Derivatives of 5-imino-13-deoxy of anthracycline and the retrieval method (variants), pharmaceutical composition, method of treatment of cancer, autoimmune disease or immunodeficiency disorders

 

The invention relates to derivatives of 5-imino-13-deoxy of anthracycline formula

where R1, R2and R3are H, HE or och3group and R4represents the following groups:

The proposed pharmaceutical composition having antitumor activity, containing at least one derivative of 5-imino-13-deoxy of anthracycline. A method of treatment of cancer, autoimmune disease or immunodeficiency disorders, including the introduction of an effective amount of at least one derivative of 5-imino-13-deoxy of anthracycline. Proposed ways (options) obtain derivatives of 5-imino-13-deoxy of anthracycline. The method works by restoring anthracyclin 13-dailydata lamborghini.com sodium in anhydrous methanol at pH around 7.0 or below in the presence of p-toluenesulfonic acid through careful boiling solution under reflux in a nitrogen atmosphere at a temperature of about 75°C. Then the reaction mixture is cooled, treated with the my gradient of chloroform/methanol, when this product is 13-deoxy of anthracycline elute with methanol. Conduct further purification methanol eluate using preparative HPLC, then the solution of purified 13-deoxy of anthracycline in a mixture of methylene chloride and water, add the bicarbonate and potassium di-tert-butyl-dicarbonate. Allocate obtained 3’-N-BOC-deoxy anthracycline, dissolving it in anhydrous methanol, the resulting solution is cooled and spend amination of 3’-N-Boc-13-deoxy of anthracycline through a saturated solution of ammonia at a temperature of about 0-4°C. to Remove the solvent and ammonia, dissolve the residue in anhydrous methanol, remove 3’-N-BOC-group and using preparative HPLC produce the target product, which is optionally converted into the hydrochloride of 5-imino-13-deoxy of anthracycline. The technical result - derivatives of 5-imino-13-deoxy of anthracycline with less side effect of cardiotoxicity. 6 N. and 7 C.p. f-crystals, 4 tab., 12 Il.

The technical field to which the invention relates.

The invention relates to derivatives of 5-imino-13-deoxy of anthracycline, medical use of 5-imino-13-deoxy of anthracycline and methods of producing proizvodstvatekhnologii type are doxorubicin and daunorubicin, which contain 13-ketogroup and 5-ketogroup. Doxorubicin is described in U.S. patent 3590028 has a wide range of use in the treatment of cancer and is used in the treatment of leukemias, lymphomas and solid tumors. Daunorubicin described in U.S. patent 3616242, is used in the treatment of acute leukemia. However, the use of these drugs is limited by serious side effect of cardiotoxicity, so the total number of medications that can be prescribed to the patient, may not exceed 550 mg/M2(E. A. Lefrak et al., Cancer, 32:302, 1973). Even if this or close to the recommended amount of the maximum total cumulative dose (430-650 mg/M2) in 60% of patients have significant and sustained cardiac dysfunction and in 14% of developing congestive heart failure (A. Dresdale et al., Cancer, 52:51, 1983). Thus, while these drugs are useful in the inhibition of growth of cancer tumors, the patient may die from congestive heart failure due to severe cardiotoxic side effect of the medication.

Some researchers believe that the present is the result of generation of free radicals quinone part of anthracycline mo is 7, 1977; J. L. Zweier, J. Biol. Chem., 259:6056, 1984). On the other hand, there is clear evidence that the generation of free radicals may not be the only mechanism of cardiotoxicity, because in the presence of traps free radicals drugs still produce cardiac damage (J. F. VanVleet c et al., Am.J.Pathol., 99:13, 1980; D. V. Unverferth et al., Am.J.Cardiol., 56:157, 1985; S. Myers et al., Seminars in Oncology, 10:53, 1983; R. H. M. Julicher c et al., J. Pharm. Pharmacol., 38:277, 1986; E. A. Porta et al., Res. Comm. Chem. Pathol. Pharmacol., 41:125, 1983).

It was also found that the inhibition of the process of generation of free radicals does not exclude the cardiotoxicity of these anthracyclines (P. S. Mushlin et al., Fed. Proc., 45:809, 1986). Instead, this study showed that the cardiotoxicity of doxorubicin and daunorubicin, which leads to reduced cardiac contractility, also depends on the metabolic recovery of 13-keto residue to 13-hydroxy metabolite. In test systems where doxorubicin was not turned into a 13-dihydro connection, cardiotoxic effects were observed only at very high concentrations (200-400 micrograms/ml) (P. S. Mushlin et al., Fed. Proc., 44:1274, 1985; R. D. Olson et al., Fed. Proc., 45:809, 1986). In contrast, 13-dihydro metabolites doxorubicinol and doorlag (1-2 micrograms/ml, R. D. Olson et al., Proceed. Am. Assoc. Cancer Res., 26:227, 1985; R. D. Olson et al., Proceed. Am. Assoc. Cancer Res., 28:441, 1987).

If doxorubicin allow you to stay in the test system, even for a very short period of time, then run some metabolic transformations and produces 13-dihydro metabolite in sufficient quantities, so that begins to develop cardiotoxicity (L. Rossini et al., Arch. Toxicol. Suppl., 9:474, 1986; M. Del Tocca et al., Pharmacol. Res. Commun., 17:1073, 1985). Thus, they have accumulated substantial evidence that the cardiotoxicity of these drugs as doxorubicin and daunorubicin is the result of strong cardiotoxic effects of their 13-dihydro metabolites ((R. Mushlin et al., Rational Drug Therapy, 22:1, 1988; S. Kuyper et al., FASEP Journal, 2:A1133, 1988; R. Boucek et al., J. Biol. Chem., 262:15851, 1987; and R. Olson et al., Proc. Natl. Acad. Sci., 85:3585, 1988).

In the present invention used the fact that the 13-deoxy form of doxorubicin and daunorubicin or other similar anthracyclines will not be metabolically transformed into cardiotoksicnae 13-dihydro forms, and thus, 5-ketogroup modify with the formation of such compounds, which will be less likely to generate free radicals, providing, thus, a means for vinoy dose.

The invention

The purpose of the present invention is new derivatives of 5-imino-13-diethoxyanthracene, which have fewer side effects.

Accordingly, an object of the present invention are methods of obtaining derivatives of 5-imino-13-deoxy of anthracycline.

In accordance with this and other objectives and advantages of the present invention in the preferred objects of the method for obtaining derivatives of 5-imino-13-deoxy of anthracycline.

Usually, the anthracyclines of formula I

where R1, R2and R3are H, HE or och3group and R4represents the balance of sugar, other than morpholinyl substituted sugar residue, easily converted into 13-tazelgettaze according to known methods. Anthracyclin 13-tazelgettaze restore the sodium lamborghini.com in acidic conditions to derivatives of 13-deoxy of anthracycline. Of course, under morpholinium refers to substituted morpholinyl residues. Products are then purified using preparative chromatography without extraction stages. Purified derivatives of 13-deoxy of anthracycline turn in the N-BOC derivative and then treated with NH3

Additional preferred objects of the present invention provide a method of obtaining derivatives of 5-imino-13-deoxy of diethoxyanthracene. The method includes obtaining an acidic solution anthracyclin 13-dailydata with lamborghini.com. The solution is gently refluxed. The reaction mixture is cooled. To the solution add a small amount of water followed by the addition of halogenated carbon solvent. The mixture is filtered. The filtrate was subjected to purification using preparative chromatography emitting derivatives of 13-deoxy of anthracycline. Cleared derivatives protect using tert-butoxycarbonyl group with the formation of 3’-N-BOC derivative, which is then treated with ammonia to obtain 3’-N-BOC-5-imino-13-diethoxyanthracene. Removal under acidic conditions, protection of the amino group leads to 5-imino-13-diethoxyanthracene, which are then purified using chromatography.

Another preferred object of the present invention provides a method for obtaining derivatives of 5-imino-13-deoxy of anthracycline. The method involves the preparation of a solution dissolved by the l of anhydrous methanol. To the solution add about 0.8 g sodium laborgerate.

The solution is heated to a temperature of from about 68°C to about 72°C. the Solution is then gently refluxed for about 1 h in nitrogen atmosphere.

The reaction mixture is concentrated to approximately 20 ml of the Reaction mixture is cooled in a freezer to a temperature of from about 0°C to about 4°C.

The reaction mixture was added 0.5 ml of water, and then 200 ml of chloroform. To the reaction mixture add anhydrous sodium sulfate. Salt is filtered off.

The filtrate is passed through a column of silica gel. Then the column is washed with a mixture of chloroform/methanol to obtain colorless eluate.

The fraction containing the product, elute with methanol. The methanol eluate is evaporated. The residue obtained after evaporation, dissolved in 30% buffered solution of acetonitrile in ammonium formate.

The product distinguish by high performance liquid chromatography (HPLC) using a column containing phenyl. The product is separated from other impurities by using a gradient of acetonitrile/ammonium formate. Fraction, purified by HPLC, and then diluted with approximately the same volume of water and the collected fraction, and this restart elute with methanol. The methanol eluate concentrate and the same product is precipitated by adding ethyl ether. The solid product produce by filtration to obtain 600 mg of 13-deoxy doxorubicin.

About 600 mg of purified 13-deoxy doxorubicin dissolved in about 60 ml of methylene chloride and 10 ml of water and treated with 180 mg of potassium bicarbonate and about 180 mg of di-tert-BUTYLCARBAMATE at room temperature for about 2 hours the Organic solution is separated, washed with water and dried with anhydrous sodium sulfate. The solution is evaporated to dryness. The residue is dissolved in about 100 ml of anhydrous methanol. The solution is saturated with ammonia, stored at a temperature of from about 0°C to about 4°C for 48 hours, the Solvent and ammonia are removed in vacuo to obtain 3’-N-BOC-5-imino-13-deoxy doxorubicin. 3’-N-BOC-5-imino-13-deoxy doxorubicin, obtained above, is treated with 60 ml of anhydrous methanol containing about 0.1 M hydrogen chloride at room temperature for about 2 h to remove 3’-N-BOC-group. The resulting solution was concentrated to about 5 ml

A concentrated solution allocate using preparative HPLC with a phenyl column. The product is separated from other impurities by using the gradient and the water, injected into preparative phenyl column for HPLC and washed with water to remove salts. Then elute the product with methanol. The methanol eluate is acidified with about 0.5 ml of hydrogen chloride in ethyl ether. Then the solution is concentrated to about 5 ml To precipitated product add about 10 ml of ethyl ether. The precipitate was filtered to give about 40% to about 60% of 5-imino-13-deoxy doxorubicin hydrochloride.

The present invention also provides compounds having the following formula

where R1, R2and R3are H, HE, or the OMe group and R4represents the balance of sugar, other than morpholinyl substituted residue of sugar. It should be clear that under morpholinium refers to substituted morpholinyl residues. Compounds that include morpholinyl substituted sugar residue, show significantly increased toxicity.

Further, the present invention is a pharmaceutical composition comprising at least one derivative of 5-imino-13-deoxy of anthracycline.

In addition, the present invention also provides a method of treating cancer, including leukemia, lymphoma and solid tumors, and this way VK the wedge.

Further objectives and advantages of the present invention will be clear and obvious to the person skilled in the art from the following description. Detailed description shows and describes only the preferred variants of the invention in order to illustrate the best form of embodiment of the invention. For those experts who will carry out the invention, the invention includes other and different ways. Details of the invention can be modified in various aspects, without deviating from the essence of the invention. Thus, the drawings and description should be regarded as illustrative in nature but not limiting the scope of invention.

Information confirming the possibility of carrying out the invention.

The present invention provides derivatives of 5-imino-13-deoxy of anthracycline, their use and methods for obtaining derivatives of 5-imino-13-deoxy of anthracycline. Table 1 presents examples of derivatives of 5-imino-13-deoxy of anthracycline, which can be obtained according to the present invention. As discussed above, compounds such as those shown in table 1, have, as you know, antitumor properties. Connections are also less pobocka 75°C. Preferably the methods carried out at a temperature from about 65°C to 75°C. More preferably, the methods carried out at a temperature from about 68°C to about 72°C.

The method according to the present invention includes a number of General conditions. For example, the reduction is preferably carried out under acidic conditions. In other words, the pH should be around 7.0 or below. Known methods for obtaining the above connections that use alkaline conditions in the reaction mixture, was found to cause decomposition of the reactant and products.

In addition, oxygen, and water should be excluded during the test recovery. Preferably the reaction is carried out in nitrogen atmosphere or an inert gas using anhydrous solvents. In addition, the amination reaction should be conducted at a temperature of about 0-4°C. a higher temperature may cause decomposition of the reactant and products.

In addition, water must be excluded from the amination reaction. Preferably the reaction is carried out in anhydrous solvents.

In accordance with the above, the present invention provides methods of making compounds of General formula AI.

The following block diagram illustrates an example of a variant of the method according to the present invention for the 5-imino-13-deoxy doxorubicin, which is 13-deoxy anthracycline derivatives.

In the following table 1 presents examples of anthracycline derivatives, the synthesis of which is described here.

In the compounds R4may be a modified version of different anthracycline analogues. In addition, the D ring may be substituted with halogen or hydroxyl group. For example, the D ring may be substituted by fluorine, iodine, or any other halogen.

Typically, the methods according to the present invention include the preparation of an acidic solution of 5-imino-13-deoxy of anthracycline. The solution is gently refluxed. Then, the reaction mixture can be cooled. According to one example, the reaction mixture is cooled to a temperature of from about 0°C to about 4°C. may Then be added a small amount of water for hydrolysis of the product. To the reaction SMEs can be cooled. For example, halogenated carbon, the solvent may be at a temperature of from about 0°C to about 4°C. an example of a halogenated carbon solvent, which can be used here, is chloroform. The reaction mixture can then be filtered after adding anhydrous sodium sulfate. Filtering can be performed at low temperature. For example, filtering can be performed at a temperature of from about 4°C to about 15°C.

The addition of water as described above, preferably initiates the hydrolysis. Any excess water can be removed by adding anhydrous sodium sulfate. Then the inorganic salt preferably is filtered off from the reaction mixture. The filtrate may be subjected to chromatographicaliy on a column of silica gel. Hydrophobic impurities can be separated by elution of less polar solvents.

13-deoxy anthracycline products can then be suirvey and the eluate is further purified.

Purified derivatives of 13-deoxy of anthracycline can be dissolved in a mixed solvent of methylene chloride and water containing a base and di-tert-butyl-dicarbonate. The solution can be mixed at a temperature of about commitology to be removed, and the residue may be dissolved in an alcohol solvent. An alcoholic solution may be saturated with ammonia at a temperature of from about 0°C to about 4°C for about 24 h, the Solvent may be removed, and then the dry residue can be processed acidified alcohol solvent at a temperature of about room temperature for about 2 hours the resulting solution may be subjected to concentration and subjected to further purification, then acidified and subjected to precipitation in ether with obtaining cleaners containing hydrochloride salt of 5-imino-13-deoxy of anthracycline.

Preferably, the methods according to the present invention include the preparation of a solution anthracyclin 13-tazingiprozem in anhydrous methanol with p-toluensulfonate and lamborghini.com sodium. The solution is gently refluxed under nitrogen atmosphere and then cooled. Water is added followed by the addition of chloroform. The precipitated salt is filtered off and the filtrate allocate on a column of silica gel. Hydrophobic impurities formed during the decomposition, elute mixed solution of chloroform and methanol. Products 13-deoxy anthracyclines elute with methanol. The methanol eluate is further cleaned using preparative HPLC.

Purified 13-the deoxy anthracyclines dissolved in a mixture of meth is ri a temperature of about room temperature for about 2 hours The organic layer was separated, washed with water and dried over anhydrous sodium sulfate. Then the solvent is removed in vacuum. The residue is dissolved in anhydrous methanol saturated with ammonia, and stored at a temperature of from about 0°C to about 4°C for about 48 hours, the Methanol and ammonia are removed from the reaction product, which is treated with diluted acid solution in alcohol at a temperature of about room temperature for about 2 h to obtain the compound 5-imino-13-deoxy of anthracycline. The connection specified in the solution is concentrated and then purified using preparative HPLC to remove impurities, and then salt. Product elute with methanol, acidified and precipitated with obtaining cleaners containing hydrochloride salt of 5-imino-13-deoxy of anthracycline.

The following description illustrates an example implementation of the method according to the present invention.

Example 1

Getting hydrochloride 5-imino-13-deoxy doxorubicin

About 1 g of doxorubicin 13-tazelgettaze hydrochloride and 2.4 g of p-toluenesulfonic acid are dissolved in about 50 ml of anhydrous methanol. To this solution add about 0.8 g laborgerate sodium. The resulting solution is heated to about 68-72°C and incubated with gentle boiling with education is up to about 20 ml and cooled in the freezer to about 0-4°C. Add about 0.5 ml of water and then adding about 200 ml of chloroform. Add anhydrous sodium sulfate and after shaking salt is filtered off.

Then the solution is passed through a column of silica gel (2,5×5 cm). Next, the column is washed with a mixture of chloroform/methanol (10/1) to obtain colorless eluate. The combined fractions containing the product, elute with methanol. The methanol eluate is evaporated and the residue is dissolved in 30% buffered solution of acetonitrile in ammonium formate (pH of 4.0, 0.5%) and allocate using preparative HPLC. Use phenyl column and separation of the product from other impurities reach a gradient of acetonitrile/ammonium formate (from 27% to 30% acetonitrile for 30 min).

Fraction, purified by HPLC, diluted with approximately an equal volume of water. The solution is passed through preparative phenyl column for HPLC. Column elute with water to remove salts. Then elute the product with methanol. The methanol eluate is concentrated and the product precipitated by addition of ethyl ether containing hydrogen chloride and collected by filtration to obtain 600 mg of 13-deoxy doxorubicin hydrochloride. The yield is 80%.

Data thin layer of chromatography the =233, 252, 293, 485 nm.

The data of mass spectrometry (MS):

530 (M+H)

Data1H NMR spectra (in methanol d4): (see below)

of 1.30 (d, 3H, 6’-CH3),

of 1.85 (m, 2H, 13-H2),

is 2.05 (m, 2H, 10-H2),

2,60 (d, 1H, 12-H),

3,05 (d, 1H, 12-H),

3,55 (m, 1H, 5’-H),

3,90 (m, 2H, 14-H2),

of 4.05 (s, 3H, O-CH3),

of 4.25 (m, 1H, 4’-H),

of 4.95 (m, 1H, 3’-H),

of 5.40 (m, 1H, 1'-N),

at 7.55 (d, 1H, 3-H),

the 7.85 (d, 1H, 2-H),

of 7.95 (d, H, 1-H).

About 600 mg of purified 13-deoxy doxorubicin dissolved in about 60 ml of methylene chloride and 10 ml of water and treated with 180 mg of potassium bicarbonate and about 180 mg of di-tert-BUTYLCARBAMATE at a temperature of about room temperature for about 2 hours the Organic solution is separated, washed with water and dried over anhydrous sodium sulfate. The solution is evaporated to dryness and the residue is dissolved in about 100 ml of anhydrous methanol. The solution is saturated with ammonia, maintained at a temperature of from about 0°C to about 4°C for 48 hours, the Solvent and ammonia are removed in vacuo to obtain 3’-N-BOC-5-imino-13-deoxy doxorubicin, which is treated with 60 ml of anhydrous methanol containing about 0.1 M hydrogen chloride, at a temperature and allocate product using preparative HPLC. Fraction, purified by HPLC, diluted with approximately an equal volume of water. The solution is injected into the preparative phenyl column for HPLC and elute with water to remove salts. Product elute with methanol. The methanol eluate is acidified hydrogen chloride in ethyl ether and the solution concentrated.

Add ethyl ether and the precipitate is filtered with getting about 360 mg (60%) 5-imino-13-deoxy doxorubicin hydrochloride.

Data thin-layer chromatography (TLC): Rf=0,35.

These UV spectra:max=222, 251, 310, 544, 585 nm.

Data1H NMR spectra: (Meon-d4).

of 1.30 (d, 3H, 6’-CH3),

of 1.85 (m, 2H, 13-H2),

of 2.10 (m, 2H, 10-H2),

2,60 (d, 1H, 12-H),

2,75 (d, 1H, 12-H),

2,95 (d, 1H, 12-H),

to 3.50 (m, H, 5’-H),

3,90 (m, 2H, 14-H2),

to 4.15 (s, 3H, O-CH3),

of 4.25 (m, 1H, 4’-H),

5,10 (m, 1H, 3’-H),

the ceiling of 5.60 (m, 1H, 1’-H),

to 7.50 (d, 1H, 3-H),

7,80 (d, 1H, 2-H),

with 8.05 (d, 1H, 1-H).

The data of mass spectrometry (MS): 529 (M+H)

The present invention also provides compounds having the following formula:

Furthermore, the present and the hydroxy anthracycline.

In addition, the present invention still provides a method of treating cancer, leukemias, lymphomas and solid tumors. The method includes a step of introducing an effective amount of at least one derivative of 5-imino-13-deoxy of anthracycline. The dosage will be similar known compounds. Due to their analogy with known compounds, a specialist in this area will be able to determine a treatment regimen, leading to similar performance, without undue experimentation. However, as discussed above, the present invention provides significantly reduced side effects, including reduced cardiotoxicity.

The present invention also provides a method of treatment of autoimmune disease or immunodeficiency disorders, such as HIV (AIDS). The method includes a step of introducing an effective amount of at least one derivative of 5-imino-13-deoxy of anthracycline.

BIOLOGICAL TESTS

Comparative evaluation of cardiotoxicity of doxorubicin, GPX 150 and GPX-100 in chronic models of rabbits

Description of the experiment

Twenty-four new Zealand white rabbit of the same age, weight 3,0 kg split randomly into 4 groups (N=6 rabbits/group). In the data g is) as a control. The dose of the drug or filler injected (iv) into the ear vein twice/week (Tuesdays and Fridays). Every week (on Mondays) take samples of serum and blood to the heart every week or next week (on Thursdays) are M-echocardiographic examination data retrieval fractional shortening of the left ventricle (LVFS). Daily estimate feed intake, and body weight was measured weekly (see Fig.6). Control rabbits receive the same amount of food as rabbits, which are doxorubicin (pair-feeding). After driving animals receive samples of the free wall of the apex of the heart and the left ventricle, which maintain in 4% glutaraldehyde (pH 7.4) for histological examination and analysis by light and transmission electron microscopy. Tissue samples of the left ventricle also get to assess cardiac levels anthracyclines and metabolites. Rabbits slaughtered at occurrence of cardiotoxicity (LVFS <30% or decreased LVFS 10% of the units, i.e., from 42% to 32% LVFS), symptoms of life-threatening or toxicity, which causes weakness (i.e., when strong mielosupression or mucositis) or 13 weeks after the start of the study.

orubicin, while optimal antitumor dose GPX 150 exceeds the optimal dose of doxorubicin in 4-8 times. Thus, were selected such doses GPX-100 and GPX-150, which twice (for GPX-100) and 7 times (for GPX 150) exceed the cumulative cardiotoxic dose of doxorubicin (17.5 mg/kg). Doxorubicin is administered at a dose of 1.25 mg/kg twice/week for 7 weeks (cumulative dose of 17.5 mg/kg), GPX-100 administered at a dose of 2 mg/kg twice/week for 10 weeks (cumulative dose of 40 mg/kg), and GPX-150 is administered in a dose of 5 mg/kg twice/week for 12 weeks (cumulative dose of 120 mg/kg).

Results

The most sensitive to the effects of doxorubicin type of blood cells appeared to be red blood cells (RBC). In Fig.9 illustrates that the control layer of the air force (upper graph - Weekly RBC levels) remained stable throughout the experiment. Within two weeks after the start of the experiment to introduce analogues of anthracycline level RBC reduced in all groups compared with control (Fig.9). The action of GPX-150 at RBC is the average between exposure to doxorubicin and GPX-100, however, is significantly different from the control throughout the experiment. The value of RBC in the group receiving doxorubicin increases after 7 weeks the week. The impact of GPX-100 on the levels of RBC and hematocrit (STC) continues to decline throughout the study (see Fig.10). By the end of the experiment, two rabbits receiving GPX-100, have a hematocrit below 10%, and one of the rabbits of this group dies during the experiment. Although the cause of death remains unknown, one of the factors contributing to his death, could be anemia.

The count of white blood cells (WBC) showed that their level is less exposed to anthracyclines than RBC (see Fig.8, upper graph - Weekly WBC levels). GPX-100 and GPX 150 lead to such a reduction in the number of WBC, which is similar to the reaction to the action of doxorubicin. Values anthracycline groups differ from control values between 3 and 7 weeks. The group receiving GPX-100 was the only group treated with anthracyclines, which had significantly lower neutrophil count than the control group (see Fig.11 - Weekly levels of neutrophils).

Summarizing, we can conclude that when used in these experiments, the doses of GPX-100 causes an equal or greater reduction in the number of blood cells than doxorubicin. Although GPX 150 also suppresses the number of blood cells compared with the control, and its value as a whole is premiato the lead to poor weight gain compared to control rabbits. The effect of doxorubicin, resulting in slower weight gain compared to control, cannot be explained by differences in feed intake, because the control group rabbits and doxorubicina group of rabbits are in terms of pair-feeding. GPX-100 causes a similar reduction in feed intake and slower weight gain compared to rabbits treated with doxorubicin. Rabbits treated GPX-150 show higher feed intake compared to control rabbits (because of the control and doxorubicina groups of rabbits receive pair feeding), although they do not have greater weight gain than the control rabbits, suggesting that GPX 150 may also intervene in the mechanisms associated with weight gain in rabbits.

Doxorubicin causes severe toxicity, which is not observed in the groups treated GPX-100 or GPX 150. In four of six rabbits treated with doxorubicin, was severe mucositis, two severely swollen and bleeding mouth and lips. These effects (mucositis, swelling, or bleeding of the lips) was not observed in any of the rabbits treated GPX-100 or GPX 150. Three of the six rabbits treated with doxorubicin was observed depletion of skeletal muscles, and the two - swelling (back the muscles, although one rabbit treated GPX-100, when driving was a small swelling of the chest wall. All rabbits treated with doxorubicin, are loss of hair, i.e. hair falls either moderately or very strongly. Only two of the five rabbits treated GPX-100, had the wool, and in four of six rabbits treated GPX-150, hair loss has been significant.

GPX-100 administered at the maximum tolerated dose, and one of the six rabbits treated GPX-100, died at the end of the experiment and was not surveyed. To the end of the experiment, the rabbits treated GPX-100, were sluggish, pale ears, nose and eyes, and 4 of the 5 rabbits were cold to the touch. Such symptoms are not observed in other rabbits treated GPX-150 or doxorubicin.

Doxorubicin also causes heart disorder that is estimated using M-echocardiography (LVFS). Weekly data of ultrasound (see Fig.7) show a progressive decrease in fractional contractions of the left ventricle (LVFS) after six weeks of treatment with doxorubicin. After driving all six animals rabbits treated with doxorubicin, have cardiac dysfunction by echocardiography, and LVFS significantly different from values in control group (P<0,001). In contrast Bo of cardiac abnormalities by echocardiography.

When driving distinguish the left atrium, divide it in half and to assess cardiac activity explore both halves in the bath tissues, where during the whole study remains constant pre-load and load after the experience. Atrium, obtained from rabbits treated with doxorubicin, characterized by deterioration of cardiac contractility (dF/dt) in comparison with control over a range of power-frequency (1, 2, and 3 Hz). For Atria obtained from rabbits treated with G-100 and GPX-150, not characterized by the deterioration of dF/dt in comparison with the control. Cardiac contractility is also assessed on the first reduction, the next 20-, 30 - and 60-second rest intervals (20-, 30 - and 60-second PRP). Enhanced airway should be the result of an increase in reserves of calcium in sarcoplasmatic reticulum (SR), which is formed during the interval of rest, and such drugs as doxorubicin and caffeine, which affect the functioning of the SR, also affect the heart breaks. As expected, doxorubicin significantly impairs contractility (dF/dt) palpitations rest period (see Fig.12), but airway (dF/dt) of the Atria obtained from rabbits treated with G-150, not worse sravnenie is s, treated GPX-100, has intermediate values that differ significantly from the values of the control and doxorubicin.

Conclusions

At the same mielosupressivne doses GPX-100, GPX 150 and doxorubicin, rabbits treated GPX-100 and GPX-150, do not have cardiac dysfunction in vivo, assessed by LVFS, and all rabbits treated with doxorubicin, have different cardiac activity in vivo. Tape the Atria obtained from rabbits treated GPX-150, when driving not have changes in contractility (dF/dt) no comparison with the control. Atrium, obtained from rabbits treated with doxorubicin, demonstrates worsened airway (dF/dt) at all frequencies heartbeat and potentiated the heartbeats rest period. Atrium, obtained from rabbits, constantly treated GPX-100, does not show any deterioration in contractility (dF/dt) throughout the investigated range of power-frequency, but it has the deterioration of contractility potentiated palpitations rest period. In addition, rabbits treated GPX-100 and GPX-150, have fewer symptoms of stomatitis (mucositis) and wasting of muscles than rabbits treated with doxorubicin.

A comparative study of antitumor activity of GPX-100, GPX 150 and doxorubicin in vivo.< in a murine model of leukemia using CD2F1 mice, which is injected P388D1 cells (derived from mouse cancer cell leukemia). CD2F1 mice intraperitoneally (ip) injected 1 million P388D1 cells in two subgroups of mice. Subgroup 1 consisted of 10 groups of 10 mice/group and subgroup 2 consisted of 7 groups of 10 mice/group. CD2F1 mice were randomly divided into experimental groups.

In subgroup 1 are investigating two doses of doxorubicin (dox) (0.8 mg/kg and 1.6 mg/kg continuously administered for 9 days on days 1-9 of the experiment), three doses of GPX-100 (1.6 mg/kg 3.2 mg/kg and 6.4 mg/kg administered according to the same scheme as dox) and four doses of GPX-150 (0.8 mg/kg, 1.6 mg/kg 3.2 mg/kg and 6.4 mg/kg administered according to the same scheme as dox). Determine the value of T/S treated mice (expressed as a percentage ratio values in treated groups to the values in the control group) for 20%, the average and 80% survival time (see tables 2-4). Dox shows the expected antitumor activity, but GPX-100 and GPX-150 also exhibit antitumor activity. Moreover, GPX-150 gives the highest value of T/S from observed during the experiment (200% at 20% survival time, PL. 2).

The experiments conducted in subgroup 2 has the aim of clarifying and distribution according to the dose-effect on the use of 3 drugs in podgruppe, which is similar to the response of the T/S on the effect of dox (see tables 2-4). Like the results obtained in subgroup 1, the best response of the T/S in the experiment are observed when using GPX 150 (at a dose of 9.6 mg/kg) in all three categories of survival times (i.e., 215%, 155% and 137% at 20%, on average and 80% survival time, respectively).

GPX-100 and GPX-150 at the doses used in this study demonstrate antitumor activity, which is equal to or greater than the activity exhibited by doxorubicin. At the doses used in this study, GPX-150 gives a higher antitumor effect compared with the effect of doxorubicin or GPX-100. Although in these experiments GPX-150 was more effective than doxorubicin or GPX-100, it was less strong than GPX-100 or doxorubicin. Thus, GPX-100 and GPX-150 have antitumor activity in models R murine leukemia.

2. Buildings and equipment.

The experiments were carried out on the basis of the following research institutions:

Mountain States Medical Research Institute

190 E. Bannock

Boise, ID 83172

Veteran Affairs Medical Center

500W. Fort Street

Boise, ID 83702

3. Sponsor. Gem Pharmaceuticals, Inc.

Pelham, AL

4. The purpose of the study. The goal was to determine whether GPX-100 and GPX 150 to show in the data cells R.

5. Methods

a) information about the materials used in the experiments:

GPX-100 (manufactured by GEM, LOT # Q200698273; may 31, 2000) was weighed for each dose daily and prepared in saline solution (0.9% NaCl). The medicinal substance was stored in the refrigerator (4°C). Doxorubicin (manufactured by GEM) was weighed for each dose daily and prepared in saline solution. The medicinal substance was stored in the refrigerator (4°C).

Cells R obtained in the collection of ATS may 22, 1998 (LOT # 946949). GPX-150 received from the company SynQuest, Inc., LOT # SMT-8-172-23-26, 26 October 2000

b) information about animals:

Male CD2F1 mice weighed 25-30 g at the beginning of the experiment. The study was conducted from March 7 through July 21, 2001

6. The test Protocol

On day 0 mice CD2F1 do intraperitoneal injection (ip) of 1.6 million freshly harvested tumor cells R. Mice are divided into 2 subgroups: subgroup 1 consists of 10 groups of 10 mice/group and subgroup 2 consists of 7 groups of 10 mice/group.

Subgroup 1 includes the following 10 groups of mice:

1) control group (100 ál saline/mouse injected ip on days 1-9, n=10);

2) dox group (0.8 mg/kg dox injected ip on days 1-9, n=10);

3) dox group (1.6 mg/kg administered ip on days 1-9, n=10);

4) GPX-100 group (1.6 mg/kg administered ip on days 1-9, n=10);

5) GPX-100 group (3.2 mg/kg of the n=10);

8) GPX-150 group (1.6 mg/kg administered ip on days 1-9, n=10);

9) GPX-150 group (3.2 mg/kg administered ip on days 1-9, n=10);

10) GPX-150 group (6.4 mg/kg administered ip on days 1-9, n=10).

Subgroup 2 includes the following 7 groups of mice:

1) control group (100 ál saline/mouse injected ip on days 1-9, n=9);

2) dox group (0.8 mg/kg dox injected ip on days 1-9, n=9);

3) GPX-100 group (2.4 mg/kg administered ip on days 1-9, n=9);

4) GPX-100 group (3.2 mg/kg administered ip on days 1-9, n=9);

5) GPX-150 group (9.6 mg/kg administered ip on days 1-9, n=10);

6) GPX-150 group (12,8 mg/kg administered ip on days 1-9, n=10);

7) GPX-150 group (25.6 mg/kg administered ip on days 1-9, n=10).

Mice are weighed every other day and for each group build a graph of body weight throughout the experiment. Throughout the experiment, daily monitor mortality. The results in each group is expressed as the percentage increase in life span of the treated mice (T) compared with the control (C); (T/C x 100).

7. Results

Investigated the antitumor activity of GPX-100 and GPX 150 and doxorubicin in vivo in a murine model of leukemia using CD2F1 mice, which were injected P388D1 cells (derived from mouse cancer cell leukemia). CD2F1 mice intraperitoneally (ip) injected 1 million P388D1 cells in two subgroups of mice. Subgroup 1 consisted of belali experimental groups.

In subgroup 1 investigated two doses of doxorubicin (dox) (0.8 mg/kg and 1.6 mg/kg continuously administered for 9 days on days 1-9 of the experiment), three doses of GPX-100 (1.6 mg/kg 3.2 mg/kg and 6.4 mg/kg administered according to the same scheme as dox) and four doses of GPX-150 (0.8 mg/kg, 1.6 mg/kg 3.2 mg/kg and 6.4 mg/kg administered according to the same scheme as dox). Defined value T/S treated mice (expressed as a percentage ratio values in treated groups to the values in the control group) for 20%, the average and 80% survival time (see tables 2-4).

The best effect of doxorubicin was obtained at a dose of 0.8 mg/kg as the dose of 1.6 mg/kg was toxic at 80% survival time values (T/S were less than 100% (82% according to table 4)). However, the values of T/s for dox dose of 1.6 mg/kg was similar to doses of dox 0.8 mg/kg at 20% and the average survival time (according to tables 2 and 3, 157% vs. 152% 135% vs. 147%, respectively).

Dose GPX-100 3.2 mg/kg and 6.4 mg/kg was also toxic and gave values of T/C less than 100% for both doses of 20%, on average and 80% survival time (table 2-4). Dose GPX-100 1.6 mg/kg resulted in values of T/S, similar to the values of T/S under the action of dox at a dose of 0.8 mg/kg (tables 2-4).

Dose GPX 150 6.4 mg/kg resulted in the largest values of T/S in the GPX-150 with an average and 80% survival time was equal to doses of 3.2 mg/kg and 6.4 mg/kg (142% versus 142% 153% 153 against%, accordingly, according to tables 3 and 4).

The experiments conducted in subgroup 2 were intended to clarify and extend the dose-effect on the use of 3 drugs in subgroup 1. As a standard for comparison we used the dose of dox 0.8 mg/kg

Dose GPX-100 2.4 and 3.2 mg/kg led to retaliatory T/S, which was similar to the response of the T/S on the effect of dox (see tables 2-4). Like the results obtained in subgroup 1, the best response of the T/S in the experiment was observed when using GPX 150 (at a dose of 9.6 mg/kg) in all three categories of survival times (i.e., 215%, 155% and 137% at 20%, on average and 80% survival time, respectively).

8. Discussion and conclusions

GPX-100 and GPX-150 at the doses used in this study demonstrate antitumor activity, which is equal to or greater than the activity exhibited by doxorubicin. At the doses used in this study, GPX-150 gives a higher antitumor effect compared with the effect when the effect of doxorubicin or GPX-100. Although in these experiments GPX-150 was more effective than doxorubicin or GPX-100, it is less strong than GPX-100 or doxorubicin. Thus, GPX-100 and GPX-150 have antitumor activity studies sabreena. In addition, disclosed in the description illustrates and describes only the preferred embodiments of the invention, but, as noted above, it should be clear that the invention can be used in various other combinations, modifications within the scope of claims of the invention as they are presented here in accordance with the above recommendations and/or experience, or knowledge in the art. Embodiments of described herein above, assume, in addition, an explanation of the best known forms of practice of the invention and enable others skilled in this area to use the invention in such, or other embodiments and with various modifications required by the particular applications or using the invention. Thus, the description is not intended to limit the invention in the form described here. In addition, it assumes that the accompanying claims can be made with the inclusion of alternative options.

Claims

1. Derivative 5b>3are H, HE or och3group;

R4represents the following groups:

2. Pharmaceutical composition having antitumor activity, containing at least one derivative of 5-imino-13-deoxy of anthracycline under item 1.

3. A method of treating cancer, autoimmune disease or immunodeficiency disorders, characterized in that it includes the introduction of an effective amount of at least one derivative of 5-imino-13-deoxy of anthracycline under item 1.

4. The method of obtaining derivatives of 5-imino-13-deoxy of anthracycline, characterized in that exercise recovery anthracyclin 13-dailydata lamborghini.com sodium in anhydrous methanol at pH around 7.0 or below in the presence of p-toluenesulfonic acid through careful boiling solution under reflux in a nitrogen atmosphere at a temperature of about 75°C, then the reaction mixture is cooled, treated with water and chloroform, are added to a solution of anhydrous sodium sulphate, the precipitate is filtered off and the filtrate is separated on a column of silica gel, washed with a gradient of chloroform/methanol eluate using preparative HPLC, then to the solution of purified 13-deoxy of anthracycline in a mixture of methylene chloride and water, add the bicarbonate and potassium di-tert-butyl-dicarbonate, allocate obtained 3’-N-BOC-deoxy anthracycline, dissolving it in anhydrous methanol, the resulting solution is cooled and spend amination of 3’-N-BOC-13-deoxy of anthracycline through a saturated solution of ammonia at a temperature of about 0-4°C, then remove the solvent and ammonia, dissolve the residue in anhydrous methanol, remove 3’-N-BOC group in an acidic environment and using preparative HPLC produce the target product, which is optionally converted into the hydrochloride of 5-imino-13-deoxy of anthracycline.

5. The method according to p. 4, characterized in that the boiling under reflux is carried out at a temperature of from about 68°C to about 72°C.

6. The method according to p. 4, characterized in that the boiling under reflux is carried out in the absence of oxygen.

7. The method according to p. 4, characterized in that the boiling under reflux is carried out in the absence of water.

8. The method according to p. 4, characterized in that the boiling under reflux is carried out in nitrogen atmosphere.

9. The method according to p. 4, characterized in that the specified boiling with Obie carried out in the absence of water.

11. The method according to p. 4, characterized in that these processes lead to the exit of the derived 5-imino-13-deoxy of anthracycline about 40%.

12. The method of obtaining derivatives of 5-imino-13-deoxy of anthracycline, characterized in that the prepared acidic solution anthracyclin 13-dailydata with lamborghini.com, gently boil the solution under reflux, the reaction mixture is cooled, add water and a halogenated hydrocarbon solvent, the reaction mixture is dried using anhydrous sodium sulfate, filtered, the resulting filtrate is passed through the column for preparative chromatography emitting derived 13-deoxy of anthracycline, then the fraction, purified by HPLC, is passed through the column HPLC with reversed phase to remove inorganic salts, then prepare an alkaline solution of 13-deoxy of anthracycline, which carry out the reaction with di-tert-BUTYLCARBAMATE, with the resulting BOC-protected derivative, the solution is treated with ammonia at a temperature of about 0-4°C, evaporated the solvent and ammonia, process the remainder of the acidic solution and the resulting solution is evaporated and passed through the column for preparative HPLC emitting 5-imino-13-desoxy to remove inorganic salts and produce the target product.

13. The method of obtaining derivatives of 5-imino-13-deoxy of anthracycline, characterized in that dissolve about 1 g of doxorubicin 13-tazelgettaze hydrochloride and 2.4 g of p-toluenesulfonic acid in 50 ml of anhydrous methanol, add about 0.8 g laborgerate sodium to the solution, the resulting solution was heated to a temperature of from about 68°C to about 72°C, gently boil the solution under reflux for about 1 h in nitrogen atmosphere, concentrate the reaction mixture to about 20 ml, cool the reaction mixture in the freezer to a temperature of from about 0°C to about 4°C, add about 0.5 ml of water to the reaction mixture, add about 200 ml of chloroform to the reaction mixture, add anhydrous sodium sulfate to the reaction mixture, is filtered salt, pass the solution through a column of silica gel, then washed the column with a mixture of chloroform/methanol to obtain colorless eluate, elute with methanol fraction containing the product is evaporated methanol eluate, dissolve the residue obtained after evaporation, 30%Mr. buffer solution of acetonitrile in ammonium formate, produce product using preparative HPLC using a phenyl column, separate the product from other impurities from the lower solution by preparative phenyl HPLC column, wash the column with water, elute the product with methanol, concentrating the solution to about 5 ml, precipitated product by adding about 10 ml of diethyl ether, filtered the precipitate to obtain 600 mg of solid 13-deoxy anthracyclines, which is dissolved in about 60 ml of methylene chloride and 10 ml of water, add about 180 mg of potassium bicarbonate and about 180 mg of di-tert-BUTYLCARBAMATE and conduct the reaction in the solution at a temperature of about room for about 2 hours, and then separating the organic solution is washed with water, dried solution with anhydrous sodium sulfate, the solvent is evaporated, dissolve the residue in about 100 ml of anhydrous methanol, maintain the solution in an atmosphere saturated with ammonia at a temperature of about 0-4°C for 48 h, evaporated solution, dissolve the residue in 60 ml of anhydrous methanol containing about 0.1 M hydrogen chloride, conduct the reaction in the solution at a temperature of about room temperature for about 2 h, concentrate the solution to about 5 ml, produce product using preparative HPLC using a phenyl column, separate the product from other impurities using a gradient of acetonitrile/ammonium formate, diluted with water fraction, adduct methanol, concentrate the solution to about 5 ml, turn the product cleaners containing hydrochloride salt precipitated product 10 ml diethyl ether and filtered precipitate with getting about 360 mg of solid 5-imino-13-diethoxyanthracene hydrochloride.



 

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