Biologically active polimerobetonie the anthracyclines, intermediate compounds, methods of preparation and pharmaceutical composition

 

(57) Abstract:

The invention relates to novel soluble synthetic polimersvarka the anthracyclines, exhibiting antitumor activity, to a method of receiving and containing pharmaceutical compositions. Describes polimerobetonie anthracyclin General formula a, obtained by the interaction of the polymer of the intermediate compounds of formula V, where n is 0 or 1, x is from 70 to 98 mol. %, w is from 30 to 2 mol.%, R1is CH2CH(OH)CH3or CH2CH2OH group, and R2is 2,4-dichlorphenoxy, p-nitrophenoxy or hydroxy-group; from a derivative of anthracycline General formula 5: H-Y-[NH-D] , where [NH-D] is the residue of anthracyclineinduced formula Q, where one of RIand RIIis hydrogen and the other is a hydroxy - group or iodine, RIIIis hydrogen or OCH3and RIVis hydrogen or hydroxy-group; Y is a peptide part selected from Gly-Phe-Gly, Gly-Leu-Gly, Phe-Leu-Gly, Gly-Phe-Leu-Gly, or Leu-Leu-Gly; if R2is a hydroxy-group, in the presence of N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; and, if necessary, carry out the processing of the received polimerbetonov anthracycline 2-hydroxypropyl 8 - 24 h; and received polimerobetonie anthracyclin has a medium molecular weight 29800-33400 g/mol and a polydispersity of 1.4 to 1.8. 5 S. and 3 C.p. f-crystals, 4 PL.

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The present invention relates to soluble synthetic polimersvarka the anthracyclines, receiving and containing pharmaceutical compositions.

Doxorubicin, 4'-epidoxorubicin and 4-demethoxygeldanamycin are examples anthracyclines, which is known in science and are currently used for clinical treatment of malignant tumors (see, for example. A. Argamon, "Doxorubicin", Medicinal Chemistry monograph, T. 17, Academic Press, 1981).

Received many polymeric derivatives of doxorubicin, endowed with antitumor activity. Among them the most promising for clinical application is soluble polimerobetonie doxorubicin, which consists of hydrophilic residues and peptide chains that are associated with doxorubicin and 2-hydroxypropylamino. It is derived polimerbetonov doxorubicin obtained by condensation of doxorubicin hydrochloride methacrylic polymer precursor containing peptide chain, activated as p-nitrophenyloctyl ether, in dioxiranes. Incubation of this material with lysosomal enzymes (tritanomaly) rats breaks the amide bond between the terminal amino acid and doxorubicin [j.Kopeček and other Biomaterials 10, 335 (1989), R. Duncan and others Biochem. Pharmacol 39, 1125 (1990); P. Donkin and other J. Controlled Release 10, 51 (1989) 18, 123 (1993) and 19, 331 (1992)].

The problem for traditional methods, for example, as described above, is that it is difficult to remove doxorubicin from doxorubicinol polymer conjugate. This is due to the formation of complexes between the binder and the free doxorubicin, it was found that the material behaves as a single unit during dialysis, molecular filtration and gel chromatography [j. Fagen and other J. Controlled Release 1, 301 (1985)].

Polimerobetonie anthracycline system of the present invention is based on methacrylic polymers bearing hydrophilic residues, peptide limit circuit, connected only with the amino group of anthracycline, and glycine residues, either in the form of the free acid or amide derivative. These systems have the advantage over existing analogue that anthracyclin can be easily released from the polymer with which it is associated. Besides polimerobetonie the anthracyclines of the invention have broader protivoop the way, the present invention relates to polimerbetona anthracycline formula A, which consists, essentially, of three units represented by General formulas 1, 2 and 3:

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where Gly is glycine, n is 0 or 1,

x - 70-98 mol.%,

y - 1-29 mol.%,

z - 1-29 mol.%,

R1-C1-6-altergroup substituted by one or more hydroxy groups

Y is the residue of an amino acid or peptide,

[NH-D] - the rest of aminoglycosidetherapy [NH2-D], and

Z is hydroxy or a residue of formula - other1as specified above.

Aminoglicozidelor, the balance of which is [NH-D], represented here as [NH2-D] , where D denotes the structure anthracyclineinduced minus the amino group of the sugar residue.

Polimerobetonie anthracyclin preferably contains from 90 to 98 mol. % of units of the General formula 1, from 0 to 10 mol.% units of the General formula 2 and from 1 to 10 mol.% units of the General formula 3.

Enzymatic in vivo hydrolysis of peptide chains leads to the release of only the active drugs D-NH2while the unit of the General formula 3 remains untouched.

Relevant accelgroup, which can be R1are C1-4-altergroup substituted one

Peptide spacer Y should be sensitive to intracellular hydrolysis. The spacer may be resistant to extracellular hydrolysis. The peptide spacer may be a length of from 1 to 10, for example, 2-4 amino acid residues. Typically, the peptide spacer is a Tripeptide or tetrapeptide.

Each of the constituent amino acid residues of the peptide spacer, y, which is chiral, may be present in the form of optical D - or L-isomer or D/L-mixture. Here we use the traditional three-letter designation scheme of amino acids, where the symbols denote the L-configuration of the chiral amino acids, unless otherwise noted. Peptide spacer y may be present as racemic mixtures or in the form of optically pure isomer.

Preferably, Y is selected from Gly-Phe-Gly (glycine-phenylalanine-glycine) Gly-Leu-Gly (glycine-leucine-glycine, Gly-Pht-Leu-Gly or Leu-Leu-Gly with a glycine residue, associated in each case with aminoglicozidelor.

Aminoglycosidetherapy balance [NH-D] is the appropriate balance of anthracyclineinduced [NH2D] the following formula Q:

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in which one of RIand RIIis hydrogen and the other is a hydroxy - group or iodine,

RIII-vador is aminoglycoside [NH2D]is doxorubicin, 4'-epidoxorubicin, 4-demethoxygeldanamycin, idarubitsin and 4'-iodo, 4'-desoxidation.

The invention also provides a method of obtaining polimerbetonov antracycline A, which consists essentially of units of the General formulas 1, 2 and 3, as defined above.

The method includes:

i) the interaction of the polymer intermediate compound B, where B is composed mainly of units of the following General formulas 1 and 4:

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where x, n and R1in the formula 1 are as defined above, w is from 30 to 2 mol.%, and R2the hydroxy-group or leaving group, with anthracycline derivatives of the General formula (5)

HY - [NH-D] (5)

in which [NH-D] and Y are as defined above,

ii) interaction of the product from step (i), where R2- leaving group, with a compound of General formula NH2R1in which R1such as defined above, in the case when you want to get polimerobetonie anthracyclin, where Z, unit of General formula 3 is other1.

The leaving group, which can represent R2is suitable phenyl-oxygraph, which is substituted on the phenyl ring with one or more electronicmusi groups. Examples of the but is a leaving group.

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in which L - elektrogorskaya group, for example, -NO2or halogen, such as fluorine or chlorine, and m is an integer from 1 to 5, usually from 1 to 3, preferably 1 or 2. Preferably, R2- p-nitrophenoxide or 2,4-dichlorphenoxy.

Compounds of General formula (5) can be easily separated from the polymer conjugate of A formula due to its high Revilest.

As described above, this approach to obtaining polimersvarka anthracyclines allows to overcome the main drawback of traditional condensation anthracyclines with polymers, namely the difficulty in separating free anthracycline from the polymer conjugate.

Polymer intermediate compound B, consisting mainly of units of the General formulas 1 and 4, as previously defined, are obtained by radical copolymerization of methacrylic compounds of the following formulas 6 and 7:

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where n, R1and R2such as defined above.

Some polymers consisting mainly of units of the General formulas 1 and 4, are known from the literature: for example, the polymer B1, consisting of units of the General formula 1 in which R1represents-CH2CH(OH)CH3n=O, and units obla deposition of N-(2-hydroxypropyl)-methacrylamide 6a: R1= CH2CH(OH)CH3n= 0 N-methacryloyloxy-p-nitrophenylamino 7a: R2=O-C6H4pNO2as described in the work of Tolkien. Kopeček, Makromol. Chem., 178; 2159 (1977). Polymer intermediate compounds comprising units of the General formulas 1 and 4, in which R2represents a hydroxyl group can be obtained by radical homogeneous polymerization.

Some monomers of General formulas 6 and 7 are known. Compounds of General formula 6 in which n=0 and R1-alkyl bearing a hydroxyl group, obtained by interaction of methacryloylamido with aliphatic alkyl bearing a secondary hydroxy-group. On the other hand, compounds of General formula 6 in which n= 0 and R1-the rest of alkyl carrying a primary hydroxyl group can be obtained from methacrylic acid and amino compounds in the presence of a condensation catalyst, such as 1-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

Peptidyl-anthracycline derivatives of the General formula 5 are an additional aspect of the present invention. Methods for their preparation are known. For example, as it is important for the interaction of the corresponding N-protected peptide with anthracyclines, N-protective piptadenia gr is Lin. An example is triphenylethylene group.

Incidentally compounds of General formula 5 can be obtained by a method that includes:

(i) interaction of the N-protected peptide of General formula 8 or 9:

R5-Y-OH (8)

R3-Y-P (9)

where R3- islamochristiana protective amino group, R is a leaving group, and Y is amino acid residue or a peptide spacer, as described above, with anthracyclineinduced NH2as mentioned above, to obtain the intermediate compounds of General formula 10:

R3- Y- [NH-D] (10)

where [NH-D], Y-R3such as defined above, and

(ii) removal of the protective group R3obtaining peptidylarginine General formula 5 in free base form. P may be a leaving group, as defined and confirmed by the examples above for R2. In addition, P can be panafcortelone - or N-hydroxysuccinimide. Examples kislotoustoytchivi protective group R3include trityl and diphenylamino.

Peptidyl derivatives of General formulas 8 and 9 are obtained following the standard methods of synthesis known from the literature about the peptides. Protection AMINOPHENYL kislotoustoytchivi GRU is 4(1982).

The reaction conditions maintained to produce compounds of General formulas 8, 9 and 10, are intended to avoid racemization, the resulting peptidyl derivatives retain therefore, the configuration of the original amino acid.

To obtain the anthracycline derivatives of the General formula 5, a compound of General formula 9 can interact with cleaners containing hydrochloride salt of anthracycline in anhydrous polar solvent such as dimethylformamide, in the presence of equivalent quantities of organic bases such as triethylamine, for example, at room temperature for 15 hours to obtain the intermediate compounds of General formula 10, which is purified by chromatography, and then is released from the derived compounds of General formula 5, for example, in water of 75% acetic acid at room temperature.

It should be noted that the reaction anthracyclines bearing a hydroxyl group in position C-14, such as doxorubicin and 4'-epidoxorubicin, in the form of cleaners containing hydrochloride salt with activated peptidyl-derivative of General formula 9 in the presence of an organic base needed to release 3'-amino anthracyclines in polar solvent, gives a mixture of the product is s-(3'-N, 14-O-peptidyl)-derivatives are removed from the mixture by chromatography.

Compounds of General formula 10 can be obtained by condensation of N-protected peptide of General formula 8 with anthracyclines in the form of cleaners containing hydrochloride salt in an anhydrous polar solvent such as dimethyl sulfoxide in the presence of an equivalent amount of a condensation catalyst, such as 1-etoxycarbonyl-2-ethoxy-1-dihydroquinoline. This method does not give the bis-peptidyl derivatives anthracyclines carrying C-14 hydroxy-group.

Condensation of the intermediate product B with pentadecatriene derivatives of General formula 5, not necessarily following the replacement of the remaining leaving groups, gives polimerobetonie the anthracyclines, consisting mainly of units of the General formulas 1, 2 and 3. It should be emphasized that this method allows to avoid the formation of ester bonds between the primary hydroxyl groups and end glycyl-activated esters.

Polimerobetonie medicine General formula A, in which the residue of Z units of the General formula 3 represents a group other1as previously described , are obtained, preferably, by the interaction of intermediate compound B, in which R2- withdrawing group, as opacom as dimethylformamide or dimethylsulfoxide. The reaction can usually go for 8 to 24 hours. The reaction is usually conducted at a temperature of from 15 to 30oC, preferably at room temperature for 15 hours, then the remaining leaving group is replaced by the interaction of the conjugate with the compound of General formula NH2R1as described above, during the period of time from 0.5 to 3 hours at room temperature.

Polimerobetonie medicine General formula A, in which the residue of Z units of the General formula 2 represents a hydroxyl group, obtained, preferably, by the interaction of the intermediate compounds of General formula B in which R2- hydroxyl group, with anthracycline derivatives of the General formula 5 in an anhydrous polar organic solvent such as dimethylformamide or dimethylsulfoxide. The reaction usually lasts for 8 to 24 hours. The reaction is usually conducted at a temperature of from 15 to 30oC, preferably at room temperature for 15 hours.

For example, to get polimerbetonov anthracycline, in which Z is a residue other1as described above, the intermediate compound of General formula B in which R2- leaving group such as p-nitrophenoxy, processed peptidases of 14% weight/volume, and the connection 5 in the amount of 2.2% weight/volume (m/o). Then added the compound of General formula NH2R1as indicated above, typically at 0.1% m/o, and the reaction mixture is aged at room temperature for 3 hours. Conjugate planted with acetone, dissolved in absolute ethanol, typically at a concentration of 8% (m/m), and planted again with acetone to obtain the desired polimerbetonov anthracycline.

In the above-described method, it is possible to avoid the formation of ester bonds between C-14 gidroksilirovanii anthracyclines and limit glycyl-activated ester due to the absence of any organic base in the condensation process.

In another example, to obtain polimerbetonov anthracycline General formula A, in which Z is a hydroxyl group, an intermediate compound of General formula B, as described above, in which R2- hydroxyl group, is treated in anhydrous dimethyl sulfoxide of peptidylarginine General formula 5 at room temperature for 15 hours. Compound B, respectively, used in 14% m/o and connection 5 with a 2.3% m/O. Conjugate is then planted with acetone, dissolved in absolute ethyl alcohol, usually when Kocak defined above.

The content of anthracycline in the conjugates of the General formula A is determined by the analysis of the aglycone released from the associated anthracycline by acid hydrolysis, for example, adriamycine is aglionby the remainder of doxorubicin, and 4'-epirubicin and 4-demethoxygeldanamycin is aglionby balance demethoxygeldanamycin.

Polimerobetonie the anthracyclines of the present invention show good solubility, biocompatibility, stability at physiological pH, and are released from the free active drugs (D-NH2after incubation with limosani enzymes (enzymes).

The compounds of formula A are increased antitumor activity in experimental samples and reduced overall toxicity compared to free anthracyclines.

Polimerobetonie the anthracyclines General formula A have antitumor activity. Man or animal can therefore be treated by the method lies in the use of a therapeutically effective amount polimerbetonov anthracycline General formula A. the condition of the patient (human or animal) can be thus improved.

Acceptable interval dosiro the nye anthracyclines General formula A is usually applied by parenteral way, for example, intramuscularly, intravenously or by the insertion of the balls. Suitable dosing interval is from 5 to 800 mg/m2anthracycline equivalent, for example, from 20 to 500 mg/m2. Suitable mode specifies the use of a solution of 25 mg anthracycline equivalent /m2intravenously at a volume of 10 ml/kg of body weight during the 2-week period through 5.9 and 15 days.

Polimerobetonie the anthracyclines General formula A can be included in the formulation of pharmaceutical compositions together with pharmaceutically acceptable carrier or diluent. Usually polimerobetonie the anthracyclines are included in formulations for parenteral use, for example, by dissolving in sterile water or in water for injection.

The following additional examples illustrate the invention. Examples 1-6 relate to methods of synthesis for the preparation of monomers of the General formulas 6 and 7 and polymer intermediates of General formula B.

Example 1

[N-(methacryloyloxy)]-2-hydroxypropylamino (6b)

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Methacryloyloxy-p-nitrophenyloctyl ester (7a: 5,28 g, 20 mmol), obtained as described in Makromol Chem. 178, 2159 (1977), dissolved in anhydrous tetrahydrofuran (20 ml) and treated 1-amino-what nom pressure, and specified in the header of the compound of General formula 6b (3.3 grams, a yield of 82.5%) is obtained after crystallization with a mixture of acetone /ethyl ether. TCX (thin-layer chromatography on Kieselgel plates F254(Merck), vinyaya system: methylene chloride/acetone (90:10 by volume), Rf=0,47.

Example 2

[N-(metabiological)]-hydroxyethylamide (6c)

(6c)

To stir a mixture of 1-etoxycarbonyl-2-ethoxy-1,2 - dihydroquinoline (37 g, 0.15 mmole) in anhydrous toluene (150 ml) dropwise over 15 minutes is added dissolved in anhydrous toluene (300 ml) metakrilovoy acid (14 ml, 0,165 mol). The reaction mixture was stirred at room temperature for 24 hours. Specified in the header of the compound of General formula 6c is obtained after planting n-hexane. TCX on Kieselgel plates F254(Merck), vinyaya system: methylene chloride/acetone (90:10 by volume) Rf=0,35

Example 3

[N-(methacryloyloxy)]2,3-dichlorphenoxy ester (7b)

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Specified in the header of the compound of General formula 7b is obtained from metareligion (2.66 g, 20 mmol), obtained as described in Makromol hem. 178, 2159, (1977), and 2,4-dichlorphenol (3,26 g, 20 mmol) in anhydrous tetrahydrofuran (50 ml) in the presence of DSS (4,2 g, 21 mmol). The compound of General formula 7B (4.7 g, yield 82%) of the fir Rf=0,47

Example 4

Copolymer of N-methacryloylamido-2-hydroxypropane and N-methacryloylamido (B2)

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N-methacryloylamido-2-hydroxypropan of 25.2 g of 0.18 mol) methacryloyloxy (2.86 g, 20 mmol) and azoisobutyronitrile (5.9 g) dissolved in anhydrous methyl alcohol (164 ml). The mixture is kept at 60oC in nitrogen atmosphere for 20 hours, then the reaction mixture is added to an acetone (2000 ml) with stirring. The precipitate is collected, washed with acetone and dried to constant weight to obtain specified in the header of polymer B2 (2.6 g). The content of carboxyl groups (N):10 mol.%

Example 5

The copolymer [N-(methacryloyloxy)-] 2-hydroxypropylamino and N -(methacryloyloxy)-2,4-dichlorophenylamino ether (B3)

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The compound of General formula 6b (14.4 g, 72 mmole) and the compound of General formula 7b (5,19 g, 18 mmol) are polymerized in anhydrous acetone (300 ml) in the presence of azoisobutyronitrile (1 g, 6 mmol), as described in operation 178, 2159 (1977), until the date stated in the title compounds of General formula B3. The polymeric material is removed by filtration from the reaction mixture, dissolved in absolute ethanol and presidets acetone. The chlorine content of: cash 6,89 mol.%, experimental - 2,84 mol.%

Example 6

The NEC polymer intermediate compound of General formula B4 is obtained from N-methacryloylamido-2-hydroxyethane (6c:23,2 g, of 0.18 mmole), metareligion (2.86 g, 20 mmol) and azoisobutyronitrile (5.9 g) in anhydrous methyl alcohol (164 ml) as described in example 2. The content of carboxyl groups (w) to 10%.

Examples 7-12 are ways to get peptidylarginine General formula 5.

Example 7

N-trimethyl-L-i.e. phenylalanyl-L-lazygirl-4-nitrophenolate

(C6H5)3C-L-Phe-L-Zeu-Gly - OC6H4pNO2(9a)

N-trityl-L-phenylanine (20,3 g, 50 mmol), obtained as described in J. Org. Chem. 47, 1324 (1982), dissolved in anhydrous tetrahydrofuran (150 ml) and thereto is added anhydrous N-hydroxybenzotriazole (8 g). The mixture is cooled at 0oC and processed 1,3-dicyclohexylcarbodiimide (11.7 g, 50 mmol) over 10 minutes to it is added dropwise a solution of 1-lazimpatnarayan-p-toluensulfonate salt (20 g, 50 mmol) in a mixture of anhydrous tetrahydrofuran (100 ml) and N-methylmorpholine (7 ml). The reaction mixture is kept at 0oC for 1 hour and at room temperature until the early hours, then filtered, and the solvent is removed under reduced pressure. The crude material dissolved in ethyl acetate, washed one after the other cooled 5% aqueous citric acid solution (g ml), ohlala by washing with a mixture of methylene chloride and methanol (99: 1 by volume) to give N - trityl-L-i.e. phenylalanyl-L-laserpitiifolium ester (18 g, 30 mmol), which is converted into the corresponding acid of General formula 8A) (17g) treatment in 95% ethyl alcohol (400 ml), 1N sodium hydroxide (30 ml) for 2 hours at room temperature. TLC on Kieselgel plates F254(Merck), vinyaya system: methylene chloride/methanol (80:20 by volume), Rf = 0.53 per share.

H1NMR (200 MHz, CDCl3).

to 0.88 (d, J =5,9 Hz, 6H +, Leu),1,2-1,2 (m, 3H, Leu), from 2.00 (DD, J= 5.7 Hz, J is 13.4 Hz, 1H, Phe), and 2.83 (DD, J=5,2 Hz, J=a 13.4 Hz, 1H, Phe), 3,51 (t, J=5,4, 1H, Phe), 3,99 (d, J=4.4 Hz, 2H, + Gly), 4,55 (m, 1H, Leu), 6,8-7,4 (m, 22H, NHgly, NHLeu, 4 - C6H5).

The compound of General formula 8a is dissolved in anhydrous tetrahydrofuran (450 ml) and thereto is added p-NITROPHENOL (5.5 g, 40 mmol). The mixture is cooled at 0oC, thereto is added dropwise a solution of 1,3-dicyclohexylcarbodiimide (8,24 g, 40 mmol) and the mixture is kept until the morning at 4oC. After that the reaction mixture is filtered, and the solvent is removed under reduced pressure. The residue is dissolved with ethyl acetate and cooled at 0oC. After one hour the mixture is filtered and the solvent removed to give, after crystallization from ethyl ether, specified in the header of the compounds of General formula 9 a ( 20 g, yield 97%) TLC on Kieselgel plate F254(Megs, vinyaya system - Adilov,4 Hz, 3H Leu), to 1.2-1.8 (m, 3H, Leu) to 1.9 (DD, J=5,9 Hz, J=13.5 Hz, 1H, Phe), 2,89 (DD, J = 4,6 Hz, J=13,5, 1H, Phe), 3,52 (DD, J = 4,6 Hz, J=5,9 Hz, 1H, Phe), of 4.0-4.4 (m, 3H, Leu, +Gly), 6,78 (t, J=5.7 Hz, 1H, NH GLY), ? 7.04 baby mortality (d, J=7.7 Hz, 1H, NH Leu), 6,8-7,4 (m, 22 Hz, 4-C6H5and 2.6

of 8.25 (m, 2H, 5,5 COO

Example 8

N-trityl-glycyl-L-i.e. phenylalanyl-L-lazygirl-p-nitrophenyloctyl ether

(C6H5)3C-Gly-L-Phe-L-Leu-Gly - OC6H4pNO2(9b)

The intermediate N-trityl-L-i.e. phenylalanyl-L-latispiculata ester (6 g, 10 mmol), obtained as described in example 7, is processed 75% aqueous solution of acetic acid at room temperature for one hour to obtain L-i.e. phenylalanyl-L-latispiculata ether, which is condensed with N - tritylglycine (3 g, 10 mm) in the presence of N - hydroxybenzotriazole and 1,3 - dicyclohexylcarbodiimide to obtain, after hydrolysis of the ethyl ester and activation of p-NITROPHENOL as described in example 7, specified in the header of the compounds of General formula (9b) (3.8 g, yield 50%) Kieselgel TLC - plate F254(Merck), vinyaya system - ethyl ester, RF =0,63. HELL-MS m/z 755 [M+H}+< / BR>
Example 9

3'-N-(glycyl-L-leucyl-L-i.e. phenylalanyl)doxorubicin (5a)

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Is the reaction of doxorubicininduced (2.9 g, 5 mmol) dissolved in anhydrous dimethylformamide is m, as described in example 7. The reaction mixture is kept until the morning, at room temperature, then visadelta mixture 1:1 of ethyl ether and n-hexane. The solid is purified on silicagel the column by washing with a mixture of methylene chloride and methanol (98:2 by volume) to give the N-protected peptidylarginine General formula 10a (4.6 g). TLC on Kieselgel plates F254(Merck), vinyaya system: methylene chloride/ methyl alcohol (95:5 by volume) , Rf=0.35 in.

HELL-MS: m/1103 [M+H]+< / BR>
The compound of General formula 10A, dissolved in 75% aqueous solution of acetic acid (250 ml) for one hour, then the solution was diluted with a mixture 1:1 of water and methylene chloride (2500 ml) and brought to pH 7 with solid sodium bicarbonate. The organic phase is separated, and the solvent is removed under reduced pressure to obtain specified in the header of the compounds of General formula 5A (3,45 g, yield 80%) TLC on Kieselgel plates F254(Merck), vinyaya system methylene chloride /methyl alcohol/ acetic acid/water (80:20:7:3) by volume, Rf = 0.5 in.

HELL-MS:m/z [M+H]+< / BR>
H1NMR (400 MHz, D)

0,8 (d, J= 6,4 Hz, 3H Leu), or 0.83 (d, J=6.4 Hz, 3H, Leu), 1,10 (d, J = 6.8 Hz, 3H, 6' CH3), between 1.3-1.6 (m, 4H, Leu and 2' EQ. H) and 1.83 (DDD, J= 2,8 Hz, J=13,2 Hz, J= 13,2 Hz, 1H, 2' Ahn) , is 2.09 (DD, J = 6.0 Hz, J =H, 3' and Phe) 4,15) (kV, J=6,4 Hz, 1H, 5' H) 4,29 (sq J =7.7 Hz, 1H, Leu), 4,55 (s, 2H, 14 CH2), 4,7-5,0 (m, 3H, 7H and 4'OH and 14-OH), 5,20 (d, J=3,4 Hz, 1H, 1'H), 5,46 (s, 1H, 90H) a 7.1 to 7.3 (m, 5H, Ar, Phe), 7,54 (d, J = 8.5 Hz, 1H, 3' NH), 7,63 (m, 1H, 3H), 7,88 (m, 2H, 1H and 2H), 8,17 (m, 4H, NH3+ NHGly), to 8.70 (d, J= 8,1 Hz, 1H, NH Leu) 13,23 (s, 1H, 11OH), 14,00 (s, 1H, 60H)

Example 10

3'-N-(glycyl-L-leucyl-L-phenylalaninol)-doxorubicin (5b)

< / BR>
Is the reaction of doxorubicininduced (2.9 g, 5 mmol) with N-trityl-glycyl-L-i.e. phenylalanyl-L-lazygirl-p-nitrophenylamino ester (9b: 3.8 g, 5 mmol), obtained as described in example 8, and then the reaction mixture is treated with 75% aqueous solution of acetic acid as described in example 9, to obtain specified in the header of the compounds of General formula 5b (4 g, yield 90%) TLC on Kieselgel plates F254(Merck), vinyaya system methylene chloride/methyl alcohol/acetic acid/water (80:20:7:3 by volume, Rf= 0.44 HELL/MS: m/938 [M+H]+< / BR>
Example 11

4 dimethoxy-3'-N(glycyl-L-leucyl-L-i.e. phenylalanyl) daunorubicin (5C)

< / BR>
Specified in the header of the compound of General formula 5C (3 g, 75% yield) is obtained from 4-dimethoxyphenylethylamine (2.9 g, 5 mmol) and N-trityl-phenylalanylglycyl-p-nitrophenylamino (9a :3.5 g, 5 mmol) analogously to the method described in example 9. TLC on Kieselgel plates F
HELL-MS: m/z 815 [M+H]+< / BR>
H1NMR (200 MHz, CDCl3)

from 0.84 (d, J=6.0 Hz, 3H, Leu), to 0.88 (d, J=6.0 Hz, 3H ' Leu) of 1.27 (d, J= 6.4 Hz, 3H, 6'CH3) of 1.4 to 1.7 (m, 3H +Leu), 1,7-2,0 (m, 2H, 2' CH2), to 2.06 (DD, J= 4,2 Hz, J= 14,9 Hz, 1H, 8 on), 2,32 (d, J=14,9 Hz, 1H, 8 EQ H), 2.40 a(s, 3H, COCH3), 2,70 (DD, J =8.6 Hz, J=13 Hz, 1H, Phe), of 3.13 (DD, J = 4,2 Hz, J= of 13.7 Hz, 1H, Phe), 2,94, 3,23 (DD, J = 19.2 Hz, 2H, + 10CH2), 3.5 to 3.8 (m, 3H, 4'H Phe and GLY) 3.9 and 4.3 (m, 4H, Gly, Leu, 5' H 3'H), 5,19 (m, 1H, 7H), the 5.45 (d, J=2.7 Hz, 1H, 1' H), the 6.9 to 8.4 (m, 12H, 1H, 2H, 3H, 4H, Ar Phe 3' NH, NH GLY Leu NH).

Example 12

4'-EPI-3'-N(glycyl-L-leucyl-L-i.e. phenylalanyl)-doxorubicin (5d)

< / BR>
Specified in the header of the compound of General formula 5d (3.25 g, yield 68%) was obtained from 4'-epidoxorubicin (2.9 g, 5 mmol) and N-trityl-phenylalanylglycyl-p-nitrophenylamino (9a: 3.5 g, 5 mmol) according to the procedure described in example 9. TLC on Kieselgel plates F254(Merck), vinyaya system methylene chloride/methyl alcohol/acetic acid/water (80:20:7:3 by volume), Rf = 0,46

HELL-MS: m/z [M + H]+< / BR>
H1NMR (400 MHz, D)

of 0.79 (d, J = 6.3 Hz, 3H, Leu), 0,81 (d, J = 6.3 Hz, 3H, Leu), 1,19 (d, J = 5,9 Hz, 3H, 6'CH3), between 1.3-1.6 (m, 4H, 2'Hax , Leu, Leu), is 1.82 (DD, J = 4,7 Hz, J = 12,5 Hz, 1H, 2'Heq), to 2.1-2.3 (m, 2H, 8-CH2), at 2.59 (DD, J = 8,2 Hz, J = of 13.7 Hz, 1H, Phe), 2,9-3,1 (m, 4H, Phe 10-CH2, h H) to 3.41 (DD, 4,7 Hz, J = 8,2 Hz, Phe), of 3.54 (DD, J = 5.5 Hz, J = 16.4 Hz, Gly), to 3.67 (DD, J = 6,2 is(d, J = 3.1 Hz, 1H, 1'H), 5,46 (s, 1H, 9-OH), a 7.1 to 7.3 (m, 5H, Ar-Phe), 7,53 (d, J = 8,2 Hz, 1H, NH-Gly), 7,60 (m, 1H, 3H), 7,86 (m, 2H, 1H, 2H, with 8.05 (d, J = 6,4 Hz, 1H, NH-Leu), 8,11 (t, J = 5,9 Hz, 1H, NH-Gly).

Example 13

Copolymer of 3-methacryloylamido-2-hydroxypropane, 3'-N(methacryloyloxy-L-i.e. phenylalanyl-L-latingirl)doxorubicin and N-methacryloylamido (A1)

< / BR>
< / BR>
The polymer precursor of the General formula B2 (7,15 g, 5 mmol-COOH), obtained as described in example 4 3'-N-(glycyl-leucyl-L-i.e. phenylalanyl)doxorubicin (5a: 2,36 g, 2.5 mmole) dissolved in anhydrous dimethylformamide (100 ml), then the solution is added N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0.7 g, 2.5 mmole). The mixture was stirred at room temperature for 24 hours, then poured into ethyl ether (800 ml). The residue is dissolved in ethanol (100 ml), planted with acetone (800 ml) and dried to constant weight to obtain specified in the header of the compounds of General formula A1 (7 g).

The content of doxorubicin HCl: 9% (m/m)

Example 14

Copolymer of 3-methacryloylamido-2-hydroxypropane, 3'-N-(methacryloyloxy-L-i.e. phenylalanyl-latingirl)doxorubicin and (N-methacryloyloxy)-2-hydroxypropylamino (A2)

< / BR>
< / BR>
The polymer precursor of the General formula B1 (10 g, 2,710-3EQ. p-nitrogene what the solution is added 3'-N(glycyl-leucyl-L-i.e. phenylalanyl)doxorubicin (5a: 1,53 g, 1,72 mmole). The mixture was stirred at room temperature for 24 hours, after which it is added L-aminopropanol (0,13 ml). After one hour the reaction mixture was added to stir the mixture acetone/ethyl ether (1.2 l 3: 1 by volume) and filtered. The residue is dissolved in ethyl alcohol (150 ml) and planted with a mixture of acetone: ethyl ether (1.2 l, 4:1 by volume) to obtain the polymer compound of General formula A2 (10 g).

The content of doxorubicin HCl: 9% (m/m)

Examples 13-18 illustrate how to obtain polimersvarka anthracyclines General formula A.

Example 15

Copolymer of 3-methacryloylamido-2-hydroxypropane, 4-dimethoxy-3'-N- (methacryloyloxy-L-i.e. phenylalanyl-L-latingirl)of daunorubicin and N-methacryloylamido (A3)

< / BR>
< / BR>
Specified in the header of the compound of General formula A3 is obtained by interaction of 4-dimethoxy-3'-N-(glycyl-L-leucyl-L-i.e. phenylalanyl)daunorubicin (5d: 1.48 g, 1,72 mmole) with the polymer precursor of the General formula B2 (10 g) in the presence of N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (0,48 g) in anhydrous dimethylformamide as described in example 13.

Example 16.

Copolymer of 3-methacryloylamido-2-hydroxypropane, 4'-EPI-3'-N-(methacryloyloxy-L-i.e. phenylalanyl-L-s is th polymer conjugate of General formula A4 (10 g) obtained by the interaction of 4'-EPI-3'-N-(glycyl-L-leucyl-L-i.e. phenylalanyl)doxorubicin (5e: 1.48 g, 1,72 mmole) with the polymer precursor of the General formula B1, and then with 1-aminopropanol (0,13 ml) as described in example 14.

The content of 4'-epidoxorubicin HCl: 9% (m/m)

Example 17

Copolymer of 3-methacryloylamido-2-hydroxypropane, 4'-EPI-3'-N-(methacryloyloxy-L-latingirl)doxorubicin and N-methacryloylamido (A5)

< / BR>
< / BR>
Specified in the header of the compound of General formula A5 is obtained as described in example 13, of the polymer of the intermediate compounds of General formula B2, 4'-EPI-3'-N-(glycylglycyl-L-i.e. phenylalanyl)doxorubicin (5e) and N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in anhydrous dimethylformamide.

Example 18.

Copolymer of N-(methacryloyloxy)-hydroxyethylamide, 3'-N-methacryloyloxy-L-i.e. phenylalanyl-L-latingirl)doxorubicin and N-methacryloylamido (A6)

< / BR>
< / BR>
Specified in the header doxorubicinol polymer prodrug of the General formula A6 is obtained by condensation polymer intermediate compounds of General formula B4 and 3'-N-(glycyl-leucyl-L-i.e. phenylalanyl)doxorubicin (5e) and N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in anhydrous dimethylformamide as described in example 13.

Example 19.

The antitumor activity of compounds of General Fort which is as follows.

Example 19

The copolymer A1

x: 90.59; y: 4.52; z: 3.87 mol.%;

M. C. = 33.400; MW/MN = 1.69.

Example 20

The copolymer A2

x: 95.56; y: 2.54; z: 1.88 mol.%;

M. C. = 33.400; MW/MN = 1.69 (polydispersity)

Example 21

The copolymer A3

x: at 90.61; y: 4.73; z: 3.64 mol.%;

M. C. = 32.900; MW/MN = 1.71.

Example 22

The copolymer A4

x: 95.56; y: 2.54; z: 1.88 mol.%;

M. C. = 32.900; MW/MN = 1.71.

Example 23

The copolymer A5

x: 90.59; y: 4.52; z: 3.87 mol.%;

M. C. = 32.900; MW/MN = 1.71.

Example 24

The copolymer A6

The content of doxorubicin in the form of cleaners containing hydrochloride salt: 8.5% (wt./weight. )

x: 95.45 area; y: 1.95; z: 1.66 mol.%;

M. C. = 29.800; MW/MN = 1.73.

Materials and methods.

1. The use of drugs.

All drug solutions are obtained immediately prior to use. The treatment is applied in a volume of 10 ml/kg body weight for 5, 9 and 15 days.

The anthracyclines dissolved in sterile water and the concentration determined spectrophotometrically.

Liofilizovannye polymers are dissolved in water to obtain the original solution of 25 mg anthracycline equivalent to 1 ml, in accordance with the concentration, further dilution is carried out in water.

2. TBE is ontiretse (5105cells/mouse) subcutaneously in C 75 B 1/6 mice to evaluate the activity of the primary tumor.

3. Evaluation of antitumor activity and toxicity.

Tumor growth is measured using a compass, and the tumor weight determined according to Guerin and others (see Cancer Chemother. Rep. 3, 1 (1972).

Antitumor activity is defined as the time (days) achieve 1 g tumor mass and is expressed as the slowing of tumor growth (SRO = TGD).

The average increase in survival time (T/C %) is calculated using the following formula:

T/C = (mean survival time (T/C %) /mean survival time of control group)100

Toxicity is assessed on the basis of weight loss and rough calculations, mainly reduction of the spleen and liver.

Neurotoxicity is defined as the number of mice with a defect in motor function.

The results are given in table 1.

Example 20.

The antitumor activity of compounds of General formula A3 M 5076.

The antitumor activity of compounds of General formula A3 described in example 15, is determined using the method and materials of example 19. The results are given in t hydroxypropane, 4'-EPI-3'-N-(methacryloyloxy-L-latingirl)doxorubicin and 1-N-(methacryloyloxy)-2-hydroxypropane) compared with 4'-epidoxorubicin HCl

Antitumor activity is determined by the same program (schedule) for 4'-epidoxorubicin HCl and the compounds of formula A4.

Against mouse reticulosarcoma compound of General formula A4 is more active than the free drug at all tested doses (see table 3).

The toxicity of the compounds of General formula A4 (copolymer of 3-methacryloylamido-2-hydroxypropane, 4'-EPI-3'-N-(methacryloyloxy-L-i.e. phenylalanyl-L-latingirl)doxorubicin and 1-N-(methacryloyloxy-2-hydroxypropane) compared with 4'-epidoxorubicin HCl.

Toxicity is assessed in healthy C 57BIF-mouse, processing intravenous, single zones 4'-HCl(13,2-16,15-19-20,6-25,2 and 33.2 mg/kg) and compound A4 (50-63-79-100-120-140 mg/kg).

To determine LD10and LD50in healthy C 57BIF-mouse analysis is used to determine the probability of the effect after 3-week recovery.

therapeutic index is calculated using the following formula:

< / BR>
Effective dose 50 is the dose that causes 50% reduction of tumor growth. Without the ENES in table 4.

Low toxicity compounds A4 allows the use of higher doses of product and get equal or better results than with 4'-HCl, and get the best therapeutic index. therapeutic index for 4'-HCl and compound A4 is equal to, respectively, 3 and 40.

3. Example formulations of compositions

Injection

Ingredients - the Number of mg per vial

The copolymer A1 - 500 (equivalent to 45 mg of doxorubicininduced)

Lactose - 250

Polysorbate - 80

Water for injection - needs to 20.0 ml.

Lactose, Polysorbate and the copolymer is dissolved with stirring in water for injection (VDI) and dearyou transmission of hydrogen ( 80% of the desired final volume of water). The resulting solution was brought to final volume by adding the required number of VDI.

The solution is filtered under sterile conditions through a microporous membrane 0.22 μm. 20 ml of the solution is aseptically placed in sterile type I ampoule of flint glass having a capacity of 50-57 ml.

The solution is frozen in vials at a temperature of from -40 to -45oC for 4-5 hours.

Then the product lyophilized and dried at the final stage in techenie sterile water, or sodium chloride for injection.

Preparative form containing copolymers A3 and A4, prepared by the above method.

1. Biologically active polimerobetonie the anthracyclines General formula A, obtained by the interaction of the polymer intermediate compound B of the formula

< / BR>
where n is 0 or 1;

x ranges from 70 to 98 mol.%;

w is 30 to 2 mol.%;

R1is CH2CH(OH)CH3or CH2CH2OH group;

R2is 2,4-dichlorphenoxy, n-nitrophenoxy or hydroxy-group,

with derivative anthracycline General formula 5

H - Y - [NH - D]

where [NH - D] is the residue of anthracyclineinduced formula Q

< / BR>
where one of RIand RIIis hydrogen and the other is a hydroxy - group or iodine;

RIIIis hydrogen or OCH3;

RIVis hydrogen or hydroxy-group;

Y is a peptide spacer selected from Gly-Phe-Gly, Gly-Leu-Gly, Phe-Leu-Gly, Gly-Phe-Leu-Gly or Leu-Leu-Gly; if R2is a hydroxy-group,

in the presence of N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, and, if necessary, carry out the processing of the received polimerbetonov anthracycline 2-hydroxypropylamino, and realamerican anthracyclin under item 1, consisting of units represented by formulas 1, 2 and 3

< / BR>
< / BR>
< / BR>
where z is from 1 to 29 mol.%;

y is up to 29 mol.%;

Z is a hydroxy-group or a residue of formula - other1where R1, R2, n, X and Y are defined in paragraph 1.

3. Polimerobetonie anthracyclin on p. 1, wherein said anthracyclineinduced formula Q represents doxorubicin, 4'-epidoxorubicin, 4-demethoxygeldanamycin, idarubitsin or 4'-iodine-4'-desoxidation.

4. Polimerobetonie anthracyclin under item 2, in which x ranges from 90 to 98 mol.%, y is from 1 to 10 mol.%, and z is from 1 to 10 mol.%.

5. The method of obtaining polimerbetonov anthracycline under item 1, which includes: i) the interaction of the polymer intermediate compounds of General formula B, where B is a

< / BR>
where n is 0 or 1; x is from 70 to 98 mol.%;

w is 30 to 2 mol.%;

R1is CH2CH(OH)CH3or CH2CH2OH group;

R2is 2,4-dichlorphenoxy, n-nitrophenoxy or hydroxy-group,

with anthracycline derivatives of the General formula 5

H - Y - [NH - D]

where [NH - D] is the residue of anthracyclineinduced formula Q

< / BR>
where one is born or OCH3;

RIVis hydrogen or hydroxy-group;

Y is a peptide part selected from Gly-Phe-Gly, Gly-Leu-Gly, Phe-Leu-Gly, Gly-Phe-Leu-Gly or Leu-Leu-Gly; if R2is a hydroxy-group,

in the presence of N-etoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, and, if necessary, carry out the processing of the received polimerbetonov anthracycline 2-hydroxypropylamino, and the reaction is carried out in an anhydrous polar solvent at a temperature of 15 - 30oC for 8 to 24 hours

6. Anthracycline derivative of General formula 5

H - Y - [NH - D]

where Y is Gly-Phe-Gly, Gly-Leu-Gly, Phe-Leu-Gly, Gly-Phe-Leu-Gly or Leu-Leu-Gly;

NH - D is the remainder of anthracyclineinduced NH2D formula Q

< / BR>
where one of RIand RIIis hydrogen and the other is a hydroxy-group or iodine;

RIIIis hydrogen or OCH3;

RIVis hydrogen or hydroxy-group.

7. The method of receiving anthracycline derivative of General formula 5

H - Y - [NH - D]

where Y is Gly-Phe-Gly, Gly-Leu-Gly, Phe-Leu-Gly, Gly-Phe-Leu-Gly, Leu-Leu-Gly;

NH - D is the remainder of anthracyclineinduced NH2-D formula Q

< / BR>
where one of RIand RIIis hydrogen and the other is hydro the hydroxy-group;

including: (i) interaction of the N-protected peptide of General formula 9

R3- Y - P,

where R3is trition;

P is n-nitrophenoxy;

Y is as defined above,

with anthracyclineinduced [NH2- D] defined above, to obtain the intermediate compounds of General formula 10

R3- Y - [NH - D]

in which [NH - D], Y, and R3defined above;

ii) removing the protective group R3obtaining peptidylarginine General formula 5 in free base form.

8. Pharmaceutical composition having antitumor activity, containing an active agent and a pharmaceutically acceptable diluent or carrier, wherein the active ingredient contains the connection PP.1 - 4 or 6 in an effective amount.

 

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