisting of polyalkyleneglycol, H, C1-6Akilov,2-6alkenyl,2-6the quinil, aralkyl and terminal branching groups, provided that both R1and R2are not H;

Z is selected from parts of the molecules are actively transported into a target cell, hydrophobic parts of the molecule bifunctional linking parts of the molecules and their combinations;

Y1-3may be the same or different and selected from O, S or NR11;

L1and L2may be the same or different bifunctional linkers selected from the group consisting of

-NR19(CR14R15)tO-;

-NR19(CR14R15)t(CR16CR17O)qNR19-;

- (CR14R15)tNR19-;

- (CR14R15)tO-;

-NR19(CR14R15)tNR19-;

-NR19(CR14R15)t(CR16CR17O)q-;

-NR19(CR16CR17O)t-;

-NR19(CR16CR17O)t(CR14R15)qNR19-;

-O(CR14R15)t-NR19-;

-O(CR14R15)tO-;

-O(CR16CR17 O)tNR19-

where R14-R17and R19independently selected from the group consisting of hydrogen, C1-6Akilov,2-6alkenyl,2-6alkinyl,3-12branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,2-6substituted alkenyl,2-6substituted alkinyl,3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic; and

R18selected from the group consisting of hydrogen, C1-6Akilov, C2-6alkenyl, C2-6alkinyl, C3-12branched Akilov, C3-8cycloalkyl, C1-6substituted Akilov, C2-6substituted alkenyl, C2-6substituted alkinyl, C3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic, NO2, haloalkyl and halogen; and

t and q are each individually selected positive integers from about 1 to 4;

R3-R11, R24and R25which may be the same or different and selected from the group consisting of hydrogen, C1-6Akilov,2-6alkenyl,2-6the quinil,3-19branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,2-6substituted alkenyl,2-6substituted alkinyl,3-8substituted cycloalkyl, arrow, substituted arrow, aralkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy and C1-6heteroatomic;

L3and L4may be the same or different and selected from the

-C(O)CR30R31OCR32R33C(O)-;

-C(O)CR30R31NR34CR32R33C(O)-;

-C(O)CR30R31SCR32R33C(O)-; or

-C(O)(CR30R31)nC(O)-,

where R30-34independently selected from H, C1-6of alkyl, C2-6alkenyl, C2-6the quinil, C1-6heteroalkyl or aryl, and

n represents a positive integer from about 2 to about 3 amine-containing biologically active agent under conditions that meet education

where a2represents the balance of aminecontaining biologically active agent;

a, b, C, d and e are independently 0 or 1,

m, n, o and p independently represent a positive integer from 1 to 6,

f and g are 0 or 1, provided that

at least one of (f+a) or (g+C) equals 2.

21. A method of obtaining a polymer of the transport system on the basis of bicine, including

a) interaction blocked bifunctional linker with the anhydride with the formation of elongated blocked bifunctional spacer of the formula

b) attaching blocked bifunctional spacer to each hydroxyl acid protected molecule bicine;

c) unlocking the resulting intermediate compound and its interaction with activated polymer in the basic environment interactions, and

d) removing the protection bichenovii acid and subsequent activation of the acid with a suitable activating group in the interface.

22. The use of compounds according to claim 1 to obtain medicines with increased period of circulation in vivo.

23. The use of compounds according to claim 1 to provide increased period of circulation of the biologically active agent in vivo.

24. The compound according to claim 1, where a is an aminecontaining biologically active molecule selected from the group consisting of oligonucleotides, proteins, enzymes and organic compounds.



 

Same patents:

FIELD: organic chemistry of natural compounds.

SUBSTANCE: invention relates to novel compounds, namely, to N'-{N-[3-oxo-lupan-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropionic acid and its salts of the formula (I) given in the invention description. This compound shows antiviral activity, in particular, anti-HIV activity, and immunostimulating activity. Compounds of the formula (I) are nontoxic and can be obtained from betulin isolated from birch bark as available raw with the high yield.

EFFECT: valuable medicinal properties of compound.

4 tbl, 9 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to compounds of the formula (I): wherein R1 and R2 are chosen independently from (C1-C4)-alkyl; R3 represents hydrogen atom or hydroxy-group; R4 represents (C1-C4)-alkyl; R5 represents hydroxy-group, or to its pharmaceutically acceptable salts, esters or amides. Also, invention relates to using these compounds as inhibitors of bile acids transfer in ileum for treatment of hyperlipemia. Also, invention describes methods for synthesis of these compounds and pharmaceutical compositions comprising thereof.

EFFECT: improved methods of synthesis, valuable medicinal properties of compounds and pharmaceutical composition.

12 cl, 2 ex

FIELD: medicine, hematology, organic chemistry, pharmacy.

SUBSTANCE: invention relates to novel peptidylarginals of the formula (I): Xaa-Xbb-Arg-H wherein Xaa means residue of alpha-substituted carbonic acid of the formula (II): Q-CH(R)-CO wherein Q means (C1-C3)-alkyloxycarbonylamino-group, methylamino-group or hydroxyl group; R means (C7-C9)-cycloalkylmethyl group or (C5-C7)-cycloalkyl group; Xbb means residue of L-proline or L- azethidine-2-carboxylic acid, and its additive acid salts formed by organic or inorganic acid. Intermediate compounds are described also. Compounds of the formula (I) possess the inhibitory effect on free thrombin, thrombin bound with a clot, Xa factor, plasmin and plasminogen activators that allows their using in pharmaceutical composition in treatment of a patient suffering from disseminated intravascular coagulation syndrome.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

17 cl, 4 tbl, 10 ex

FIELD: synthesis of biologically active compounds.

SUBSTANCE: invention provides 1,5-benzothiazepines of general formula I (formulae presented below), in which Rv and Rw are independently selected from hydrogen and C1-C5-alkyl; one of Rx and Ry represents hydrogen or C1-C6-alkyl and the other hydroxy or C1-C6-alkoxy; Rz is selected from halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1 of invention; v is a number from 0 to 5; one of R4 and R5 represents group of general formula IA; R3 and R6 and the second from R4 and R5 are independently selected from hydrogen, halogen, nitro, cyano, hydroxy, amino, carboxy, carbamoyl, mercapto, sulfamoyl, C1-C6-alkyl, and other residues indicated in claim 1; R3 and R6 and the second from R4 and R5 being optionally substituted by one or several R16 groups at their carbon atoms; D represents -O-, -N(Ra)-, -S(O)b- or -CH(Ra)-, wherein Ra is hydrogen or C1-C6-alkyl; and b=0-2; ring A represents aryl or heteroaryl and is optionally substituted by one or several substituents selected from R17; R7 represents hydrogen, C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R18; R8 represents hydrogen or C1-C4-alkyl; R9 represents hydrogen or C1-C4-alkyl; R10 represents hydrogen or C1-C4-alkyl, carbocyclyl, or heterocyclyl and is optionally substituted by one or several substituents selected from R19; R11 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORc)(ORd), -P(O)(OH)(ORc), -P(O)(OH)(Rd), or -(O)(ORc)(Rd), wherein Rc and Rd are independently selected from C1-C6-alkyl; or R11 represents group of general formula IB, in which X is -N(Rq)-, N(Rq)C(O)-, -O-, or -S(O)a, wherein a=0-2; and Rq is hydrogen or C1-C4-alkyl; R12 represents hydrogen or C1-C4-alkyl; R13 and R14 are independently selected from hydrogen, C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R23 can be optionally independently substituted by one or several substituents selected from R20; R15 represents carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORe)(ORf), -P(O)(OH)(ORe), -P(O)(OH)(Re), or -P(O)(ORe)(Rf), wherein Re and Rf are independently selected from C1-C6-alkyl; or R15 represents group of general formula IC, in which R24 is selected from hydrogen and C1-C4-alkyl; R24 is selected from hydrogen, C1-C4-alkyl carbocyclyl, heterocyclyl, and R27, of which C1-C4-alkyl, carbocyclyl, heterocyclyl, or R27 can be optionally independently substituted by one or several substituents selected from R28; R26 is selected from carboxy, sulfo, sulfino, phosphono, tetrazolyl, -P(O)(ORg)(ORh), -P(O)(OH)(ORg), -P(O)(OH)(Rg), or -P(O)(ORg)(Rh), wherein Rg and Rg are independently selected from C1-C6-alkyl; p=1-3; wherein meanings for R13 can be the same or different; q=0-1; r=0-3; wherein meanings for R14 can be the same or different; m=0-2; wherein meanings for R10 can be the same or different; n=1-3; wherein meanings for R7 can be the same or different; z=0-3; wherein meanings for R25 can be the same or different; R16, R17, and R18 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N,N-(di-C1-C4-alkyl)amino, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, and N,N-(di-C1-C4-alkyl)sulfamoyl; wherein R16, R17, and R18 can be optionally independently substituted by one or several of R21 at their carbon atoms; R19, R20, R23, R27, and R28 are independently selected from halogen, nitro, cyano, hydroxy, carbamoyl, mercapto, sulfamoyl, C1-C4-alkyl, C2-C4-alkenyl, C2-C4-alkynyl, C1-C4-alkoxy, C1-C4-alkanoyl, C1-C4-alkanoyloxy, N-(C1-C4-alkyl)amino, N.N-(di-C1-C4-alkyl)amino, C1-C4-alkanoylamino, N-(C1-C4-alkyl)carbamoyl, N,N-(di-C1-C4-alkyl)carbamoyl, C1-C4-alkyl-S(O)a (wherein a=0-2), C1-C4-alkoxycarbonyl, N-(C1-C4-alkyl)sulfamoyl, N,N-(di-C1-C4-alkyl)sulfamoyl, carbocyclyl, heterocyclyl, sulfo, sulfino, amidino, phosphono, -P(O)(ORa)(ORb), -P(O)(OH)(ORa), -P(O)(OH)(Ra), or -P(O)(ORa)(Rb), wherein Ra and Rb are independently selected from C1-C6-alkyl and wherein R19, R20, R23, R27, and R28 can be optionally independently substituted by one or several of R22 at their carbon atoms; R21 and R22 are independently selected from halogen, hydroxy, cyano, carbamoyl, mercapto, sulfamoyl, trifluoromethyl, trifluoromethoxy, methyl, ethyl, methoxy, ethoxy, vinyl, allyl, ethynyl, methoxycarbonyl, formyl, acetyl, formamido, acetylamino, acetoxy, methylamino, dimethylamino, N-methylcarbamoyl, N,N-dimethylcarbamoyl, methylthio, methylsulfinyl, mesyl, N-methylsulfamoyl, N,N-dimethylsulfamoyl; or pharmaceutically acceptable salt thereof, solvate, or salt solvate. Described are also method for preparing compounds of formula I, pharmaceutical compositions based on compounds I, and a method for achieving inhibiting effect relative to interscapular brown adipose tissue (IBAT), and intermediates. (I), (IA), (IB), (IC).

EFFECT: expanded synthetic possibilities in the 1,5-benzothiazepine series.

36 cl, 121 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to novel 4-(dipeptidylamino)-piperidine-1-carboxamidines of the general formula (1): or their optical isomers or pharmaceutical acceptable salts wherein R1 is chosen from hydrogen atom (H), lower alkyl, R4-CO wherein R4 means -OCCH2, R5-OCO wherein R means -SO2; R2 is chosen from lower alkyl, cycloalkyl, (C5-C12)-cycloalkylalkyl, phenylalkyl and others; R3 is chosen from H, -OH and group O-lower alkyl; R4 is chosen from H, lower alkyl and phenyl; R5 is chosen from lower alkyl, phenyl and benzyl. Proposed compounds are inhibitors of kallikrein and can be used in treatment of intestine inflammatory disease, arthritis, septic shock, hypotension or cancer.

EFFECT: valuable medicinal properties of compounds.

12 cl, 2 tbl, 60 ex

FIELD: organic chemistry, peptides, biochemistry.

SUBSTANCE: invention relates to compounds of the formula (I): , wherein R means hydrogen atom; R1 means carbocyclic aryl substituted optionally with halogen atom or (carbocyclic aryl or biaryl)-(lower)-alkyl substituted optionally with trifluoromethyl group or halogen atom; alk means (lower)-alkylene; R3 means hydrogen atom or acyl substituted optionally with alkoxy-group, morpholinyl, triazolyl or piperazinyl groups; R4 means hydrogen atom substituted optionally with methoxy-(lower)-alkyl or oxacycloalkyl group; R5 means hydrogen atom or (lower)-alkyl; R6 means (lower)-alkyl; R7 means (lower)-alkyl; or R6 and R7 in common with carbon atom to which they are bound mean 3-10-membered cycloalkylidene; X means -O-,-S(O)n-, -NHCO- wherein n = 0 or 2; -COOR2 means carboxyl or derivative of carboxyl group as a pharmaceutically acceptable ester wherein R2 is chosen from (lower)-alkyl, morpholine carbonylmethyl, dimethylaminocarbonylmethyl or diethylaminocarbonylmethyl, or its pharmaceutically acceptable salt. Proposed compounds are used as dual inhibitors of angiotensin-converting enzyme and neutral endopeptidase and as inhibitor of endothelin-converting enzyme also.

EFFECT: valuable medicinal and biochemical properties of dipeptides.

8 cl, 7 ex

FIELD: organic chemistry, medicine, endocrinology, pharmacy.

SUBSTANCE: invention relates to novel compounds of the formula (I): or their pharmaceutically acceptable salts and composition containing thereof that possess activity with respect to release of growth hormone. In the formula (I) R1 represents hydrogen atom or (C1-C6)-alkyl; R2 represents hydrogen atom or (C1-C6)-alkyl; L represents compound of the formula: wherein R4 represents hydrogen atom or (C1-C6)-alkyl; p = 0 or 1; r = 1; q, s, t and u = 0, 1, 2, 3 or 4 being independently of one another; the sum q + r + s + t + u = 0, 1, 2, 3 or 4; R9, R10, R11 and R12 represent independently of one another hydrogen atom or (C1-C6)-alkyl; Q represents >N-R12 wherein R13 represents hydrogen atom or (C1-C6); or L represents compound of the formula: , wherein r = 0 or 1; q, s, t and u independently of one another = 0, 1, 2, 3 or 4; the sum q + r + s + t + u = 0, 1, 2, 3 or 4; R9, R10, R11 and R12 represent independently of one another hydrogen atom or (C1-C6)-alkyl; Q represents >N-R13 or compound of the formula: , wherein o = 0, 1 or 2; T represents -N(R15)(R16) or hydroxyl; R13, R15 and R16 represent independently of one another hydrogen atom or (C1-C6); R14 represents hydrogen atom; G represents compounds of the formula or wherein R17, R18, R19, R20 and R21 represent independently of one another hydrogen atom or aryl; J represents compounds of the formula or wherein R22, R23, R24, R25 and R26 represent independently of one another hydrogen or halogen atom; a = 0, 1 or 2; b = 0, 1 or 2; c = 0, 1 or 2; d = 0 or 1; e = 0, 1, 2 or 3; f = 0 or 1; R5 represents hydrogen atom; R6 and R7 represent independently of one another hydrogen atom or (C1-C6)-alkyl; or R6 and R7 can form -(CH2)i-U-(CH2)j- optionally wherein i and j = 1, 2 or 3 independently one another, and U represent a valence bond; R8 represents hydrogen atom or (C1-C6)-alkyl; M represents arylene or -CR27=CR28- wherein R27 and R28 represent independently one another hydrogen atom or (C1-C6)-alkyl. Also, invention relates to a pharmaceutical composition comprising compounds of the formula (I) or its pharmaceutically acceptable salt as an active component in common with a pharmaceutically acceptable carrier or excipient. Also, invention relates to a method for stimulation of growth hormone secretion from mammalian hypophysis that involves administration to mammal the effective dose of compound or its pharmaceutically acceptable salt. Invention provides synthesis of novel compounds and their composition containing thereof that can be used in treatment of diseases associated with the growth hormone deficiency.

EFFECT: improve stimulating method, valuable medicinal properties of compounds and pharmaceutical composition.

13 cl, 29 ex

FIELD: pharmaceutical dosage forms.

SUBSTANCE: invention relates to compositions and methods for delivery of drugs. Composition according to invention represents powdered composite containing polymer, therapeutical agent, and complexing agent, said polymer containing one or more cyclodextrin fragments. Polymer interacts with complexing agent in a host/guest or guest/host manner resulting in formation of inclusion complex. Both polymer of composite and complexing agent may be used in a way to introduce functionality into therapeutical composition. Invention also discloses a method for preparing composition and to a method for delivering therapeutical agent.

EFFECT: widened choice of drug delivery methods.

26 cl, 31 dwg, 1 tbl, 65 ex

Peptide compounds // 2281955

FIELD: chemistry of peptides, medicine, pharmacy.

SUBSTANCE: invention relates to compound of the formula (I): wherein R1 represents benzofuranyl substituted with halogen atom or styryl substituted with halogen atom; R2 represents substituted hydroxyl substituted with mercapto-group or substituted sulfonyl, or its pharmaceutically acceptable salts. Compound of the formula (I) and its pharmaceutically acceptable salts possess the strong inhibitory effect on production of nitrogen oxide (NO) and can be useful in prophylaxis and/or treatment of NO-mediated diseases in humans and animals.

EFFECT: valuable medicinal and biochemical properties of compounds.

FIELD: chemistry of peptides, microbiology, biotechnology.

SUBSTANCE: L-alanyl-L-glutamine is prepared by incubation of a mixture containing a microorganism able to produce L-alanyl-L-glutamine from L-alanine ester and L-glutamine, L-alanine ester and L-glutamine, and isolation of the end product. Using the invention allows simplifying the process for preparing L-alanyl-L-glutamine. Invention can be used in pharmacy and food processing industry.

EFFECT: improved preparing method of dipeptide.

3 cl, 3 tbl, 1 ex

FIELD: medicine; pharmacology.

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

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

25 cl, 1 tbl, 2 ex

FIELD: organic chemistry, biochemistry.

SUBSTANCE: invention describes heterocyclic compounds represented by the general formula (I): and possessing elastase-inhibitory activity, and intermediate compounds for synthesis of such compounds. In the formula (I) R1 represents heterocyclic group represented by the formula (II): wherein A represents presence or absence of benzene ring; X represents oxygen atom, sulfur atom or -NH; Y represents nitrogen atom or -CH. Indicated heterocyclic group can be substituted with 1-3 substitutes that can be similar or different and they are chosen from group consisting of lower alkyl, lower alkoxy group and phenyl that can be optionally substituted with halogen-containing lower alkyl, lower alkoxy group or halogen atom; each among R2 and R3 represents hydrogen atom or hydroxyl, or R2 and R3 can be combined to form oxo group under condition that both are not hydrogen atoms.

EFFECT: valuable biochemical property of compounds.

8 cl, 7 tbl

FIELD: organic chemistry of natural compounds.

SUBSTANCE: invention relates to novel compounds, namely, to N'-{N-[3-oxo-lupan-28-oyl]-9-aminononanoyl}-3-amino-3-phenylpropionic acid and its salts of the formula (I) given in the invention description. This compound shows antiviral activity, in particular, anti-HIV activity, and immunostimulating activity. Compounds of the formula (I) are nontoxic and can be obtained from betulin isolated from birch bark as available raw with the high yield.

EFFECT: valuable medicinal properties of compound.

4 tbl, 9 ex

FIELD: medicine, biochemistry.

SUBSTANCE: invention describes compounds that inhibit function of NS3-protease encoded by hepatitis C virus.

EFFECT: valuable medicinal properties of inhibitors.

6 cl, 2 tbl, 472 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to compounds of the formula (I): wherein R1 and R2 are chosen independently from (C1-C4)-alkyl; R3 represents hydrogen atom or hydroxy-group; R4 represents (C1-C4)-alkyl; R5 represents hydroxy-group, or to its pharmaceutically acceptable salts, esters or amides. Also, invention relates to using these compounds as inhibitors of bile acids transfer in ileum for treatment of hyperlipemia. Also, invention describes methods for synthesis of these compounds and pharmaceutical compositions comprising thereof.

EFFECT: improved methods of synthesis, valuable medicinal properties of compounds and pharmaceutical composition.

12 cl, 2 ex

FIELD: pharmaceutical chemistry, chemistry of peptides, hormones.

SUBSTANCE: invention relates to a method for preparing analogs of adrenocorticotropic hormone (ACTH) (4-10) possessing neurotropic activity. Method for preparing analogs of adrenocorticotropic hormone (ACTH), a sequence (4-10), of the general formula (I): A-Glu-His-Phe-Pro-Gly-Pro-OH (I) wherein A means hydrogen atom (H), Met, Met(O), Lys, Ser, Trp, Ala, Gly, Thr is carried out by liquid-phase method by step-by-step splicing peptide chain beginning from C-terminal protected tetrapeptide of the formula: H-Phe-Pro-Gly-Pro-OH (II) wherein X means a protective group and using corresponding fully protected amino acids in activated form followed by removal of protective groups at each step and purification of the end product by liquid chromatography. Method provides simplifying the process and to enhance the yield of the end product.

EFFECT: improved preparing method.

5 cl, 1 tbl, 5 ex

FIELD: medicine, hematology, organic chemistry, pharmacy.

SUBSTANCE: invention relates to novel peptidylarginals of the formula (I): Xaa-Xbb-Arg-H wherein Xaa means residue of alpha-substituted carbonic acid of the formula (II): Q-CH(R)-CO wherein Q means (C1-C3)-alkyloxycarbonylamino-group, methylamino-group or hydroxyl group; R means (C7-C9)-cycloalkylmethyl group or (C5-C7)-cycloalkyl group; Xbb means residue of L-proline or L- azethidine-2-carboxylic acid, and its additive acid salts formed by organic or inorganic acid. Intermediate compounds are described also. Compounds of the formula (I) possess the inhibitory effect on free thrombin, thrombin bound with a clot, Xa factor, plasmin and plasminogen activators that allows their using in pharmaceutical composition in treatment of a patient suffering from disseminated intravascular coagulation syndrome.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

17 cl, 4 tbl, 10 ex

FIELD: biochemistry.

SUBSTANCE: invention relates to dipeptide mimetic selected from glutaminyl thiazolidine or glutaminyl pyrrolodine and salts thereof as well as to using of such compounds in treatment of abnormal glucose tolerance, glucoseuria, diabetes mellitus and other disordered as well as complications associated with diabetes mellitus in mammalians.

EFFECT: new effectors of dipeptidyl peptidase IV.

18 cl, 3 tbl, 2 ex

FIELD: biochemistry.

SUBSTANCE: invention relates to method for tripeptide production of formulae Ac-D-2Nal-D-4ClPhe-D-3Pal-OH and Boc-D-2Nal-D-4ClPhe-D-Pal-OH, which represent intermediates for synthesis of LHRH analogs in combination with acceptable heptapeptides in particular P1-Ser(P2)-NMeTyr(P3)-D-Lys(Nic)-Leu-Lys(iPr,P4)-Pro-D-AlaNH2 and P1-Ser(P2)-NMeTyr(P3)-D-Asn-Leu-Lys(iPr,P4)-Pro-D-AlaNH2 heptapeptides.

EFFECT: new synthetic intermediates for LHRH antagonists.

7 cl, 8 ex

FIELD: biotechnology, medicine, oncology.

SUBSTANCE: invention proposes peptide of the structure Tyr-Ser-Leu and a pharmaceutical composition based on thereof that is used for stimulating antitumor immune response. Also, invention proposes methods for treatment of mammal and for modulation of the immune response. Proposed inventions expand assortment of agents used in treatment of cancer diseases.

EFFECT: valuable medicinal properties of peptide and pharmaceutical composition.

20 cl, 48 tbl

FIELD: medicine.

SUBSTANCE: method involves detecting duodenum wall integrity disorders in performing endoscopic examination of duodenal ulcer after removing necrotic detritus from ulcer bottom. Available wall defect is subjected to endoscopic probing with catheter introducing water-soluble radiopaque solution through the probe and followed with fistulographic examination. The radiopaque solution being observed outside of duodenum, duodenal fistula or perforated ulcer are diagnosed.

EFFECT: high reliability of early stage diagnosis.

3 dwg

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