Oligonucleotide polymeric prodrugs

FIELD: medicine.

SUBSTANCE: invention refers to polymer conjugates of formula (I) comprising nucleotide or oligonucleotide residue, which can be applied for treatment of cancer and methods for their obtaining. where R1 and R2 independently represent H or polyalkylenoxyde, not necessarily having capping group selected from OH, NH2, SH, CO2N, C1-6 alkyls, compounds of formula (II) X2-(L2)n-(L1)o- and the formula (III) -(L4)p-(L3)m-X3, and when (o+n)≥2 each of n and o is a positive integer, each p and m are equal to zero, and R2 represents H, and when (p+m)≥2 each p and m is a positive integer, each n and o are equal to zero and R1 represents H; X1, X2, X3 are independently selected from a single-stranded or double-stranded oligonucleotide residue; L1 and L4 independently represent released link fragments; L2 and L3 are independently selected from bifunctional spacer groups.

EFFECT: developing of new nucleotide conjugates with antitumor activity.

21 cl, 25 ex, 7 tbl, 12 dwg

 

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of provisional patent application U.S. No. 60/462070, filed April 13, 2003, the contents of which are incorporated herein by reference.

The technical FIELD

The present invention relates to polymeric nucleotide prodrugs, suitable as therapeutic agents. Also proposed compositions and methods of using such prodrugs.

The LEVEL of TECHNOLOGY

It is well known that for most medical conditions multicellular organisms, including most painful conditions, is influenced by proteins. Such proteins through direct exposure or through their enzymatic or other functions to a large extent contribute to many diseases and regulatory functions in animals and humans. In the case of painful States of classical therapy is usually focused on the interaction with these proteins in an attempt to weaken their function, causing disease or aggravating disease. In the latest therapeutic approaches, it is desirable to adjust the existing production of such proteins. Affecting the production of proteins, it is possible to achieve maximum therapeutic effect with minimal side effects. Thus, the overall goal of such therapy is approach is to influence or modify another method of gene expression, which lead to the formation of undesirable protein.

One method of inhibiting the expression of a specific gene is to use oligonucleotides, particularly oligonucleotides, which are complementary to a specific sequence of the messenger RNA (mRNA) targets. Typically, the nucleic acid sequences, comlementary products of gene transcription (e.g., mRNA) is designated as "antisense", and nucleic acid sequences containing the same sequence as the transcript, or receive as a transcript denoted here as "semantic". See, for example, Crooke, 1992, Annu. Rev. Pharmacol. Toxicol.,32: 329-376. Antisense oligonucleotide can be selected for hybridization with the whole genome or a part of it, therefore, to modify the expression of this gene. Transcription factors interact with double-stranded DNA during transcription regulation. Oligonucleotides can serve as competitive inhibitors of transcription factors to modify their actions. Several recent messages describe such interactions (see Bielinska, A., et al., 1990, Science,250: 997-1000; and Wu, H., et al., 1990, Gene89: 203-209).

Develop molecular strategies down-regulating the expression of unwanted genes. Recently, the use of modified is the R oligonucleotide compounds has evolved into a promising method of treatment, directed against diseases such as viral infections, inflammatory and genetic diseases and, to a large extent, cancer. Antisense DNA were first proposed as alkylating complementary oligodeoxynucleotide against natural nucleic acids (Belikova, et al., Tetraxedron Lett.37: 3557-3562, 1967). Zamecnik and Stephenson were the first who suggested the use of synthetic antisense oligonucleotides for therapeutic purposes. (Zamecnik & Stephenson, 1978, Proc. Natl. Acad. Sci. U.S.A.,75: 285-289; Zamecnik & Stephenson, 1978, Proc. Natl. Acad. Sci. U.S.A.,75: 280-284). They reported 13-a measure of the oligonucleotide, complementary to RNA of rous sarcoma virus to inhibit the growth of virus in cell culture. Since that time, it was published many other studies showing the efficacy ofin vitroantisense oligonucleotides in inhibiting the growth of viruses, such as viruses, vesicular stomatitis (Leonetti et al., 1988, Gene, 72: 323), herpes simplex viruses (Smith et al., 1987, Proc. Natl. Acad. Sci. U.S.A., 83: 2787) and influenza virus (Seroa, et al., 1987, Nucleic Acids Res.,15: 9909).

Oligonucleotides have also found the use of, among others, in diagnostic tests, reagents for research, such as primers in PCR technology (PCR) and other laboratory operations. Oligonucleotides can be synthesized specifically to include functions, the cat is who adapted to be suitable for the desired application. Thus, the oligomeric compounds were implemented numerous chemical modifications to enhance suitability for diagnosis, as reagents for research and therapy elements.

Although oligonucleotides, particularly antisense oligonucleotides, show promise as therapeutic agents, they are extremely susceptible to the action of nucleases and can be quickly destroyed before and after they get into target cells, which makes unmodified antisense oligonucleotides unusable inin vivosystems. Because the enzymes responsible for their destruction, are present in most tissues, were modifications of oligonucleotides in attempts to stabilize the connection and fix this problem. The most widely studied modifications belong to the skeleton of oligonucleotide compounds. In General, see Uhlmann and Peymann, 1990, Chemical Reviews,90pages 545-561 and cited there links. Among the many received the frames, only phosphorothioate showed a significant antisense activity. See, for example, Padmapriya and Agrawal, 1993, Bioorg. & Med. Chem. Lett.3, 761. Although the introduction of sulfur atoms in the backbone slows down the rate of the enzymatic destruction, at the same time it increases the toxicity. Other deficiencies the introduction of sulfur atoms is what it does achiral chiral skeleton, which leads to 2nthe diastereomers. This may cause additional side effects. Other drawbacks of existing antisense oligonucleotides are that they can have a negative charge on the phosphate group, which inhibits their ability to pass through predominantly lipophilic cell membrane. The longer the connection remains outside the cell, the more it is destroyed and the less active compound is delivered to the target. An additional shortcoming of existing antisense compounds is that the oligonucleotides are prone to the formation in solutions of secondary structures and structures of higher order. Once these patterns are formed, they become targets for binding of various enzymes, proteins, RNA and DNA. This leads to non-specific side effects and reduces the number of active compounds that bind to the mRNA. Other attempts at improving nucleotide therapy has included the addition of the linker fragment and polyethylene glycol. See, for example, Kawaguchi et al., Stability, Specific Binding Activity, and Plasma Concentration in Mice of Oligonucleotide Modified at the 5'-Terminal with Poly(ethylene glycol), Biol. Pharm. Bull.,18(3)474-476 (1995), and U.S. patent No. 4904582. In both of these examples, the modifications relate to the use of the linker fragments on an ongoing basis with the aim of stabilizes and of the oligonucleotide from damage and increase cell permeability. However, both of these attempts have failed.

Because of the inadequacy of these methods there is a need to improve stability and resistance to degradation by nucleases, as well as reduce toxicity and increase binding affinity to mRNA oligonucleotide compounds. Used oligonucleotide therapy is extremely expensive. Mainly, this is due to the problem of destruction. Thus, there is a real need for protection antisense oligonucleotide compounds from destruction, preventing the formation of structures of high order and, at the same time, in the delivery of sufficient quantities of active antisense oligonucleotide compounds to the target. In the present invention include improvements.

The INVENTION

In one aspect of the present invention offers oligonucleotide prodrugs of formula (I):

in which

R1and R2independently represent H or a polymeric residue;

L1and L4independently represent released linker fragments;

L2and L3independently represent a spacer elements of the group;

X1represents a nucleotide residue or oligonucleotide residue;

m, n, o the R independently represent zero or a positive integer, provided that either (o+n), or (p+m)≥2.

Another aspect of the present invention includes bifunctional compounds that are formed when R1and/or R2are polymeric residues, which consist of both alpha - and omega - terminal of the linker group as described here, so that two oligonucleotide associated with a contemplated polymeric delivery systems. Examples of this variant implementation include oligonucleotides that are associated with the polymer systems through their respective 3'-, 5'-terminal group, such as 3'-bis-oligonucleotide conjugates or 5'-bis-oligonucleotide conjugates or conjugates formed by binding of the first oligonucleotide 3'-end to the 5'-end of the second oligonucleotide. Examples of such polymer conjugates is illustrated below as formula (i), (ii), (iii) and (iv):

bis-3'-oligonucleotide,

bis-5'-oligonucleotide,

bis-5',3'-oligonucleotide and

bis-3',5'-oligonucleotide,

where all variables are the same as described above.

For the purposes of the present invention, the term "residue" shall be understood to refer to a fragment of biologically active compounds, i.e. of the oligonucleotide, more specifically, antisense of Oleg the nucleotide which remains after he joined the substitution reaction, which was attached to the carrier prodrugs.

For the purposes of the present invention, the term "residue of a polymer or residue PEG" should be understood to refer to that part of the polymer or polyethylene glycol (PEG), which remains after he has entered into reaction with the modified oligonucleotide connection.

For the purposes of the present invention, the term "alkyl" should be understood as including an unbranched, branched, substituted, for example, halogen-, alkoxy-, nitro-, With1-12alkali,3-8cycloalkyl or substituted cycloalkyl etc.

For the purposes of the present invention the term "substituted" should be understood as including the addition or substitution of one or more atoms contained in the functional group or compound with one or more different atoms.

For the purposes of the present invention substituted alkali include carboxyacid, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethyl; replaced alkenyl include carboxyaldehyde, aminoalkyl, dialkanolamine, hydroxyalkyl and mercaptoethanol; substituted alkinyl include carboxykinase, aminoalkyl, dialkanolamine, hydroxyalkyl and mercaptoethanol; substituted cycloalkyl include fragments, such as 4-chlorocyclohexane is; arily include fragments such as naphthyl; substituted arily include fragments, such as 3-bromophenyl; arylalkyl include fragments such as toluyl; heteroalkyl include fragments such as ethylthiophene; substituted heteroalkyl include fragments, such as 3-methoxythiophene; alkoxy include fragments, such as methoxy; and phenoxy include fragments, such as 3-nitrophenoxy. Halogen is to be understood as comprising fluorine, chlorine, iodine and bromine.

The term "sufficient amount" or "effective amount" for purposes of the present invention is to indicate a quantity, which leads to a therapeutic effect, such as the effect implied by the experts in this field of technology.

Some of the main advantages of the present invention include novel polymeric oligonucleotide prodrugs that exhibit increased stability and resistance to degradation by nucleases, increased solubility, increased cell permeability and reduced toxicity.

Another advantage of the compounds of the present invention is that the modified oligonucleotide compounds added with the ability to release a variety of polymer proletarienne platform. This advantage allows the practitioner to develop a conjugate drugs, which which can be manipulated to enable different portions between the polymer residue and the attached oligonucleotide, thus, to influence the rate of hydrolysis of the prodrug. Specialist, thus, has the ability to include substituents that allow you to change the speed of hydrolysis of the prodrug.

Presents methods of production and application of these compounds, such as treatments for cancer or malignant tumors, as well as conjugates described herein. It is also assumed that the polymer oligonucleotide prodrugs according to the invention was administered, together (simultaneously and/or sequentially) any other suitable anti-cancer agent.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows schematically the means of obtaining polietilenglikolya oligonucleotides compounds 3 and 5.

Figure 2 shows schematically the means of obtaining polietilenglikolya oligonucleotides compounds 7 and 9.

Figure 3 shows schematically the means of obtaining polietilenglikolya oligonucleotides compounds 11, 12 (SEQ ID NO: 1) and 14 (SEQ ID NO: 1).

Figure 4 schematically shows the method of obtaining polietilenglikolya of the oligonucleotide compound 16 (SEQ ID NO: 1).

Figure 5 shows schematically the means of obtaining polietilenglikolya oligonucleotides compounds 17 (SEQ ID NO: 2), 18 (SEQ ID NO: 3) and 19 (SEQ IDNO: 4) from AS1 (SEQ ID NO: 2), AS2 (SEQ ID NO: 3) and AS3 (SEQ ID NO: 4).

6 schematically shows the means of obtaining polietilenglikolya oligonucleotides compounds 21 (SEQ ID NO: 1) and 22 (SEQ ID NO: 2).

7 schematically shows the means of obtaining polietilenglikolya oligonucleotides compounds 24 (SEQ ID NO: 1) and 26 (SEQ ID NO: 1).

Fig schematically shows the means of obtaining polietilenglikolya oligonucleotides compounds 28 (SEQ ID NO: 1), 29 (SEQ ID NO: 2), 30 (SEQ ID NO: 3) and 31 (SEQ ID NO: 4) of AS1 (SEQ ID NO: 2), AS2 (SEQ ID NO: 3) and AS3 (SEQ ID NO: 4).

Figure 9 shows schematically the means of obtaining polietilenglikolya oligonucleotides compounds 33 (SEQ ID NO: 1) and 35 (SEQ ID NO: 1).

Figure 10 shows the inhibitory effect of compound 14 and compound 28 on the growth RS cells. 0,4×104cells were planted in 96-well plates, were treated with complexes of compound 14 or connection 28 (400 nm) and lipofectin (15 μg/ml) for 24 hours in Opti-MEM and then complete environment without complexes. Cell viability was determined daily and the absorption was determined at 570 nm. Data are presented as mean ± standard deviation; n=4. The curves represent the following:

the control marked with ♦ and dotted line;

the connection 28 at 400 nm labeledand solid line;

the connection 14 at 400 nm marked with ▲ and dashed line;

the connection 28 at 200 nm labeled and a dashed line;

the connection 14 at 200 nm indicated ■ dotted line.

11 And represents the analysis of ROS production (from running cytometrical analysis) using oligonucleotides connection 14 and the connection 28, the determination of the oxidation penetrating into the cell 2',7'-dehydrochlorination diacetate to fluorescent 2',7'-dichlorofluorescein (DCF). RS cells were treated with oligonucleotide complexes (400 nm)/lipofectin (15 μg/ml) for 24 hours and analyzed after 3 days, as described. Multiple increase in the average channel fluorescence were normalized by comparison with untreated cells. Experiments were performed three times and data are presented as mean ± standard deviation (n=3).

Figv presents the analysis of ROS production (from running cytometrical analysis) using oligonucleotides connection 14 and the connection 28, the determination of the oxidation hydrohalide (NOT) to ethidium (E), which is then included in the DNA, fluorescently-defined flow cytometry. RS cells were treated with oligonucleotide complexes (400 nm)/lipofectin (15 μg/ml) for 24 hours and analyzed after 3 days, as described. Multiple increase in the average channel fluorescence were normalized by comparison with untreated cells. Experiments were performed three times and data are presented as mean ± standard on klonnie (n=3).

Fig represents the results of Western Blot confirming the inhibition of protein expression of bcl-2 compound 14 in the presence of lipofectin. RS-cells were treated with oligonucleotide compound 14 (200, 400 and 800 nm) in the presence (+Lipo) and in the absence (-Lipo) lipofectin in 24 hours in Opti-MEM and then for an additional 67 hours in complete medium. The protein samples (30-40 μg protein/lane) were analyzed by Western blotting as described in "Materials and methods", tubulin, used as a control protein samples. "C" denotes control.

DETAILED description of the INVENTION

Thus, the present invention provides polymer-linked oligonucleotide prodrugs having a large number of practical applications, including use as a diagnostic and analytical reagents as tools for research and scientific studies asin vitroandin vivoand as therapeutic agents. In order to more fully appreciate the scope of the present invention, defines the following terms. The specialist will be understood that the terms "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), single-stranded or double-stranded, unless otherwise provided, and to any chemical which is a mini-versions. "Oligonucleotide" generally represents a relatively short polynucleotide, for example, with a size ranging from about 2 to about 200 nucleotides, or more preferably, from about 10 to about 30 nucleotides in length. The oligonucleotides according to the invention are synthetic nucleic acids are single-stranded, unless otherwise indicated. As the synonyms can also be used the terms "polynucleotide" and "poliolefinovoy acid".

Modification of oligonucleotides according to the invention optionally include, for example, addition to or replacement of selected nucleotides functional groups or fragments, which makes it possible covalent binding of the oligonucleotide with a specific polymer, and/or the addition or substitution of functional fragments that tell the oligonucleotide additional charge, polarizability, hydrogen bond, electrostatic interaction, and functionality. Such modifications include, but are not limited to, modification of the 2'-position sugar modification, 5-position pyrimidine, modifications, 8-position purine modifications ekzoticheskih amines, substitution of 4-thiouridine, substitution of 5-bromo - or 5-iodouracil, modification of the side chain, methylation, a combination of complementary mating on the ground of nucleic acids, such as Sosnovaya socitey and isoguanine and similar combinations. Oligonucleotide modifications can also include 3'and 5'modifications such as capping.

The term "antisense" is used here to refer to nucleotide sequences that are complementary to a specific DNA or RNA sequence that encodes a gene product or the coding of the control sequence. The term "antisense chain" refers to a chain nucleic acid that is complementary to the "sense" circuit. During normal functioning of cellular metabolism semantic chain of the DNA molecule is a chain, which encodes the polypeptides and/or other gene products. Semantic chain serves as a matrix for the synthesis of the transcript of the messenger RNA ("mRNA") (antisense chain), which, in turn, directs the synthesis of any of the encoded gene product. Antisense molecules of nucleic acids can be obtained by any method known in the art, including synthesis by ligating the gene(s)that represent(s) of interest in a reverse orientation to a viral promoter, which makes possible the synthesis of a complementary chain. After introduction into the cell transcribed this circuit is combined with the natural sequences, synthesized the diversified cell with the formation of duplexes. These duplexes then block further transcription or translation. Thus can be generated mutant phenotypes. Indicate "negative" or "-" is also known in the art and relate to antisense chain and "positive" or "+" is also known in the art and refer to a semantic chain.

For example, if you need different regulation of expression of mRNA transcripts in a cell or cells, in the cell administered antisense oligonucleotide. After introduction into the cell antisense nucleotide hybridizes with the corresponding mRNA sequence by binding Watson-Crick, forming heteroduplex. Once formed duplex, inhibited protein translation of bound mRNAs encoded by the sequence. Thus, the antisense oligonucleotides are also used in the art as probes, such as probe hybridization normally associated with the identifier or label, as used to provide precision down-regulating the expression of certain cellular products or genetic regulatory elements, both for research and for therapeutic purposes.

For the purposes of the present invention using the singular or the plural does not mean limitation is to a certain numerical value in a given point or object. Thus, the use of the singular in relation to the cell, the polymer or the drug does not mean that only handle one cell, only one molecule, receive or use and/or use only one medication, and the use of the plural does not exclude the use of only one specified object, if not specified.

For the purposes of the present invention, the term "residue" shall be understood as part of a biologically-active compound, such as an oligonucleotide probe, which is stored after it has entered into reaction, in which a portion of the carrier prodrugs joined by modifying, for example, available hydroxyl or amino groups with the formation of, for example, ester or amide groups, respectively.

A. DESCRIPTION of OLIGONUCLEOTIDES

One of the features of the invention consists in the ability to provide enhanced nucleotide or oligonucleotide polymer conjugates. Described here polymeric transport system is not limited to one type of oligonucleotide, but, on the contrary, are designed to work with a wide variety of such fragments, implying that the polymer transport system can be attached to one or more 3'- or 5'-ends usually RHO4or SO4groups of the nucleotide. Sequence is lnasty nucleotides depicted here using accepted nomenclature, in which sequence reads from left to right, from the 5'-end to 3'-end(5'- → 3'-).

X1-3are the same or different nucleotide or oligonucleotide residues, which for the purposes of the present invention include oligodeoxynucleotide residues. More preferably X1-3are independently selected antisense oligonucleotide residues or oligodeoxynucleotide remains.

A non-limiting list of potential nucleotides, which can be used solely or as part of the oligonucleotide (10-1000 nucleotides include

,,

,,,

,

,and

in which

M represents O or S;

B1and In2independently selected from the group consisting of A (adenine), G (guanine), C (cytosine), T (thymine), U (uracil), and modified bases;

R100and R101independently selected from the group consisting of H, OR', where R' represents H, C1-6alkyl, substituted alkyl, nitro, halogen and the reel.

Some of these oligonucleotides and oligodeoxynucleotides used in the methods of the invention include, but are not limited to, the following:

oligonucleotides and oligodeoxynucleotides with natural postradiation skeleton or phosphorothioate skeleton, or any other modified backbone analogues;

SNK (locked nucleic acid);

NCP (peptide nucleic acid);

tricyclo-DNA;

false ONE (double-stranded oligonucleotide);

RNA (catalytic RNA sequence);

ribozymes;

mirror-targeted oligonucleotides (spiegelmers) (oligonucleotides with L-conformation);

CpG oligomers and the like, such as oligomers described in:

Tides 2002, Oligonucleotide and Peptide Technology Conferences, 6-8 may 2002, Las Vegas, NV, and

Oligonucleotide and Peptide Technology, 18 and 19 November 2003, Hamburg, Germany, the contents of which are incorporated herein by reference.

The oligonucleotides according to the invention can also optionally include any suitable well-known in the art nucleotide analogues and derivatives, including nucleotide analogs and derivatives are listed in table 1 below.

Table 1
Representatives of nucleotide analogues and derivatives
4-acetylcytidine 5-methoxyaminomethyl-2-thiouridine
5-(carboxyhydroxymethyl)uridinebeta, D-mannosidosis
2'-O-methylcytidine5-methoxycarbonylmethyl-2-thiouridine
5-carboxymethylaminomethyl-2-thiouridine5-methoxycarbonylmethylene
5-carboxymethylaminomethyl5-methoxyuridine
Dihydrouridine2-methylthio-N6-isopentenyladenosine
2'-O-methylpseudouridineN-((9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl) carbarnoyl)threonine
D-galactosylceramideN-((9-beta-D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine
2'-O-methylguanosineMethyl ester of uridine-5-exucuse acid
Inosinethe uridine-5-oxiana acid
N6-isopentenyladenosinewybutosine
1-methyladenosinepseudouridine
1-m is terpsitone cousin
1-methylguanosine2-thiocytidine
1-methylinosine5-methyl-2-thiouridine
2.2-dimethylguanosine2-thiouridine
2-methyladenosine4-thiouridine
2-methylguanosine5-methyluridine
3-methylcytidineN-((9-beta-D-ribofuranosylpurine-6-yl)-carbarnoyl)threonine
5-methylcytidine2'-O-methyl-5-methyluridine
N6-methyladenosine2'-O-methyluridine
7-methylguanosinewybutosine
5-methylaminomethyl3-(3-amino-3-carboxypropyl)uridine

Preferably the antisense oligonucleotide is an oligonucleotide which reduces expression of a protein that contributes to the resistance of tumor cells to anticancer therapy. For example, BCL-2 inhibits the release of cytochrome C and factor causing apoptosis, mitochondria and, thus, the pre is atitvout implementation apoptosis.

Cancer cells that have high levels of BCL-2, thus, is highly resistant to both chemotherapy and radiotherapy. U.S. patent No. 6414134, incorporated herein by reference, describes antisense oligonucleotides reduce the expression of the protein Bcl-2, which is associated with resistance to anticancer therapy in a large number of cancer cells, for example cells including prostate cancer, myeloma cells and other tumor cells. According to the above-mentioned U.S. patent is believed that the gene bcl-2 contributes to the pathogenesis of cancer, primarily increasing the survival rate of tumor cells, and not accelerating cell division. U.S. patent No. 6414134 in General describes antisense oligonucleotides ranging in length from 17 to 35 bases that are complementary to the mRNA of bcl-2 and which includes a nucleic acid molecule having the sequence TACCGCGGCGACCCTC (SEQ ID NO: 5). They preferably include at least one phosphorothioate connection.

Other known in the art of cellular proteins, which are examined by various companies as targets for decreasing their expression antimyeloma the oligonucleotides for cancer therapy, are listed in the following table 2.

Table 2
Protein-target
Affinitak (ISIS 3521)PKC-alpha
ISIS 112989 (OGX 011)Secretory protein clusterin
ISIS 23722Survivin
AP 12009TGF-Beta2
GEM 231Protein kinase a
GEM 240MDM2
IGF-1R/AS oneInsulin-like growth factor
MG98DNA methyltransferase
LErafAONC-raf-1
Ki-67 antisense oligonucleotideKi-67
GTI-2040Ribonucleotides
ISIS 2503H-ras
AP11014TGF-Beta1

The antisense oligonucleotides suitable for use in down-regulating the expression of proteins associated with the survival of cancer cells, such as expression of bcl-2, include oligonucleotides, who have from about two to two hundred nucleotide codons, more preferably from ten to forty codons, and most preferably from about 17 to 20 codons. These oligonucleotides are preferably chosen from oligonucleotides, complementary to the strategic sites in the chain precursor mRNA bcl-2, such as sites of translation initiation, donor or splicing sites, or sites of transfer or destruction.

Blocking broadcast in these strategic sites prevents the formation of functional bcl-2 gene product. It should be understood, however, that any combination or subcombination antibodiesa oligomers, including oligonucleotides, complementary to or substantially complementary to the bcl-2 precursor mRNA or mRNA that inhibit cell proliferation, suitable for use in the invention. For example, oligodeoxynucleotide, complementary parts of posledovatelnosti on adjacent or non-contiguous fragments of bcl-2 RNA can inhibit cell proliferation and are, therefore, suitable for use in the methods according to the invention.

Oligonucleotides suitable for down-regulating the expression of bcl-2, also include oligonucleotides, complementary to or substantially complementary to portions of the sequences flanking the strategic and other sites along the bcl-2 mRNA. Part of the flanking sequence is lnasty preferably range from about two to about one hundred and grounds before and after the previously mentioned sites along the bcl-2 mRNA. The length of these sites is preferably in the range from about five to about twenty codons. Also preferably, the oligonucleotides were complementary parts of the sequence of the precursor mRNA or mRNA, which is usually not contained in the precursor mRNA or mRNA of other genes, in order to minimize the homology of the oligonucleotide precursor mRNA or mRNA encoding the chains of other genes.

A number of preferred antisense or complementary oligonucleotides to down-regulation of bcl-2 are given in table 3.

Table 3
antisense oligonucleotide of translation initiation (TI-AS)3'...CCCTTCCTACCGCGTGCGAC...5' (SEQ ID NO: 6)
bcl-25'...CTTTTCCTCTGGGAAGGATGGCGCACGCTGGGAGA...3' (SEQ ID NO: 7)
antisense oligonucleotide donor splicing (SD-AS)3'...CCTCCGACCCATCCACGTAG...5' (SEQ ID NO: 8)
bcl-25'...ACGGGGTAC...GGAGGCTGGGTAGGTGCATCTGGT...3' (SEQ ID NO: 9)
antisense oligonucleotide acceptor splicing (SA-AS)3'...GTTGACGTCCTACGGAAACA...5' (SEQ ID NO: 10)
bcl-2 5'...CCCCCAACTGCAGGATGCCTTTGTGGAACTGTACGG...3' (SEQ ID NO: 11)

It will be clear that can be used antisense oligonucleotides that include more or less substituted nucleotides and/or who have more stretch along the bcl-2 mRNA chain or a 3'- or 5'-direction, relative to the oligonucleotides listed in table 3 above.

Preferably, the antisense oligonucleotides used in the prodrugs according to the invention, have the same, or substantially similar nucleotide sequence as has Genasense (a/k/oblimersen sodium produced by Genta Inc., Berkeley Heights, NJ). Genasense is an 18-dimensional phosphorothioate antisense oligonucleotide TCTCCCAGCGTGCGCCAT (SEQ ID NO: 1), which is complementary to the first six codons of the initiating sequence of bcl-2 mRNA man (human bcl-2 mRNA are well known in the art and described, for example, as SEQ ID NO: 19 in U.S. patent No. 6414134, incorporated herein by reference). The management under the control over products and medicines (FDA) gave Genasense the status of "Orphan" in August 2000 and has accepted the Application for a new drug (NDA) for Genasense for the treatment of cancer. NDA proposes the introduction of Genasense in combination with dacarbazine for the treatment of patients with generalized melanoma, which had not been previously treated with chemotherapy. Additionally, the FDA gave this C the turnout priority status examination (priority review), what is the purpose of the activity no later than June 8, 2004 Cm. also the publication of Chi et al., 2001, Clinical Cancer Research, vol. 7, 3920-3927 included here as a reference, confirming the activity of Genasense in combination therapy for prostate cancer in early clinical trials. Prodrugs of the present invention have the same uses as identified for the native (unmodified) 18-measure.

It was shown that Genasense is characterized by a decrease in the production of the protein Bcl-2 and enhances the sensitivity of tumor cells to therapy and ultimately causes cell death. A number of studies have shown promising results in the treatment of several types of cancer Genasense in combination with anticancer agents. Phase I/II trials of Genasense in combination with dacarbazine in patients with melanoma showed promising activity and phase III multicenter trial is underway. Additionally, Genasense, used in combination with mitoxantrone in patients with hormonerefractory prostate cancer have shown promising results. Kim et al., 2001, ibid.

The combination of antisense oligonucleotides, such as Genasense, with polymers is an example of one preferred variant of the invention.

In alternative embodiments, the implementation of additional suitable ant the sense oligonucleotides include:

T-C-T-C-C-C-A-G-C-G-T-G-C-G-C-C-A-T (junction 13, SEQ ID NO: 1);

T-C-T-C-C-C-A-G-C-A-T-G-T-G-C-C-A-T (junction 36, SEQ ID NO: 2);

A-T-C-C-T-A-A-G-C-G-T-G-C-G-C-C-T-T (compound 37, SEQ ID NO: 3); and

T-C-T-C-C-C-A-G-X-G-T-G-X-G-C-C-A-T (compound 38, SEQ ID NO: 4),

as well as oligonucleotides presented in other examples.

Century FORMULA (I)

In one preferred version of the invention are provided oligonucleotide prodrugs of formula (I):

in which

R1and R2independently represent H or a polymeric residue;

L1and L4are independently selectable released linker fragments;

L2and L3are independently selected spacer elements of the group;

X1represents a nucleotide residue or oligonucleotide residue;

m, n, o and p are independently selected from zero or a positive integer, provided that (o+n) or (R+m)≥2.

Polymer transport system of the present invention is based, at least in part, on one R1and R2preferably represents a polymer residue, optionally having kapperud group, denoted here as A. Suitable capirola groups include, for example, HE, NH2, SH, CO2H, C1-6alkali and oligonucleotides groups, such as

in which X2and X3or the same as X1or are other nucleotide or oligonucleotide residue.

Preferred capirola group (II) or (III) allow to form a composition with the formula (i), (ii), (iii) and (iv)shown below.

bis-3'-oligonucleotide,

bis-5'-oligonucleotide,

bis-5',3'-oligonucleotide and

bis-3',5'-oligonucleotide,

in which all variables are the same as described previously.

In another preferred embodiment of the invention L4is a released linker fragment selected from the formula

and

in which

Y1-25independently selected from the group consisting of O, S, or NR9;

R6-7, R9-13, R16-25and R27-41are independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl,1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy and C1-6/sub> heteroatomic;

Ar represents a fragment, which forms multiplexing aromatic hydrocarbon or multiplexing heterocyclic group;

L5-12independently represent a bifunctional spacers;

Z is chosen from the fragments, actively trasportare in the target cell, hydrophobic fragments, bifunctional linker fragments and their combinations;

c, h, k, l, r, s, v, w, v', w', c' and h' are independently selected positive integers;

a, e, g, j, t, z, a', z', e' and g' independently represent or zero, or a positive integer; and

b, d, f, i, u, q, b', d' and f' independently represent zero or one.

In another preferred embodiment, L1is a released linker fragment selected from the formulas:

and

in which

Y1'-25'are independently selected from the group consisting of O, S, or NR9;

R6'-7', R9'-13', R16'-25'and R27'-41'are independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, aryl is of kilov, With1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy and C1-6heteroatomic; and

L5'-12'are independently selected bifunctional spacers.

In some preferred versions of the invention, the L5-12independently represent a bifunctional spacers selected from the

and

and L5'-12'independently represent a bifunctional spacers selected from the

and

in which:

R55-R59and R55'-59'independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl,1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy and C1-6heteroatomic, and

R60and R60'selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl, the 1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy,1-6heteroatomic, NO2, halogenoalkane and halogen; and

s' and t'each represents a positive integer.

In another preferred variant of the invention, the L2and L3independently represent a spacer elements group having from about 1 to about 60 carbon atoms and from about 1 to 10 heteroatoms. Preferably L2and L3independently represent a spacer elements group having from about 2 to about 10 carbon atoms and from about 1 to about 6 heteroatoms. Most preferably L3choose from

and

and

most preferably L2choose from

and

in which

Q and Q' independently are selected from O, S and NH;

R50-53and R50'-53'independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl,1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy,1-6heteroatomic;

R54and R54 independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl,1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy,1-6heteroatomic, NO2, halogenoalkane and halogen; and

q' and r'each represents a positive integer.

In terms of other variables contained in the formula of the present invention, preferred are the following:

Y1-25and Y1'-25'are independently selected from the group consisting of O, S, or NR9;

R6-7, R9-13, R16-25, R27-41and R6'-7', R9'-13', R16'-25'and R27'-41'are independently selected from the group consisting of hydrogen, C1-6Akilov,3-8cycloalkyl, arrow, arylalkyl and C1-6heteroalkyl;

c, h, k, l, r, s, v, w, v', w', c' and h' represent the unit;

a, e, g, j, t, z, a', z', e' and g' independently represent or zero or one; and

b, d, f, i, u, q, b', d' and f' independently represent zero or one.

In another preferred embodiment, the invention provides compounds of formula (Ia):

in which/p>

L2is a spacer elements group;

X1represents a nucleotide or oligonucleotide residue;

u' is a positive integer;

T is a branched polymer, which is preferably selected from the compounds described in the application filed in the usual manner PCT publication number WO 02/065988 and WO 02/066066, each of which is incorporated herein by reference. Within this General formula, the following are preferred:

and

in which D' is one of the

and

in which R61represents a polymeric residue, such as defined for R1realizing that the polymer may be bifunctional, if R61shown with substituents at both ends; and all other variables are the same as described above.

To illustrate a non-limiting compound of formula (Ia) is:

in which all variables are the same as described above.

Another aspect of formula (Ia) include bifunctional compounds to the categories established in the case, if the polymer residue (R61includes both the alpha and the omega-terminal of the linker group, so delivered at least four of the oligonucleotide. Examples of such polymer conjugates is illustrated below as formula (vi) and (vii)

in which all variables are the same as described above.

In another preferred variant of the invention, the L2and L3independently represent a spacer elements group having from about 1 to about 60 carbon atoms and from about 1 to about 10 heteroatoms. Preferably L2and L3independently represent a spacer elements group having from about 2 to about 10 carbon atoms and from about 1 to about 6 heteroatoms. Most preferably L3choose from

and

and

most preferably L2choose from

and

,

in which

Q and Q' independently are selected from O, S or NH;

R50-53and R50'-53'independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, aryl is of kilov, With1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy,1-6heteroatomic;

R54and R54'independently selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl,1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl,1-6heteroalkyl, substituted C1-6heteroalkyl,1-6alkoxy, phenoxy,1-6heteroatomic, NO2, halogenoalkane and halogen; and

q' and r'each represent a positive integer.

In terms of other variables contained in the formula of the present invention, preferred are the following:

Y1-25and Y1'-25'are independently selected from the group consisting of O, S, or NR9;

R6-7, R9-13, R16-25, R27-41and R6'-7', R9'-13', R16'-25'and R27'-41'are independently selected from the group consisting of hydrogen, C1-6Akilov,3-8cycloalkyl, arrow, arylalkyl and C1-6heteroalkyl;

c, h, k, l, r, s, v, w, v', w', c' and h' represent the unit;

a, e, g, j, t, z, a', z', e' and g' independently represent or zero or one; and

b, d, f, i, u, q, b', d' and f' independently represent zero or one.

C. DESCRIBE THE S Ar FRAGMENT

In certain aspects of the invention can be seen that Ar fragment is a fragment, which when included in formula (I) forms politeley aromatic hydrocarbon or multiplexing heterocyclic group. The main characteristic is that Ar fragment is inherently aromatic. Usually, to be aromatic, the PI electrons must be distributed in the cloud above and below the plane of the circular molecule. Moreover, the number of PI electrons must satisfy the hückel rule (4n+2). Average experts in this field will understand that there are a countless number of fragments that will satisfy the requirements for aromaticity fragment in the formula (I) and, thus, are suitable for use here.

Some particularly preferred aromatic groups include:

in which R62-67independently selected from the same group, which determines R6.

Other preferred aromatic hydrocarbon fragments include, but are not limited to

in which E and E' independently represent CR68or NR69; and J represents O, S or NR70in which R68-70selected from the same group, as defined DL is R 6or cyano, nitro, carboxyl, acyl, substituted acyl or carboxyethyl. Also covered isomers five and six-membered cycles as benzo-and dibenzo-system and related related compounds. Specialist without special skills in this area will also be understood that the aromatic rings can optionally be substituted with heteroatoms such as O, S, NR9and so on, provided that the hückel rule is still observed. Moreover, aromatic or heterocyclic structures may also optionally be substituted by halogen(s) and/or the side chains, as these terms are generally understood in the art.

D. POLYALKYLOXY

Referring to the formula (I), one can see that R1and R2represent polymer fragments, such as polyalkylated. Suitable examples of such polymers include polyethylene glycols, which, essentially, are not antigenic. Also suitable polypropylenglycol, such as those described in the application filed in the usual way the U.S. patent nos 5643575, 5919455 and 6113906. Other PEG fit in the methods according to the invention described in Shearwater Polymers, Inc. catalog "Polyethylene Glycol and Derivates 2001". A description of each of these is included here as a reference. R1and R2preferred are PEG derivatives, such as-O-(CH2 CH2O)x-. In this aspect, R1-2are independently selected from

J-O-(CH2CH2O)n'-

J-O-(CH2CH2O)n'-CH2C(O)-O-,

J-O-(CH2CH2O)n'-CH2CH2NR48-,

J-O-(CH2CH2O)n'-CH2CH2S-,

-O-C(O)CH2-O-(CH2CH2O)n'-CH2C(O)-O-,

-NR48CH2CH2-O-(CH2CH2O)n'-CH2CH2NR48-,

-SCH2CH2-O-(CH2CH2O)n'-CH2CH2S-,

in which

n' represents the degree of polymerization, selected in such a way that srednevekovaja molecular weight is at least 2,000 Da to about to 136,000 Yes;

R48selected from the group consisting of hydrogen, C1-6Akilov,3-12branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl, C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and C1-6heteroatomic, and

J represents kapperud group, such as methyl or an optional linker group, which allows to obtain a bifunctional polymer.

Although RAO and PEG can largely differ in molecular weight, preferably R1The R 2independently have srednevekovoy molecular weight of from about 2000 Da to about to 136,000 Yes in most aspects of the invention. More preferably R1and R2independently have srednevekovoy molecular weight of from about 3,000 Da to about 100,000 Da, with the most preferred srednevekovoi molecular weight of from about 5,000 Da to about 40,000 Da.

Polymeric substances included herein are preferably water-soluble at room temperature.

A non-limiting list of such polymers include homopolymers of polyalkylated, such as polyethylene glycol (PEG) or polypropylenglycol, polyoxyethylenated a polyalcohol, their copolymers and block copolymers, providing, to maintain the solubility of the block copolymer in water.

E. SYNTHESIS of CONJUGATES of OLIGONUCLEOTIDES WITH POLYMERS

Usually prodrugs get

(a) the interaction of the compounds of formula:

R2-L4-withdrawing group

with the compound of the formula

N-L3-X1

under conditions sufficient for the formation of prodrugs of formula

R2-L4-L3-X1,

in which

R2represents a polymeric residue;

L4is a released linker fragment.

L3is a spacer elements group;

X1pre what is a nucleotide or oligonucleotide residue.

In this aspect of the invention, it is preferable to use activated polymers that include attached released linkers. A non-limiting list of suitable combinations include a disposable transport system based on PEG, as described in the application filed in the usual way U.S. patents№№ 6624142, 6303569, 5965119, 6566506, 5965119, 6303569, 6624142 and 6180095, the contents of each of which is incorporated herein by reference.

Specific examples include, but are not limited to

of course, it is understood that the molecular weight of the polymer part may vary according to the needs of the manufacturer.

Then releasing the polymer linker, shown above, is introduced into reaction with modified oligomer under conditions that allow the formation of the conjugate.

Any of the nucleotides or oligonucleotides described above can be functionalized by one of the 5'- or 3'-terminal phosphate or phosphorothioate using standard methods, such as phosphoramidate ways to attach the specified alkylamine group or the other to the terminal phosphate. For example, attach blocked (Fmoc) aminoalkyl, the compound obtained oxidize, remove protection and clean.

The synthesis of certain conjugates of oligonucleotides with polymers or prodrugs of Slagelse in the examples. Alternatively, the prodrug can be obtained:

1) introduction to reaction of the activated PEG polymer with a protected bifunctional released linker group under suitable conditions, the combinations for the formation of the first intermediate product,

2) removing the protection and activation of the intermediate product from stage 1) suitable activating group such as NHS ester, and

3) the introduction of the activated intermediate product from stage 2) in the reaction with modified oligonucleotide in PBS buffer system for a given oligonucleotide-polymer prodrugs.

A non-limiting list of activated polymers include bis-Succinimidyl-carbonate-activated PEG (SC-PEG), bis-thiazolidin-2-tion-activated PEG (T-PEG), N-hydroxyphthalimide-carbonate-activated PEG (BSC-PEG) (see submitted in the usual way USSN 09/823296, the description of which is incorporated herein by reference), Succinimidyl-succinate-activated PEG (SS-PEG) and monooctylamine PEG, such as those described in, for example, in the above-mentioned 2001 Shearwater Catalog.

Conjugation of activated PEG polymer with a protected bifunctional released linker group can be carried out in the presence of a condensing agent. A non-limiting list of suitable condensing agents include 1,3-diisopropylcarbodiimide (DIPC),any suitable dialkylammonium, halides 2-halogen-1-alkylpyridine (reagents Mukaiyama), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), a cyclic anhydride papapostolou acid (PPACA) and phenyldichlorophosphine, etc. that are available, for example from commercial sources such as Sigma-Aldrich Chemical, or can be synthesized using known methods.

Preferably the substituents introduced into the reaction in an inert solvent, such as tetrahydrofuran (THF), acetonitrile (CH3CN), methylene chloride (DCM), chloroform (CHCl3), dimethylformamide (DMF) or their mixtures at temperatures from 0°C to about 22°C (room temperature).

Conjugation of the modified oligonucleotide with PEG-releasing linker can be carried out in PBS-buffer system at pH in the range of approximately 7.4 to 8.5. The person skilled in the art will understand that described in the synthesis of prodrugs also includes the use of typical laboratory conditions, such as solvent, temperature, binding agents, etc. such as described in the examples.

Regardless of the synthesis of some preferred compounds which is obtained by using the synthetic methods described herein include

and

<> in which

represent oligonucleotide and place modification of the terminal phosphate, and mPEG is a CH3-O-(CH2-CH2-O)X-, in which x is a positive integer selected from about 10 to about 2300.

More preferred compounds according to the invention include:

and

G. TREATMENT

Another aspect of the present invention provides methods of treatment of various diseases in mammals. The methods include administration to a mammal in need of such treatment, an effective amount of oligonucleotide prodrugs, which was obtained as described here. Compositions used for the treatment of other diseases, tumors, reduction of the severity of the tumor, preventing metastasis of neoplasms and preventing recurrences of tumor growth/tumors, liver disease, viral diseases such as HIV, in mammals. Prodrugs of the present invention can be used in all indications for which use native oligonucleotide and antisense oligonucleotides, i.e. in the treatment of cancer, etc. only as examples of prodrugs according to the invention Ave is delagoutte for use in the treatment of multiple myeloma, chronic lymphocytic leukemia, non-small cell lung cancer, small-cell lung cancer, prostate cancer and other tumors or cancers that are too numerous to list.

The number of input prodrug will depend on the initial constituent molecules and the diseases being treated. Normally, the amount of prodrug used in the treatment represents a quantity that effectively reaches a given therapeutic effect in mammals. Naturally, different doses proletarienne substances will be slightly different depending on the source connection, the speed of hydrolysis in vivo (in vivo), the molecular weight of the polymer, etc. the Range specified above is provided for illustrative purposes, and specialists in this field will determine the optimal dose selected prodrugs based on clinical experience and evidence of treatment. The actual dose will be clear to the specialist without excessive experimentation.

Prodrugs of the present invention can be included in one or more suitable pharmaceutical compositions for the introduction of mammals. The pharmaceutical compositions can be in the form of solution, suspension, tablet, capsule, etc. obtained according to methods well known in the village is authorized engineering. It is also assumed that the introduction of such compositions can be performed oral or parenteral routes, depending on the need of a specialist. The solution and/or suspension of the composition can be applied as the basis of the carrier for injection or administration of any composition known in the art methods, for example intravenously, intramuscularly, subcutaneously, injection, etc.

Such introduction can be carried out by infusion in the departments or the body cavity, as well as by inhalation and/or internasals. However, in preferred aspects of the invention, the prodrug is administered mammal in need of them, parenteral.

It is also assumed that the prodrugs according to the invention can be introduced in combination (e.g., simultaneously and/or sequentially) with other known in the field of anticancer agents. Suitable anti-cancer agents include, only as an example, to list a few such agents: (Paclitaxel; Briston Myers Squibb); Camptosar® (Irinotecan; Pfizer); Gleevec® (Imatinib mesilate; Novartis); Rituxan® (Rituximab; Genentech/IDEC); Fludara® (fludarabine; Berlex Labs); ytoxan® (cyclophosphamide; Bristol Myers Squibb); Taxotere® (Docetaxel; Aventis Pharmaceuticals); Mylotarg® (Gemtuzumab ozogamicin; Wyeth-Ayerst); Cytosine arabinoside and/or dexamethasone.

EXAMPLES

The following examples serve to ensure dalneishih the understanding of the invention, but in no way limit the effective scope of the invention. The underlined and bold the numbers listed in the examples correspond to the numbers shown on the drawings. In each of the drawings sugar fragment and phosphate backbone is represented in the form

Designation mPEG should be understood as representing

The General procedure.All the conjugation reaction between PEG-linkers and oligonucleotides was performed in PBS buffer system at room temperature. Extraction with an organic solvent is usually removed unreacted oligonucleotides, followed by anion-exchange chromatography separated the PEG-oligoanuria from unreacted excess PEG linkers to obtain pure products.

The HPLC methods.The reaction mixture and the purity of the intermediates and final products were analyzed by HPLC Beckman Counter System Gold® when using BOND® 300 SB C-8 column with reversed phase (150×4.6 mm) or a Phenomenex Jupiter® 300A C18 column with reversed phase (150×4.6 mm) with multivalvular UV detector, using a gradient of 30 to 90% acetonitrile in 0.5% triperoxonane acid (TFA) and 25-35% acetonitrile in 50 mm TEAA buffer with 4 mm TBACl at a flow rate of 1 ml/min Anion-exchange chromatography was performed on a Bio-Cad 700E Perfusion Chromatography Workstation from Applied Biosystems use or Poros 50HQ strong anion-exchange resin from Applied Biosystems or DEAE Sepharose Fast flow weak anion-exchange resin from Amersham Biosciences, Packed in AP-empty glass column from Waters. Salting out was performed using a HiPrep 26/10 or PD-10 vasilevousa columns from Amersham Biosciences.

Example 1

The connection 3. A solution of compound 1 (440 mg, being 0.036 mmol) and 2 (5 mg, 3.6 μmol) in PBS buffer (10 ml, pH 7.4) was stirred at room temperature for 12 hours. The reaction solution was extracted with methylene chloride (DCM, 3×10 ml) and the combined organic layers were dried (MgSO4), filtered and the solvent evaporated under reduced pressure. The residue was dissolved in double-distilled water (1.5 ml per 100 mg) was loaded in HQ/10 Poros strongly anion-exchange column (10 mm × 60 mm, the volume of the layer of ~6 ml). Unreacted PEG linkers were suirable water (3~4 volume of the column) and then the product was suirable 0.2 M solution of NH4HCO3(~2 volume of the column). The fractions containing pure product were combined and liofilizirovanny obtaining pure 3 (19 mg, 1.44 mmol, 40%).

Examples 2-6

Compounds 5, 7, 9, 11 and 12 were obtained and purified in the same manner as 3 with outputs in the range from 30% to 50%.

Example 7

The connection 14. To a solution of compound 13 (10 mg, 1.7 μmol) in PBS buffer (5 ml, pH 7.4) was added 10 (175 mg, 85 μmol) in five equal parts every hour and stirred at room temperature for 12 hours. The reaction solution was extracted with DCM (3×10 ml) and saturated saline (10 m is) and the combined organic layers were dried (MgSO 4), filtered and the solvent evaporated under reduced pressure. The residue was dissolved in double-distilled water (1.5 ml) and was loaded onto DEAE Fast flow, weak anion-exchange column (10 mm × 60 mm, the volume of the layer of ~6 ml), which was pre-calibrated 20 mm Tris-HCl buffer, pH 7.4. Unreacted PEG linkers were suirable water (3 to 4 column volumes) and then the product was suirable with a gradient from 0 to 100% 1 M NaCl in 20 mm Tris-HCl buffer 7.4 for 10 minutes, followed by 100% of 1 M NaCl for 10 minutes, with a flow rate of 3 ml/min Fractions containing pure product were combined and vysalivanie on PD-10 vasilevousa column with 0.2 m solution of NH4HCO3(~2 volume of the column) and the resulting solution liofilizirovanny obtaining pure 14 (25 mg, 0.95 mmol, 57%).

Example 8

Compound 16 was obtained and was purified in the same manner as 14 with output of 60%.

Example 9

The connection 17. To a solution of AS1 (5 mg, 0.85 mmol) in phosphate buffer (2 ml, pH of 7.8) was added 10 (175 mg, of 0.085 mmol), which was divided into five equivalent parts, for 2 hours and the resulting solution was stirred at room temperature for additional 2 hours. The reaction solution was extracted with DCM (3×6 ml) and saturated brine (5 ml) and the combined organic layers were dried (MgSO4), filtered and the solvent evaporated under reduced pressure. The remainder of the solution is whether in double-distilled water (5 ml) and was loaded onto DEAE fast flow, weak anion-exchange column (10 mm × 60 mm, the volume of the layer of ~6 ml), which was pre-calibrated 20 mm Tris-HCl buffer, pH 7.4. Unreacted PEG linkers were suirable water (3 to 4 column volumes) and then the product was suirable with a gradient from 0 to 100% 1 M NaCl in 20 mm Tris-HCl buffer, pH 7.4 for 10 minutes, followed by 100% of 1 M NaCl at a flow rate of 3 ml/min for 10 minutes. The fractions containing pure product were combined and vysalivanie on PD-10 vasilevousa column and the resulting solution liofilizirovanny obtaining pure 17 (15 mg, or 0.57 mmol, 67%).

Examples 10-11

Compounds 18 and 19 were obtained and purified in the same manner as 17 with yields of final products 67%, when using AS2 and AS3 instead of AS1.

Examples 12-15

Compound 21 was obtained and was purified in the same manner as 14, by replacing 12 at 20 with output equal to 90%.

Compound 22 was obtained and was purified in the same manner as 14, by replacing 12 at 20 with the release of 65%.

Compound 24 was obtained and was purified in the same manner as 14, by replacing 12 at 23 and vysalivaniya used water (~2 volume of the column) for elution of the product instead of a 0.2 M solution of NH4HCO3. The final yield was 30%.

Compound 26 was obtained and was purified in the same manner as 24, by replacing 23 to 25. The yield was 30%.

Example 16

The connection 27. To a solution of 13 (10 mg, 1.7 μmol) in phosphate buffer (5 ml, pH 8.5) is obavljale 27 (180 mg, 0,084 mmol), which was divided into ten equivalent parts and the resulting solution was stirred at room temperature for 4 days. The reaction solution was extracted with DCM (3×10 ml) and saturated saline (10 ml) and the combined organic layers were dried (MgSO4), filtered and the solvent evaporated under reduced pressure. The residue was dissolved in double-distilled water (1.5 ml) and was loaded onto DEAE fast flow, weak anion-exchange column (10 mm × 60 mm, the volume of the layer of ~6 ml), which was pre-calibrated 20 mm Tris-HCl buffer, pH 7.4. Unreacted PEG linkers were suirable water (3 to 4 column volumes) and then the product was suirable with a gradient from 0 to 100% 1 M NaCl in 20 mm Tris-HCl buffer, pH 7.4 for 10 minutes, followed by 100% of 1 M NaCl with a flow rate of 3 ml/min for 10 minutes. The fractions containing pure product were combined and vysalivanie on PD-10 vasilevousa column and the resulting solution liofilizirovanny obtaining 14 (105 mg, 0,0102 mmol, 60%). Product purity was determined by HPLC.

Examples 17-21

Compounds 29, 30 and 31 was obtained and was purified in the same manner as 28, except that 13 was replaced by AS1, AS2 and AS3, respectively. For each of the final products was obtained, yield 65%.

Compound 33 was obtained and was purified in the same manner as 14, by replacing 12 to 32, which led to the exit 76%.

Compound 35 was obtained and chiwele in the same way, 14, except that instead of 10 used activated PEG 34. The final yield was 30%.

BIOLOGICAL DATA

Somein vitroproperties of PEG-oligoanuria presented below:

Table 4
In vitroproperties of PEG-oligoanuria
Connectiont1/2(buffer)t1/2(rat plasma)t1/2(PE I)t1/2(PE II)t1/2(nuclease P1)
3>>24 h0,7 h<5 min>24 h<5 min
7>>24 h0.5 h<5 min>24 h<5 min
5>>24 h0.5 h<5 min>24 h<5 min
9>>24 h3.7 p<5 min360,3 h
11>>24 h2,3 h<5 min630,3 h
12>>24 h10,6 h<5 min1,7<5 min
14>>24 h14,7 h<5 min38,90,3 h
16>>24 h13,8 h<5 min42,70,3 h
21>>24 h6.5 h>24 h>24 h<5 min
24>>24 h11 h18,7 h 36,8 h<5 min
28>>24 h>48 h>24 h>48 h<5 min
33>>24 h>48 h>24 h>48 h<5 min
35>>24 h4,1 h5,6 h>48 h<5 min

PE I = phosphodiesterase I, 5'-exonuclease, the kinetics was investigated at pH 8.8, TEAA buffer at 37°C; PE II = phosphodiesterase II, 3'-exonuclease, the kinetics was investigated at pH 6.5 TEAA buffer at 37°C; Nuclease P1 = endonucleases, the kinetics was investigated at pH 5.3 TEAA buffer at 37°C; for all enzymes 1 unit releases 1 μmol of oligonucleotide.

Pharmacokinetics RED oligoanuria ICR mice

The General procedure

1) the keeping of animals: mice were placed at 6 in the cell, in crates for breeding. The size of the cells selected in accordance with the “Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council.

2) diet: mice had access to water supply the water and were fed with commercially available feed for laboratory animals enough ( ad libitum).

3) preparation of the compounds: compound 13 was dissolved in 4.0 ml of salt solution and compound 14 was dissolved in 4.1 ml of saline solution.

4) the Place of administration: connections 13 and 14 was administered as a single dose (day 1) through tail vein.

The research plan

Sixty (60) mice were chosen, injected drug and bled according to the following plan, shown in table 5 below.

Table 5
GroupTxNDose (mg/kg)Dose* (mg/kg)InjectionThe time of bleeding (h)Volume (μl)
11431204iv0,031000
21431204iv0,251000
31431204iv0,51000
41431204iv1 1000
51431204iv31000
61431204iv61000
71431204iv 241000
81431204iv481000
91431204iv721000
1014 31204iv961000
1113344iv0,031000
1213344iv0,25 1000
1313344iv0,51000
1413344iv11000
1513344iv 31000
1613344iv61000
1713344iv241000
181334 4iv481000
1913344iv721000
2013344iv96

iv = intravenous

*oligoelement

Three (3) untreated mice were bled by heart puncture in tubes containing EDTA to collect untreated control plasma.

The mice were injected intravenously original connection 13 or 14 100 ál of each mouse. After sedation 0,09% of Avertin mice were completely eviscerated ~1000 ál by puncture of the heart. Blood was collected in vials containing EDTA. Plasma was collected after centrifugation of blood and immediately frozen at -80°C on dry ice.

Clinical studies

Mice were inspected visually every day after infusion of the analyte on mortality and signs of reaction to treatment. Noted any mortality and clinical manifestations. Body weight was measured just before the day of injection.

Pharmacokinetic studies of compounds 28 and 33 carried out in the same way.

Results studies

The pharmacokinetic results are presented in the following table 6 below.

Table 6
In vivoproperties of PEG-oligoanuria
ConnectionCmax (mg/ml)The plasma half-life (h)AUC (h·µg/ml)
1314,90,194,1
14of 54.80,6651,8
28491,21,05730,6
33556,70,25191,0

Examples 22-25

Confirmationin vitroactivity of antisense PEG conjugates

As it has been shown that bcl-2 has significant anti-apoptotic activity in prostate cancer cells. Decreasing regulation of expression of the protein bcl-2 in cancer cells of the prostate gland was confirmed by cell death and the induction of cell death bcl-2 antimyeloma PEG-conjugates were used to confirm successful intracellular delivery of antisense oligonucleotides.

Materials and methods for examples 22-25.

The investigated compounds are presented in table 7 below

Table 7
ConnectionDescription
14 5'G aromatic released
285' - constant
335'G → AND aromatic released
2424k-mPEG-BCN3-5' aliphatic released
3520k-mPEG-RNL9 5' released (intermediate t1/2in rats)

Were obtained as described above.

Cell culture

Not containing Mycoplasma RS cells, obtained from American Type Culture Collection (Rockville, MD), were grown in Roswell Park Memorial Institute medium (“RPMI”) (Invitrogen, Grand Island, NY) supplemented with 10% amniotic calf serum (FBS)containing 10% (vol./about.) V / V heat inactivated (56°C) FBS plus 1% nonessential amino acids, 1% pyruvate, 25 mm HEPES (N-2-hydroxyethylpiperazine-N'-2-econsultancy acid) buffer, 100 u/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. Uterine cultures were maintained at 37°C in an incubator with humidified 5% atmosphere of CO2.

Reagents

FBS and lipofectin (liposomal composition of the cationic lipid N-[1-(2,3-dialerace)propyl]-n,n,n-ammonium chloride) was purchased from Invitrogen (Grand Island, NY). Anti-bcl-2 monoclonal antibody was from Dako (Carpinteria, CA). Anti-α-tubulin monoclonal antibody and 3-(4,5-dime iltiazem-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). Phosphorothioate oligonucleotides were synthesized and purified using standard methods.

Transfection of olignucleotides

Cells were sown on the day before the experiment in 6-hole tablets with a density of 25×104cells per well, with 60-70% confluence on the day of the experiment. All transfections were performed in Opti-MEM medium in the absence of FBS and antibiotics, as described in the instructions of the manufacturers. Suitable amounts of reactants were diluted in 100 μl Opti-MEM medium with a final concentration lipofectin and the oligonucleotide. The solution was carefully mixed and pre-incubated at room temperature for 30 minutes, allowing the formation of complexes. Then was added 800 μl of Opti-MEM solution was mixed and applied to cells that were pre-washed with Opti-MEM. The incubation period for oligonucleotide complexes/lipofectin in Opti-MEM was 24 hours, followed by incubation in complete medium containing 10% FBS. The total incubation time before cell lysis and protein isolation was usually 72 hours at 37°C.

Western blot analysis

Cells treated with complexes of oligonucleotide-lipid, washed in PBS and then were extracted in lytic buffer [50 mm Tris-HCl pH to 7.4, 1% NP-40, 0.25% of desoxycholate sodium, 150 mm NaCl, 1 mm EGTA, 50 μg/ml Pefabloc SC, 15 μg/ml Aprotinin, leupeptin, hemostasia, pepstatin And 1 mm Na3 VO4, 1 mm NaF] at 4°C for 1 hour. Cell fragments were removed by centrifugation at 14000 g for 20 minutes at 4°C. protein Concentration was determined using Bio-Rad system for protein analysis (Bio-Rad Laboratories, Richmond, CA). Aliquots of cell extracts containing 25-40 μg of protein, were separated by SDS-PAGE and transferred to Hybond ECL filter paper (Amersham, Arlington Heights, IL) and the filters were incubated at room temperature for 1-2 hours in 5% BSA in PBS containing 0.5% Tween-20. The filters are then examined the 1:500 diluted anti-bcl-2 antibody in 5% BSA in PBS containing 0.5% Tween-20 at 4°C over night. After washing in PBS containing 0.5% Tween-20, the filters were incubated for 1 hour at room temperature in 5% milk in PBS containing 0.5% Tween, diluted 1:3000 peroxidase conjugated secondary antibody (Amersham). After washing (C minutes) was performed electrochemical luminescence (“ECL”) according to the manufacturer's instructions.

Determining the speed of cell proliferation

The influence of PEG conjugates on cell viability was determined by MTT assay. Briefly, 15-20×104cells were sown in 6-hole tablets and allowed to settle over night. Then cells were treated with appropriate concentrations of complexes of oligonucleotides with lipofectin for 24 hours at 37°C, followed by incubation in complete medium (100 μl)containing the th 10% FBS. Cell viability was determined daily. 10 μl of 5 mg/ml MTT in PBS was added to each well followed by incubation for 4 hours at 37°C. Then, to each well was added 100 μl of 10% SDS in 0.04 M HCl, followed by incubation overnight at 37°C to dissolve crystals formazan. The absorption was determined at 570 nm using a Benchmark plus Microplate spectrophotometer (Bio Rad, Hercules, CA). Experiments were performed 6 times and data were presented as the mean +/- S.D. (standard deviation).

Quantitative analysis of intracellular levels of ROS

2',7'-DICHLORODIFLUOROMETHANE diacetate (H2DCF-DA) and dihydrouridine (NOT) used for the determination of reactive oxygen species (“ROS”) and superoxide. Both dyes are afluorescent and can freely diffuse into cells. When NOT oxidized to amidine (E), is embedded in the cellular DNA and fluorescent. Oxidation of H2DCF-DA leads to the formation of 2',7'-dichlorofluorescein (DCF), which is also fluorescent, and he and the other can be determined by flow cytometry. The cells were collected by treatment with trypsin, washed with PBS and stained with 50 µm H2DCF-DA or 50 μm'T phenol containing no red DMEM for 2 hours at 37°C. the Average number of fluorescent channels DCF and E were analyzed using about the internal cytometry in FL-1 and FL-2 channels respectively. For each sample, it was necessary minimum of 10,000 cells and data were analyzed using CELLQuest software (Becton Dickinson). The histogram plotted in logarithmic scale.

Example 22

Inhibition of protein expression of bcl-2

Three PEG of the oligonucleotide (compound 14, 28, and 33), the object of which was the expression of bcl-2, was transfectional in RS cells and their ability to inhibit protein bcl-2 were analyzed by Western blot testing.

Influence lipofectin

At the beginning of the degree of inhibition of protein expression of bcl-2 in RS cells induced by compound 14 was determined in the presence and in the absence of lipofectin. RS cells were treated with compound 14 (200, 400 and 800 nm) in the presence or in the absence of lipofectin in 24 hours in Opti-MEM and then additional 67 hours in complete medium. The protein samples (30-40 μg protein/lane) were analyzed by Western blotting as described in "Materials and methods", using tubulin as a control of protein samples. The percentage of inhibition compared to untreated control cells was determined using a laser-scanning densitometry. The results of the Western blot shown in Fig.

The expression of α-tubulin and RKS-α did not change, confirming that lipofectin useful to achieve penetration of compound 14 in PC-3 cells, and confirming that ponie the maintenance regulation of expression of the protein bcl-2 was caused solely by the connection 14.

Additional studies have shown that compounds 14 and 28 were most active at 400 nm. RS cells were treated with complexes of compound 14, compound 28 and compound 23 at 400, 800 and 1000 nm and lipofectin in 24 hours in Opti-MEM and then additional 67 hours in complete medium. The protein samples (30-40 μg protein/lane) were analyzed by Western blotting as described in "Materials and methods", using tubulin as a control of protein samples. The percentage of inhibition compared to untreated control cells was determined using a laser-scanning densitometry.

Compound 14 was led to 86% down-regulation and the connection 28 resulted in 78% down-regulation.

Example 23

Analysis of the protein expression of bcl-2, depending on the dose of PEG oligonucleotides

To further confirm the inhibitory effect of compounds 14 and 28 on the expression of the protein bcl-2, RS cells were treated with increasing concentrations (25, 50, 100, 200 and 400 nm) of compound 14, compound 28 and compound 13 as a positive control in complex with lipofectin in 24 hours in Opti-MEM and then additional 67 hours in complete medium. The protein samples (30-40 μg protein/lane) were analyzed by Western blotting as described in "Materials and methods".

Inhibition of protein expression of bcl-2, depending on what concentratie observed using Western blotting for compounds 14 and 28 relative to the control. Approximately 1-2% inhibition was observed at 50 nm, increasing to 99% and 75% at a concentration of 400 nm. To connect 24 at 50 nm was not observed any inhibition, but at a concentration of 400 nm inhibition was increased to 77%. Transfection of compound 13 was used as a positive control. None of the oligonucleotides is not inhibited expression of α-tubulin.

The above experiment as a control were repeated for the connection 24. RS cells were treated with increasing concentrations of compounds 35 and 24 (25, 50, 100, 200 and 400 nm) in complex with lipofectin in 24 hours in Opti-MEM and then additional 67 hours in complete medium. The protein samples (30-40 μg protein/lane) were analyzed by Western blotting as described in "Materials and methods", using α-tubulin as a control of protein samples. The percentage of inhibition compared to untreated control cells was determined using a laser-scanning densitometry.

Example 24

The influence of PEG oligonucleotides on the growth RS cells

Also investigated the effect of compound 14 and compound 28 on the growth of cancer cells of the prostate gland RS,in vitro. RS cells were treated with oligonucleotide complexes/lipofectin. As shown in figure 10 transfection of antisense oligonucleotide compound 14 at 400 and 200 nm strongly in what has ebervale cell growth, while the connection 28 is only slightly influenced the rate of cell proliferation.

Example 25

The influence of PEG oligonucleotides on the production of reactive forms of oxygen in RS cells

The production of reactive oxygen species or ROS in RS cells were evaluated by flow cytometry in two ways. The first was based on the oxidation of hydroidolina (NOT) to amidine (E), which is then embedded in DNA fluorescence determined through flow cytometry. The second method used the oxidation of penetrating into cells 2',7'-dehydrochlorination diacetate to fluorescent 2',7'-dichlorofluorescin (DCF). In RS cells processing complexes connection 14/lipofectin (14 nm/15 mcg/ml) for 24 hours in Opti-MEM led to ROS three days after the determination of flow cytometry as F (1,9 fold increase compared with control untreated cells)and DCF (2-fold increase compared with control untreated cells) fluorescence. As confirmed by the data presented figure 11, the connection 28 does not lead to any increase in ROS production compared to control untreated cells. Moreover, the production of ROS is tightly linked to the rate of cell proliferation; cells stop growing after treatment with 400 nm of compounds 14 and this oligonucleotide also causes uvelichenie production of ROS (DCF).

1. Oligonucleotide prodrug of formula (I):

in which R1and R2independently represent H or polyalkylated with a molecular weight of from 2000 up to 136,000 Yes, with optional kapperud group selected from the group consisting of HE, NH2, SH, CO2H, C1-6Akilov, formula (II) and formula (III)
and,
provided that when (o+n)≥2, n and o each is a positive integer, p and m are each equal to zero, and R2is N, and when (p+m)≥2, p and m each is a positive integer, n and o are each equal to zero, and R1represents H;
X1X2X3independently represent single-stranded or double-stranded oligonucleotide residue in length from 10 to 1000 nucleotides;
L1and L4independently represent released linker fragments,
where L1selected from the group consisting of
,


and

L4selected from the group consisting of
,

,


where Y1'-25'The Y 1-25are independently O, S or NR109;
Ar is
or;
R6'-7', R9'-13', R16'-25', R27'-41', R6-7, R9-13, R16-25, R27-41, R62-67and R109independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov and C1-6alkoxy;
L5'-12'independently selected from the group consisting of
-(CH2)3-,
-(CH2)3NHC(O)-,
-(CH2)3NH-,
-C(O)(CR57'R58')s'O(CR55'R56')t'-,
-NH(CH2CH2O)s'(CH2)t'NR59'-,
-NH(CH2CH2O)s'-,
-NH(CR55'R56')s'O-,
-C(O)(CR55'R56')s'NHC(O)(CR57'R58')t'NR59'-,
-C(O)O(CH2)s'O-,
-C(O)(CR55'R56')s'NR59'-
-C(O)NH(CH2CH2O)s'(CH2)t'NR59'-,
-C(O)O(CH2CH2O)s'NR59'-,
-C(O)NH(CR55'R56')s'-O-,
-C(O)O(CR55'R56')s'O-,
-C(O)NH(CH2CH2O)s'-,
and
;
or L5'-6'selected from-C(O)CH2(Och2CH2)2NH-;
L5-12independently selected from the group consisting of
-(CH2)3-,
-C(O)NH(CH2)3-, -NH(CH2)3-,
-(CR55R56)sO(CR57R58)tC(O)-
-NR59(CH2)(OCH2CH2)NH-,
-(OCH2CH2)sNH-,
-O(CR55R56)sNH-,
-NR59(CR57R58)tC(O)NH(CR55R56)sC(O)-,
-O(CH2)sOC(O)-,
-NR59(CR55R56)sC(O)-
-NR59(CH2)t(OCH2CH2)sNHC(O)-,
-NR59(OCH2CH2)sOC(O)-,
-O(CR55R56)sNHC(O)-,
-O(CR55R56)sOC(O)-,
-(OCH2CH2)sNHC(O)-,
and
;
or L5-6selected from-NH(CH2CH2O)2CH2C(O)-;
where R55'-59'and R55-59independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov and C1-6alkoxy;
R60and R60'independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov and C1-6alkoxy;
s' and t' are each equal to 1;
C, h, k, l, r, s, v, w, v', w', C' and h' are each equal to 1;
a, e, g, j, t, z, a', z', e' and g' are each independently equal to or 0 or 1;
b, d, f, i, u, q, b', d' and f' each independently equal to or 0 or 1;
L2and L3independently selected from bifunctional spacer elements of groups, each of which comprises from 2 to 10 carbon atoms,
where L2select the n group, consisting of
-(CR50'R51')q'Q',
-(CR52'R53')r'O(CR50'R51')q'Q',
-(CR52'R53')r'(OCH2CH2)Q'-,
-(CR52'R53')r'C(O)NH(CR50'R51')q'Q',
and
-(CH2)-S-S-(CH2)q'Q'-;
L3selected from the group consisting of
-Q(CR50R51)q'-,
-Q(CR50R51)q'O(CR52R53)r',
-Q(CH2CH2O)q'(CR52R53)r'-,
-Q(CR50R51)q'NHC(O)(CR52R53)r'-,
and
-Q(CH2)q'-S-S-(CH2)r'-;
and
where Q and Q' are independently selected from O, S or NH, where Q and Q' are attached to the L4and L1, respectively;
R50'-54'and R50-54independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov, substituted C1-6Akilov and C1-6alkoxy, where the substituted alkali include carboxyacid, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethyl; and
q' and r' each independently is from 1 to 9.

2. The prodrug according to claim 1, wherein said nucleotide selected from the group consisting of
,,

,,,

,and
in which M represents O or S;
B1and In2independently selected from the group consisting of A (adenine), G (guanine), C (cytosine), T (thymine), U (uracil), and modified bases;
R100and R101independently selected from the group consisting of H, OR', where R' represents H, C1-6alkyl, substituted alkali, nitro, halogen and aryl.

3. The prodrug according to claim 2, in which M is S.

4. The prodrug according to claim 1, in which the oligonucleotide balance is an oligonucleotide residue with fosfomifira skeleton or phosphorothioate skeleton.

5. The prodrug according to claim 1, in which the oligonucleotide balance is an antisense oligonucleotide residue or oligodeoxynucleotides the rest.

6. The prodrug according to claim 5, wherein said antisense oligonucleotide residue or oligodeoxynucleotides residue selected from the group consisting of oligonucleotides and oligodeoxynucleotides with phosphodieterase the cores or phosphorothioate the cores, closed nucleic acids, peptide nucleic acids, tricyclo-DNA, false oligodeoxynucleotides, R Is K, mirror-oriented oligonucleotides and CpG oligomers.

7. The prodrug according to claim 5, wherein said antisense oligonucleotide has a sequence selected from the group consisting of SEQ ID NO:
1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

8. The prodrug according to claim 1, having the formula (I)

where R2represents hydrogen,
p and m are 0, and
R1is polyalkylated with capirola group of the formula (II):

where X1and X2independently represent a 3'-oligonucleotide, or 5'-oligonucleotide.

9. The prodrug according to claim 1, in which R1is a polyethylene glycol.

10. The prodrug according to claim 1, in which R1selected from the group consisting of:
J-O-(CH2CH2O)n'-
J-O-(CH2CH2O)n'-CH2C(O)-O-,
J-O-(CH2CH2O)n'-CH2CH2NR48-,
J-O-(CH2CH2O)n'-CH2CH2S,
-O-C(O)CH2-O-(CH2CH2O)n'-CH2C(O)-O-,
-NR48CH2CH2-O-(CH2CH2O)n'-CH2CH2NR48-,
-SCH2CH2-O-(CH2CH2O)n'-CH2CH2S,
in which n' is an integer positive number such that srednevekovaja molecular weight of the polymer portion of the prodrug according to claim 1 ranged from 2000 to 13600 Yes;
R48selected from the group consisting of hydrogen, C1-6Akilov,
With3-12branched Akilov,3-8cycloalkyl, C1-6substituted Akilov,
With3-8substituted cycloalkyl, arrow, substituted arrow, arylalkyl,
C1-6heteroalkyl, substituted C1-6heteroalkyl, C1-6alkoxy, phenoxy and
With1-6heteroatomic; and
J represents kapperud group selected from the group consisting of HE, NH2, SH, CO2H, C1-6Akilov, formula (II) and formula (III)
and

11. The prodrug according to claim 1, in which R1includes
-O-(CH2CH2O)x-,
where x is a positive integer selected so that srednevekovaja molecular weight ranged from 2000 up to 136,000 Yes.

12. The prodrug according to claim 1, in which R1has srednevekovoy molecular weight component from 3000 to 100000 Da.

13. The prodrug according to claim 1, in which R1has srednevekovoy molecular weight component from 5000 to 40000 Da.

14. The prodrug according to claim 6, wherein said antisense oligonucleotide has the sequence of SEQ ID NO:1.

15. Oligonucleotide prodrug of the formula:

in which L2represents a bifunctional spacer elements group, containing the s from 2 to 10 carbon atoms;
X1represents a single-stranded or double-stranded oligonucleotide residue, where the length of the oligonucleotide is from about 10 to 1000 nucleotides;
u' is a positive integer; and
T is a:
or
in which D' is one of the
,
,,
,
,,
,
,
,
,
,,
,
and
,
where R61is polyalkylated;
L2selected from the group consisting of
-(CR50'R51')q'Q',
-(CR52'R53')r'O(CR50'R51')q'Q',
-(CR52'R53')r'(OCH2CH2)Q'-,
-(CR52'R53')r'C(O)NH(CR50'R51')q'Q',
and
-(C 2)-S-S-(CH2)q'Q'-;
where Q' is independently selected from O, S and NH;
R50'-54'independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov, substituted C1-6Akilov and C1-6alkoxy, where
substituted alkali include carboxyacid, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethyl; and
q' and r' each independently is an integer from 1 to 9.

16. The prodrug according to claim 1, selected from the group consisting of:

and

all of them include oligonucleotide SEQ ID NO:1.

17. The way to obtain prodrugs, including:
the interaction of the compounds of formula:
R2-L4-withdrawing group
with the compound of the formula
H-L3-X1
under conditions sufficient for the formation of prodrugs of formula
R2-L4-L3-X1,
in which R2are polyalkylene with a molecular weight of from 2000 up to 136,000 Yes;
L4is a released linker fragment selected from the group consisting of:
,

,


where Y1-25is independently O, S or NR109;
Ar is
or;
R6-7, R9-13, R16-25, R27-41, R62-67and R109independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov and C1-6alkoxy;
L5-12independently selected from the group consisting of
-(CH2)3-,
-C(O)NH(CH2)3-,
-NH(CH2)3-,
-(CR55R56)sO(CR57R58)tC(O)-,
-NR59(CH2)(OCH2CH2)NH-,
-(OCH2CH2)sNH-,
-O(CR55R56)sNH-,
-NR59(CR57R58)tC(O)NH(CR55R56)sC(O)-,
-O(CH2)sOC(O)-,
-NR59(CR55R56)sC(O)-
-NR59(CH2)t(OCH2CH2)sNHC(O)-,
-NR59(OCH2CH2)sOC(O)-,
-O(CR55R56)sNHC(O)-,
-O(CR55R56)sOC(O)-,
-(OCH2CH2)sNHC(O)-,
and

or L5-6selected from-NH(CH2CH2O)2CH2C(O)-;
where R55-59independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov and C1-6alkoxy;
R60selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov and C1-6alkoxy;
s' and t' are each equal to 1;
with that , k, l, r, s, v, w, v', w', C' and h' are each equal to 1;
a, e, g, j, t, z, a', z', e' and g' are each independently equal to or 0 or 1;
b, d, f, i, u, q, b', d' and f' each independently equal to or 0 or 1;
L3represents a bifunctional spacer elements group containing from 2 to 10 carbon atoms;
and L3selected from the group consisting of
-Q(CR50R51)q'-,
-Q(CR50R5l)q'O(CR52R53)r'-,
-Q(CH2CH2O)q'(CR52R53)r'-,
-Q(CR50R51)q'NHC(O)(CR52R53)r'-,
and
;
where Q is independently selected from O, S and NH, where Q is attached to the L4;
R50-54independently selected from the group consisting of hydrogen, C1-6Akilov branched C3-12Akilov, substituted C1-6Akilov and C1-6alkoxy, where the substituted alkali include carboxyacid, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethyl; and
q' and r' each independently is an integer from 1 to 9;
X1represents a single-stranded or double-stranded oligonucleotide residue, where the length of the oligonucleotide is from about 10 to 1000 nucleotides.

18. The method of delivery of the oligonucleotide to the organs, tissues and cells comprising the administration to a mammal in need of such delivery, the effective number ol gonucleotide prodrug according to claim 1.

19. The method according to p in which delivery is made to treat cancer.

20. The method according to p, wherein said antisense oligonucleotide has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

21. The prodrug according to claim 1 of formula (I)

where R1represents hydrogen,
n and o are 0, and
R2is polyalkylated with capirola group of the formula (III):

where X1and X3independently represent a 3'-oligonucleotide, or 5'-oligonucleotide.



 

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FIELD: biology.

SUBSTANCE: invention relates to nucleic acid encoding of chromophore and/or fluorescence protein having amino acid sequence with at least 80 % similarity with amino acid sequence represented in SEQ ID NO: 02, 04, 06, 08, 10, 12, 16, 18, 20, 22, 24, 26, or 28, or complementary nucleic acid hybridizing under hard conditions with abovementioned nucleic acid/ Also disclosed are nucleic acid encoding of protein having amino acid sequence SEQ ID NO: 02, 04, 06, 08, 10, 12, 16, 18, 20, 22, 24, 26, or 28 and kit for protein production.

EFFECT: new chromophore and/or fluorescence protein.

7 cl, 21 dwg

FIELD: chemistry.

SUBSTANCE: present invention pertains to ester lipids of halogenated adenine nucleotides with formula I, which can be used in treating cancerous diseases. (I), where R1 - C1-C20-alkyl, can be substituted with C1-C6-alkoxyl radical, C1-C6-alkylmercaptan radical, C1-C6-alkylsulfenyl or C1-C6-alkylsulfonyl groups, R2 - C1-C20-alkyl, which can be substituted with C1-C6-alkoxyl radical, C1-C6-alkylmercaptan radical or C1-C6-alkylsulfonyl group, R3 - amino group, X - sulphur atom, sulfenyl or sulfonyl group, Y - oxygen atom.

EFFECT: obtaining new biologically active compounds.

3 cl, 9 ex, 1 tbl

FIELD: radiolabeled compounds, biochemistry.

SUBSTANCE: invention relates to a novel labeled analogue of the physiologically active compound, namely, tritium-labeled acyl-coenzyme A of the general formula (I): wherein Acyl means [5,6-3H]-(5,6-dihydro)-arachidonoyl or [6,7-3H]-linoleyl.

EFFECT: valuable medicinal and biochemical properties of compound.

2 dwg, 4 ex

FIELD: medicine, pharmacology, bioorganic chemistry, pharmacy.

SUBSTANCE: invention relates to the effective using amount of β-L-2'-deoxynucleoside of the formula (I) or (II) used in manufacturing a medicinal agent used in treatment of hepatitis B, pharmaceutical compositions containing thereof, and methods for treatment of hepatitis B. Proposed agent shows the enhanced effectiveness in treatment of hepatitis B.

EFFECT: enhanced and valuable medicinal properties of agent.

83 cl, 6 tbl, 11 ex

FIELD: organic chemistry, chemical technology, medicine.

SUBSTANCE: invention relates to acyclic nucleoside phosphonate derivatives of the formula (1): wherein means a simple or double bond; R1 means hydrogen atom; R2 and R3 mean hydrogen atom or (C1-C7)-alkyl; R7 and R8 mean hydrogen atom or (C1-C4)-alkyl; R4 and R5 mean hydrogen atom or (C1-C4)-alkyl possibly substituted with one or more halogen atoms, or -(CH2)m-OC(=O)-R6 wherein m means a whole number from 1 to 5; R6 means (C1-C7)-alkyl or 3-6-membered heterocycle comprising 1 or 2 heteroatoms taken among the group consisting of nitrogen (N) and oxygen (O) atoms; Y means -O-, -CH(Z)-, =C(Z)-, -N(Z)- wherein Z means hydrogen atom, hydroxy-group or halogen atom, or (C1-C7)-alkyl; Q (see the claim invention); its pharmaceutically acceptable salts or stereoisomers. Also, invention proposes methods for preparing compounds of the formula (1) and their using in treatment of hepatitis B or preparing a medicinal agent designated for this aim.

EFFECT: improved preparing method, valuable medicinal properties of compounds and agent.

16 cl, 10 tbl, 87 ex

FIELD: organic chemistry, medicine, virology.

SUBSTANCE: invention relates to technology of organic compounds, namely, to 5'-aminocarbonylphosphonates d4T that are inhibitors of the human immunodeficiency virus reproduction. Invention describes 5'-aminocarbonylphosphonates d4T of the general formula: wherein R' means hydrogen atom (H), alkyl, aryl; R'' means hydrogen atom (H), alkyl, aryl; R', R'' mean cyclic alkyl; R means alkyl. These compounds are inhibitors of the human immunodeficiency virus reproduction. Invention provides preparing new compounds eliciting valuable biological properties.

EFFECT: valuable medicinal properties of compounds.

2 dwg, 1 tbl, 5 ex

The invention relates to nucleoside analogs of formula (1) in which R1represents H or a group protecting the hydroxyl, R2represents H, a group protecting the hydroxyl group of phosphoric acid, a protected group, phosphoric acid or a group of the formula P(R3R4in which R3and R4are the same or different and represent a hydroxyl group, a protected hydroxyl group, alkoxygroup, allylthiourea, cyanoacetylurea, amino group, substituted alkyl group; And represents alkylenes group containing from 1 to 4 carbon atoms, and a represents a substituted purine-9-ilen group or substituted 2-oxopyrimidine-1-ilen group containing at least one Deputy, selected from hydroxyl groups, protected hydroxyl groups, amino groups, protected amino groups, alkyl groups

The invention relates to methods for treating diseases caused by hepatitis B virus (also known as HBV and Epstein-Barr (also known as EBV, which include the introduction of an effective amount of one or more of the active compounds disclosed here, or farmatsevticheskii acceptable derivatives or prodrugs of one of these compounds

The invention relates to new nukleotidfosfatazu derived from the remnants of lipid esters of General formula I, in which R1, R2represent a linear or branched saturated alkyl chain containing 1-20 carbon atoms; R3, R5represent hydrogen, hydroxyl group; R4represents a hydroxyl group; X represents a sulfur atom, sulfinyl or sulfonyloxy group; Y represents an oxygen atom; b is a purine and/or pyrimidine base, provided that at least one of the residues R3or R5represents hydrogen; their tautomers, their optically active forms and racemic mixtures, or their physiologically acceptable salts with inorganic and organic acids and/or bases, and also to processes for their preparation and medicines containing the above-mentioned connection

The invention relates to pharmacology, in particular ORGANOMETALLIC compounds possessing biological activity, which can find application in drug development for the prevention and treatment of coronary heart disease

FIELD: chemistry.

SUBSTANCE: invention relates to 5'-phosphorus-containing derivatives of 2',3'-dideoxy-3'-thiacytidine with general structural formula given below , where R=H, NH2-C(O)-. The invented compounds can be used for inhibiting reproduction of the human immunodeficiency virus type 1 in an MT-4 cell culture.

EFFECT: increased antiviral activity of the compounds.

1 cl, 2 tbl, 4 ex

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