A method of treating a mammal in need of cancer treatment and the method of obtaining derivatives of 13 - diethoxyanthracene

 

The invention relates to medicine, in particular to Oncology, and for the treatment of cancer in a mammal. I propose to use certain derivative anthracycline, in particular, containing 13-ketogroup, for example 13-dezoksiguanosin. The introduction of such substances is carried out without restrictions total cumulative dose. This technique provides the possibility of treating cancer without side effects, particularly cardiotoxic. Suggested methods of obtaining these substances. 2 N. and 14 C.p. f-crystals, 14 tab., 22 Il.

The technical field to which the invention relates

The invention relates to derivatives of 13-diethoxyanthracene as derivatives, which do not show cardiotoxic side effects, and methods of obtaining data derived 13-diethoxyanthracene.

The level of technology

The anthracyclines have the widest spectrum of activity against cancers of the person in comparison with the rest of cancer chemotherapy. The most well-known anthracycline anticancer drug is doxorubicin and daunorubicin, which contain 13-ketogroup. Among these drugs doxorubicin, on the effective medicinal substances in sarcomas and carcinomas, in addition to leukemia, lymphomas and solid tumors.

Close structural analogue, daunorubicin, described in U.S. patent No. 3616242, is not effective in sarcomas and carcinomas, and this difference is apparently due to the absence of group 14-HE's in the molecule of daunorubicin. However, daunorubicin used in the treatment of acute leukemias.

Nevertheless, the cumulative cardiotoxicity limits the use of these drugs. In this regard, cardiotoxicity usually limits the duration of treatment with doxorubicin to approximately 9 months at obecniho. The total cumulative dose of doxorubicin or daunorubicin usually may not exceed 550 mg/m2(see E. A. Lefrak et al., Cancer, 32:302, 1973). Even at the recommended maximum total cumulative dose or near it (430-650 mg/m2) in 60% of patients there is a significant and permanent cardiac dysfunction and in 14% of developing congestive heart failure (see A. Dresdale et al., Cancer, 52:51, 1983). Thus, when using these drugs to inhibit the growth of cancerous tumors, the patient may die from congestive heart failure due to the strong cardiotoxic side effect l is tracycline compounds have a relatively low outputs of the order of approximately 30% (see article Smith et al., J. Med. Chem., 21:280-283, 1978).

Successful action of doxorubicin in the elimination of tumors and limit its clinical application was the basis for researchers around the world in attempts to create improved doxorubicin. In this regard, there was long a tangible need in the analog of doxorubicin, which would have no restrictions on cumulative irreversible cardiotoxicity. Despite the fact that in the past more than 25 years was synthesized more than 2000 counterparts, none of them did not provide significant advantages relative to doxorubicin (see R. D. Weiss. The anthracyclines. Will there ever was found superior doxorubicin? The anthracyclines: Will we ever find a better doxorubicin?), Seminars in Oncology, 19:670-686, 1992).

In the last more than 25 years there have been extensive studies on the elucidation of the mechanism of cardiotoxicity anthracyclines. A popular theory, which was developed, is the free-radical theory. According to this theory cardiotoxicity anthracyclines is the result of the formation of free radicals quinone molecule structure of anthracycline (see J. Dorowshow et al., J. Clin. Invest., 68:1053, 1981; D. V. Unverferth et al., Cancer Treat. Rev., 9:149, 1982; J. Goodman et al., Biochem. to prevent cumulative cardiotoxicity using traps free radicals and antioxidants (see the work of D. Garreg and E. Maser, Recovery of carbonyl daunorubicin in the liver and heart rabbit (Carbinyl reduction of daunorubicin in rabbit liver and heart), Pharmacology and Toxicology, 80:240-245, 1997; J. F. VanVleet 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 et al., J. Pharm. Parmacol., 38:277, 1986 and E. A. Porta et al., Res. Comm. Chem. Pathol. Pharmacol., 41:125, 1983).

In other words, it was found that the inhibition of free radical formation does not eliminate the present data anthracyclines (see P. S. Mushlin et al., Fed. Proc., 45:809, 1986). Dr. Richard D. Olson and Dr. Phillip S. Mushlin the last 15 years devoted to the study of the mechanism of anthracyclineinduced cardiotoxicity and has developed "theory of metabolites, which, as expected, will now become the prevailing theory (see R. D. Olson, and P. S. Mushlin, cardiotoxicity of doxorubicin. Analysis of the prevailing hypotheses (Doxorubicin cardiotoxicity: an Analysis of prevailing hypotheses), FASEB Journal, 4:3076-3086, 1990). According to this theory cardiotoxicity anthracyclines mediated 13-HE-metabolite of the parent compound.

This study shows that the cardiotoxicity of doxorubicin and daunorubicin, which is manifested as a decrease in the contractility of the myocardium depends on metabolic recovery 13-metastructure to 13-digit the care, cardiotoxic effects are observed only at very high concentrations (200-400 µg/ml) (see P. S. Mushlin et al., Fed. Proc., 44:1274, 1985; R. D. Olson et al. Fed. Proc., 45:809, 1986).

If doxorubicin allow you to stay in test systems even for short periods of time, there is some metabolic transformation and produces 13-dihydroimidazole in sufficient quantity in order to begin to develop cardiotoxicity (see article L. Rossini et al., Arch. Toxicol. suppl., 9:474, 1986; M. Del Tocca et al., Pharmacol. Res. Commun., 17:1073, 1985). Thus, the accumulated evidence that the cardiotoxicity of these drugs as doxorubicin and daunorubicin, is the result of strong cardiotoxic effects, provided their 13-dihydroimidazolium (see article 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 contrast to the above 13-dihydroimidazole doxorubicinol and daunorubicinol cause cardiotoxicity in the same test systems at relatively low concentrations (1-2 mg/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).

In light of the above, the torus 13-ketogroup restored to alcohol, as shown below:

This theory offers an explanation for the delayed nature of cardiotoxicity anthracyclines. Recently, a review was published studies Olson and Mushlin and shows the number of points of view (D. Garreg and E. Maser, Recovery of carbonyl daunorubicin in the liver and heart rabbit (Carbonyl reduction of daunorubicin in rabbit liver and heart, Pharmacology and Toxicology, 80:240-245, 1997).

For example, studies confirm the existence of a direct link between intracardiac accumulation of 13-alcohol metabolite and violation of both contractility and relaxation of the heart muscle. In addition, if regular long introduction doxorubicin his 13-alcohol metabolite, doxorubicinol, selectively accumulates in cardiac tissue of a rat or rabbit. Studies additionally demonstrate that the cardiotoxic effect daunorubicinol in vitro significantly exceeds the cardiotoxic effect of the parent drug.

Moreover, studies show that doxorubicinol was 30 times more active than doxorubicin in inhibiting cardiac contractility in the papillary muscles of the rabbit. Moreover, studies have demonstrated that the mechanism of cardiac dysfunction is associated with inhibition of ATPase, since the definition of reticulum, MD2+-ATPase of mitochondria and Na+-K+-ATPase activity sarcolemma. In addition, daunorubicinol, but not daunorubicin was detected in cardiac tissue two days after injection of daunorubicin in animal studies.

Recently, the mechanism underlying alcohol induced metabolites anthracyclines cardiotoxicity was explained in the work Minotti et al. Secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/ultrahosting protein-1 in cytosolic fractions infarction person (The secondary alcohol metabolite of doxorubicin-1 irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium), FASEB Journal, 12, in press, 1998. Minotti et al. demonstrated that doxorubicinol, but not doxorubicin violates the metabolism of iron and irreversibly inactivates ultrahosting protein-1 (IRP-1). As a result, the iron becomes unavailable for enzymes that require iron. Inactivation of these enzymes leads to cardiotoxicity.

With these discoveries agrees that the use of chelator of dexrazoxane to reduce the cardiotoxicity of doxorubicin (see G. Weiss et al., Modulation of the expression of the transferrin receptor with dexrazoxane (ICRF-187) through asset is:1419-1424, 1997). Dexrazoxane, apparently, stimulates activity of IRP-1, which counteracts the effect doksorubitsinola.

Olson and Mushlin formulated the idea, namely, that similar 13-CountryLine, which cannot form an alcohol metabolite, should not be cardiotoksicnae. The most attractive option was the restoration of 13-ketogroup to a methylene group. There are no known enzymes that will metabolize this group to alcohol. The results obtained with anthracyclines aclarubicin, consistent with this idea.

Aclarubicin has many variations compared with doxorubicin, including the lack of 14-Oh group. This drug was not effective against sarcoma or carcinoma, which is consistent with the absence of 14-Oh group. However, it was effective against acute leukaemia. Aclarubicin also does not contain a 13-metastructure, but it includes 13-methylene group.

This drug is commercially used in France and Japan. Aclarubicin, apparently, there is no irreversible cumulative cardiotoxicity. Patients received up to 3,000 mg/m2in the absence of manifestations of the m myeloblastic leukemia (Phase II study of aclarubicin in acute myeloblastic leukemia, American Journal of Clinical Oncology, 10:523-526, 1987). Specialists in this area did not have a correct view about the lack of cardiac toxicity aclarubicin and incorrectly assumed that this effect is due to its distribution and pharmacokinetics. However, Olson and Mushlin thought it was a confirmation of the importance of missing 13-metastructure for cardiotoxicity.

The figure 1 presents a diagram that illustrates the path leading to cardiotoxicity.

The invention

The purpose of this invention to provide evidence that derivatives of 13-diethoxyanthracene not show cardiotoxicity.

Another object of this invention is the development of improved methods for obtaining such derivatives 13-diethoxyanthracene.

The next object of this invention is to provide precursors of certain derivatives of 13-diethoxyanthracene and methods of obtaining predecessors.

In accordance with these and other objectives and advantages, objects of this invention are derivatives of 13-diethoxyanthracene having the formula I:

where R1- H or HE; R2- N., HE, or OMe; R3- H or HE; R4Mr. acceptable salts of compounds of formula I. Pharmaceutically acceptable salts include salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric (salt), Hydrobromic, sulphuric, nitric, perchloro, fumaric, malic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methansulfonate, formic, benzoic, malonic, naphthalene-2-sulfonic, triperoxonane and benzosulfimide acid. Salts derived from appropriate bases include alkali, such as sodium and ammonium.

The object of the present invention also presents methods of treatment of a mammal host, which requires anti-cancer treatment. Effective anti-cancer amount of at least one of the compounds of formula I, below, is administered to the host in an effective amount.

where R1- H or HE; R2- N., HE, or OMe; R3- H or HE; R4- H or HE and R5-carbohydrate or substituted carbohydrate.

Other objects of this invention are a method of obtaining derivatives of 13-diethoxyanthracene. The method includes the formation of acidic rastakraut in the flask with reflux condenser. The reaction mixture is cooled. To the solution was added saturated aqueous Panso3and the solvent is then presented halogen-substituted hydrocarbon. The mixture is filtered. The filtrate is acidified. The filtrate is subjected to preparative chromatography for separation of derivatives 13-diethoxyanthracene.

In addition, the purpose of this invention is to overcome the above disadvantages of the known processes for obtaining derivatives of 13-diethoxyanthracene.

Accordingly another object of this invention is the development of improved methods of obtaining derivatives of 13-diethoxyanthracene, which provide an increased output compared with the known processes.

According to this further aspect of the present invention are a method of obtaining derivatives of 13-diethoxyanthracene.

In General, the anthracyclines have the formula I,

in which R1, R2, R3, R4and R5such as described above. The anthracyclines are easily converted into 13-tazelgettaze in accordance with known methods. 13-tazelgettaze anthracyclines to restore derivatives 13-diethoxyanthracene lamborghini.com sodium in acidic conditions. is from about 70% to about 80%.

Additional objects of this invention are a method of obtaining derivatives of 13-diethoxyanthracene. The method includes the formation of an acidic solution of 13-tailgatenation with lamborghini.com. The solution is gently heated in a flask with reflux condenser. The reaction mixture is cooled. To the solution was added a saturated aqueous solution Panso3and the solvent is then presented halogen-substituted hydrocarbon. The mixture is filtered. The filtrate is acidified, the Filtrate is subjected to preparative chromatography for separation of derivatives 13-diethoxyanthracene.

The following objects of this invention are a method of obtaining derivatives of 13-diethoxyanthracene. The method includes obtaining a solution by dissolving about 1 g of the hydrochloride 13-tailgatenation and approximately 2.4 g of p-toluenesulfonic acid in approximately 50 ml of anhydrous methanol. To the solution add approximately 0.8 g of laborgerate sodium. The solution is heated to a temperature of from about 68With up to approximately 72C. the Solution is then gently boiled in a flask with reflux condenser for about one hour under a nitrogen atmosphere. Reature from approximately 0With up to approximately 4C. Approximately 2 ml of a saturated aqueous solution of sodium bicarbonate added to the reaction mixture. The reaction mixture was added about 200 ml of chloroform. The reaction mixture was added anhydrous sodium sulfate. Salt is filtered off. The filtrate is acidified with hydrogen chloride in diethyl ether. The solution passed through a column of silica gel. Then the column was washed with chloroform/methanol up until the eluate becomes colorless. The fraction containing the product, elute with methanol. The methanol eluate is evaporated. The residue obtained by evaporation, dissolved in 30% acetonitrile in ammonium formatnum buffer. The product distinguish preparative HPLC (high performance liquid chromatography), using a column containing phenyl groups. The product is separated from other impurities, using a gradient of acetonitrile/ammonium formate. Fraction, purified by HPLC, then lyophilizer, receiving approximately 600 mg 13-deoxydoxorubicinol hydrochloride.

List of drawings and other materials

In Fig.1 presents a diagram illustrating the biosynthesis pathway leading to cardiotoxicity.

In Fig.2 shows a graph representing the end of the program, suitable for the invention, or doxorubicin, depending on the time.

In Fig.3 shows a graph representing the absorption of [3H]-thymidine by the cells of HL-60 in the presence of option connection corresponding to this invention, or doxorubicin and showing the inhibition of cell growth.

In Fig.4 shows a graph representing the absorption of [3H]-thymidine cells R in the presence of option connection corresponding to this invention, or doxorubicin and showing the inhibition of cell growth.

In Fig.5 shows a graph representing the absorption of [3H]-thymidine MCF7 cells in the presence of option connection corresponding to this invention, or doxorubicin and showing the inhibition of cell growth.

In Fig.6 shows a graph representing the absorption of [3H]-thymidine cells MDA-MB-231 in the presence of option connection corresponding to this invention, or doxorubicin and showing the inhibition of cell growth.

In Fig.7 is a graph illustrating the effect of Connection suitable for the invention, and daunorubicin on the contractile function.

In Figures 8.1, 8.2 and 8.3 are presented respectively microphotograph who were treated within 20-23 weeks option connection corresponding to this invention, rabbits were treated with doxorubicin, or control sample and showing vacuolation of myocytes and loss of myofibrils in the sample using doxorubicin, not noted when using the Connection corresponding to this invention, or in the control samples.

In Figures 9-13 shows the graphs representing the percentage survival of mice with leukemia, with the introduction of compounds of the invention, in various doses.

In Figures 14-20 shows the graphs and charts that reflect the status of the rabbits in the study on these models, the cardiotoxicity of doxorubicin, GPX 150 and GPX-100.

Information confirming the possibility of carrying out the invention

The present invention uses the fact that the 13-Depository doxorubicin, daunorubicin and other similar anthracyclines will not be metabolically transformed into cardiotoxic 13-digitiform, thus presenting the possibility of introducing the compounds of this invention, in nocardioides quantities without limitation the total cumulative dose.

The invention includes an improved doxorubicin, having the formula:

In the following in vitro experiments investigated the biotransformation of improved doxorubicin suitable for the invention. The aim of the study was to establish metabolized whether the Connection And, corresponding to this invention, up From 13 hydroxymetabolites in isolated preparations of the heart muscle of the rabbit. Doxorubicin, doxorubicinol and Connection And tested on the strips of the outer wall of the right atrium and right ventricle, isolated from new Zealand white rabbits using techniques based on fluorescence HPLC. Thin strips of Atria and ventricle were incubated in trays muscles (30(C) containing oxygenated bicarbonate buffer Krebs. Doxorubicin (175 μm) or Compound a (175 μm) was added to the bath and removed from them strips atrial or ventricular intervals of 30 minutes for 210 minutes. Each strip was quickly washed in normal saline solution, dried by soaking, cut in half and weighed the ina, doksorubitsinola and Connections A. Tissue concentrations of doxorubicin, doksorubitsinola and Connections And was determined by the standard curve in three independent experiments and expressed as mean ±SEM (standard error). It was noted gramasevaka increasing concentrations of Compounds a and doxorubicin in the atrium and the ventricle. After 210 minutes of incubation between the concentrations of Compounds a and doxorubicin (ng/mg wet weight) in tissue of the Atria no significant differences (Compound a: 743±89; doxorubicin: 617±35). Concentration of the Compound in the ventricle were significantly higher than the concentration of doxorubicin in the ventricle after 210 minutes of incubation (Connection: 626±31; doxorubicin: 407±30; P<0,05). However, only doxorubicin metabolisable to-13-hydroxymetabolites doksorubitsinola. No metabolism of the Compounds was not determined. These experiments indicate that the Connection And does not form a s-13-hydroxymetabolites in isolated heart preparations.

The aim of the study was to determine metabolized whether the Connection a to C-13-hydroxymetabolites in isolated preparations of the heart muscle of the rabbit.

Tests were conducted using the Compounds And corresponding isologo atrium, the outer wall of the right ventricle, bicarbonate of Krebs buffer (pH 7.4), normal (of 0.9%) saline, (NH4)2SO4, isopropyl alcohol, chloroform, daunorubicin, doxorubicin, doksorubitsinola and methanol.

The test Protocol consisted of incubation of thin strips (each 80-100 mg) of the outer wall of the right ventricle and atrium NZW (new Zealand) rabbit in the bath for isolated muscles (30(C) containing oxygenated bicarbonate Krebs buffer (pH 7.4) of the following composition: 127 mm NaCl, 2.5 mm l2, 2.3 mm KCl, 25 mm Panso3, 1.3 mm KH2PO4, 0.6 mm MgSO4and 5.6 mm glucose, as was previously published (see P. S. Mushlin et al., Br. J. Pharmacol., 110:975-982, 1993).

In accordance with the synthesis doksorubitsinola doxorubicinol synthesized by the method Takanashi and Bachur (see S. Takanashi and N. R. Bachur, Drug Metab. Disp., 4:17-87, 1976) with minor modification (see P. S. Mushlin et al., Br.J.Pharmacol., 110:975-982, 1993).

According to the statistical analysis of the results of experiments concentrations in tissues (ng/mg wet weight) of doxorubicin, doksorubitsinola and Connections And was determined in three different experiments and expressed as mean ±SEM (standard error). Dogfuck the Amma Prizm (GraphPad).

The results of observations and studies include the observation breathalysing of increasing concentrations of Compounds a and doxorubicin in the tissues of the outer wall as the Atria (table 1) and the right ventricle of the rabbit (table 2) (figure 1). Concentration of the Compound And in the Atria and the ventricles were equal to or exceeded the concentration of doxorubicin in the Atria and in the ventricles. However, only doxorubicin metabolisable to-13-hydroxymetabolites doksorubitsinola and gramasevaka cumulation doksorubitsinola was observed in tissues such as the Atria (table 1), and ventricle (table 2) rabbit (figure 2). No metabolism of the Compound And was not observed (figure 2).

Based on the discussion and conclusions from the experiments conclude that the results of the experiments show that the Connection And does not form a s-13-hydroxymetabolites in isolated preparations of the rabbit heart. However, doxorubicin, structurally similar compound, metabolizability to doksorubitsinola, With-13-hydroxymetabolites. In addition, the experimental results indicate that the metabolism of doxorubicin to doksorubitsinola, seems to be higher in the tissue of the auricle, h the points (V), incubated at 175 microns Connection corresponding to this invention, or doxorubicin over time. As can be seen from Fig.2, the connection corresponding to this invention, is present in much lower concentrations compared with conventional doxorubicin.

Other in vitro studies have shown that the Connection And, corresponding to this invention, had the same efficiency as doxorubicin in inhibiting the growth of cancer cells.

In experiments demonstrating the effectiveness of the compounds suitable for the invention, in terms of inhibiting the growth of cancer cells compared the effects of Compounds corresponding to this invention, and doxorubicin on cell proliferation in vitro.

According to the experiments showing the effectiveness of the compounds of the invention, the antiproliferative effect of the Compounds As compared with doxorubicin in cultured cell lines derived from human and murine leukemia cells (HL60 and R) and cancer human breast (MCF7 and MDA-MB 231).

The inhibition of proliferation of cancer cells was studied by measuring the incorporation into cells [3H]-thymidine. Antiprolifera the tion, resulting in 50% increase of the maximum inhibition (IC50), were obtained using the analysis using tracing the empirical curve. Average values IC50(in nm) and 95% confidence intervals are presented below. Average values were determined from 3 or 4 independent assays in triplicate (see table A).

Both Compound a and doxorubicin, completely stopped the inclusion of [3H]-thymidine into cells of each of the four studied cell lines. These studies demonstrate that Compound a and doxorubicin are strong inhibitors of proliferation of cancer cells in vitro, although, as shown by the ratio of the values of the IC50(the ratio of activities), doxorubicin was somewhat more active than Compound a in the inhibition of incorporation of [3H]-thymidine into cells of all four lines (P<0,05).50for the test compounds in each of the four cell lines and to compare this value with the value obtained for doxorubicin.

Dilution of test compounds were made in specific cell environments in the following intervals (see table).

Cultivation was introduced into each well in triplicate in the form of an aliquot of 50 μl and the cells were grown in the presence of test compounds for 24 hours.

Statistical analysis included unpaired t-test, where it was acceptable. Was selected as the significance level of P<0,05.3H]-thymidine. Concentration providing 50% of the maximum responses were obtained through analysis using tracing the empirical curve. The values of the IC50(in nm) are shown below and reflect the average value (95% confidence inte the

The results show that Compound a is less active than doxorubicin in inhibiting cell proliferation in all four cell lines in vitro. However, both compounds are equally effective.

Discussion and conclusions from the results of in vitro studies have shown that as the Compound a, and doxorubicin completely block the inclusion of [3H]-thymidine into cells of each of the four studied cell lines. As shown, the ratio of the values of the IC50(the ratio of activities) doxorubicin was more active than Compound a in the inhibition of incorporation of [3H]-thymidine into cells of all four lines (P<0,05) (see Fig.3-6). These studies demonstrate that Compound a and doxorubicin are strong inhibitors of proliferation of cancer cells in vitro. In Fig.8.1-8.3 shows micrographs that illustrate the effects on the heart muscle connection corresponding to this invention, in comparison with the known compound doxorubicin and control sample.

In vivo studies showed that Compound a was effective in terms of increasing life expectancy on a murine model of leukemia at a lower si in mice are described below.

The male CDF1 mice intraperitoneally was inoculable 106cells of mouse leukemia L on day 0. In the days 1-9 mice intraperitoneally injected doxorubicin or connection A. Daily measured the masses of bodies and recorded survival. In one of these studies, the mice were injected doxorubicin or Compound a at a dose of 0.8 mg/kg/day. On day 22 was 0/8 surviving animals in the group, which has introduced the media, 7/8 in the group which was administered doxorubicin, and 5/8 in the group, which was introduced by the connection A. the Values obtained for doxorubicin and Connections And, significantly different from the values obtained for the media, but not from each other.

In another study on the same model of murine leukemia doxorubicin were injected with the dose of 0.8 mg/kg/day, and the Connection And were injected with the dose of 1.6, 2.4 or 3.2 mg/kg/day (see table 3).

On day 19 the Compound a at a dose of 1.6 mg/kg/day was as effective as doxorubicin in terms of suppression of weight gain, resulting from the growth of leukemia cells in combination with associated ascites. Both doses of Compound A, 2.4 and 3.2 mg/kg/day were more effective than doxorubicin in terms of suppression of weight gain. On day 25, all doses of the Compounds And were n is 2 mg/kg/day was effective in prolonging survival as compared with the carrier and doxorubicin. The dose of doxorubicin used in this study is the maximally effective dose for this model. Higher doses of doxorubicin in fact reduced survival. Thus, although the Connection And is less active than doxorubicin, it is more effective at higher doses than doxorubicin in terms of increasing life expectancy.

It was also shown that Connection, 13-desexualise of daunorubicin, no cardiotoxic properties of daunorubicin in respect of myocardial contractility in vitro model using the rabbit heart, described Mushlin et al., supra. This is illustrated in Fig.7.

It was also shown that Connection, 13-desexualise of daunorubicin, no cardiotoxic properties of daunorubicin on the model in vivo in rats, as described below. The following discussion demonstrates the absence of cardiotoxic effects Connection corresponding to this invention, in rats after intravenous administration.

Of daunorubicin hydrochloride or Compound In hydrochloride in water were injected with an intravenous dose of 5 mg/kg/day for three days with an interval of one day (total dose 15 mg/kg) to male rats Spraque Dawle the first dose rats were narcoticyou by intraperitoneal administration of a 50 mg/kg of phenobarbital sodium. In the trachea entered the tube, and the rat was breathing 100% oxygen. Body temperature was maintained at a level 37With by means of a heating lamp and a temperature sensor. The catheter was placed in the right carotid artery and was introduced into the aorta to record mean arterial pressure (SBP) and heart rate (ES), using a Statham pressure transducer and recording device Gould. Then the catheter is pushed into the left ventricle to record the systolic pressure in the left ventricle (LISD), the maximum value of dP/dt of the left ventricle (dP/dt) and end diastolicheskoe pressure in the left ventricle (CLID).

In rats treated with daunorubicin, MAP, LIST and dP/dt were significantly and substantially suppressed compared to controls with the introduction of media (table 4). In rats treated with daunorubicin, was also significantly reduced body weight. On the contrary, MAP, LIST, dP/dt and body weight were similar in rats treated with the carrier and the Compound In (table 5). Data show that the Connection not present at the dose where daunorubicin causes a significant reduction in contractility and heart activity. The results illustrate the possibility of introducing Connections at terapeuticas the t violation cardiac function.

The values shown in table 4 represent the average values ± standard errors; rats were injected with an intravenous connection at a dose of 5 mg/kg/day for 3 days with an interval of one day; the measurements were carried out on day 7 after the first injection. BW1 = body weight at day 0, BW2 = body weight at day 7. *p<0,05 relative to the carrier.

Rats were injected with an intravenous connection at a dose of 5 mg/kg/day for 3 days with an interval of one day; the measurements were carried out on day 7 after the first injection. BW1 = body weight at day 0, BW2 = body weight at day 7.

Connection And evaluated on the model of cardiotoxicity at regular administration of doxorubicin in rabbits. In this model, doxorubicin caused disturbance of cardiac function and histopathological changes similar to the observed in people with regular long-term treatment with doxorubicin. Histopathological and/or functional abnormalities in heart rabbit was observed in 5/6 rabbits treated with doxorubicin. In the same conditions, the Compound a did not cause relevant clinical cardiotoxicity.

Nocardioides the nature of the compounds suitable for the invention, the PNA model continuous injection in rabbits.

In accordance with this study, twenty-four male new Zealand white rabbits randomly divided into four groups. Six rabbits were injected with 1 mg/kg doxorubicin in the marginal ear vein 2 times a week for 8 weeks. Six additional rabbits were injected with 1 mg/kg Compound And marginal ear vein 2 times a week for 8 weeks. Feed intake of rabbits in groups treated with doxorubicin and treated with Compound a, monitorrole daily and the same amount of feed given appropriate by age and sex rabbits, employees pairwise power control, which were injected with the vehicle (0.9% NaCl) in the marginal ear vein 2 times a week for 8 weeks. Acceleration of aortic root monitorrole weekly method of Doppler ultrasonography (Doppler method) during the study. The decrease in the fraction of exile was determined once in two weeks M-echocardiography, starting from the tenth week of the study and continued throughout the study. Rabbits painlessly cut, starting from 20 weeks after the start of the study or when reducing fractions was less than 25% or remained at the level of 25-29% for at least 3 weeks. Figurative rabbit were prepared for histological analysis and processed by histopathology blind. Lesions classified as weak, medium or strong on the basis of the degree of vacuolization, degeneration of myofibrils, inflammation caused by mononuclear cells, and necrosis. An abnormal decrease in the fraction of expulsion was observed in 4/6, 0/6, 0/6, 1/6 and the rabbits treated with doxorubicin, Connection And control for doxorubicin and control to connect And groups, respectively. Anomalous acceleration of the aortic root (values lower than 9 m/s/s) was observed in 3/6, 0/6, 0/6 and 0/6 of rabbits treated with doxorubicin, Connection And control for doxorubicin and control to connect And groups, respectively. All 6 rabbits in the group treated with doxorubicin, was abnormal histopathology, lying in the range from weak to strong, from 2/6 rabbits in group Connections were weak And histological abnormalities. No histopathological lesions were observed in the cardiac tissue of rabbits from both control groups. Total cardiac status was defined as abnormal when at least 2 of the 3 tests for cardiotoxicity were abnormal. When using these criteria 5/6 rabbits in the group treated with doxorubicin had abnormal total cardiac status, 0/6 had anomalies in the other 3 groups (amoxicot at the level of the tested doses. In addition, the Connection And had no noticeable effect on Hematology and weight gain, whereas doxorubicin significantly changed the Hematology and suppressed weight gain. Under existing schemes dosing Connection And causes lower cardiotoxicity and systemic toxicity in rabbits in comparison with doxorubicin.

The purpose of the evaluation experiments cardiotoxicity was to compare the cardiac toxicity of the Compounds And doxorubicin on the model of the regular introduction of rabbits.

In 67% (4/6) of rabbits treated with doxorubicin, developed abnormal shortening fraction of the left ventricle. Three of the six treated with doxorubicin rabbits (50%) developed abnormal acceleration of the aortic root. In contrast, none of the rabbits treated with Compound A, no functional evidence of cardiotoxicity. The most sensitive indicator of cardiac toxicity was histopathology. All rabbits treated with doxorubicin, appeared histopathological damage, characterized primarily by vacuolization of myocytes and loss of microfibril. In four of six rabbits was shown weak cardiotoxicity, 1 rabbit was medium damage and 1 rabbit were Denia (see figure 8). When the results of all three tests for cardiotoxicity were combined, abnormal cardiac status was observed in 5 out of 6 rabbits in the group treated with doxorubicin, but in 0 of 6 rabbits in the group treated with Compound A (P<0.02; the accuracy criterion Fisher). Total cardiac status was defined as abnormal if at least 2 of the 3 tests for cardiotoxicity were abnormal. During the eighth week of the study, collected the blood samples from the marginal ear artery to obtain the formula of blood. Doxorubicin treatment caused a significant decrease in white cells, red cells, platelets, hemoglobin, mean concentrations of erythrocyte hemoglobin and amplitude distribution of red cells compared to animals treated with media or Compound a, P>0,05. Connection And did not modify the data variables compared to media with the exception of a slight increase in the amplitude distribution of red cells. In addition, treatment with doxorubicin inhibited weight gain compared with treatment with Compound A. the Rabbits treated with the compound And had a body mass 3,17±0.06 kg in the beginning of the study and 4.10±0,10 kg at the end of the study, whereas rabbits treated with docks the analyses anova, New multilateral range criterion Duncan (Dunkan''s New Multiple Range test)).

The anomalous acceleration of the aortic root is defined as values below 9.0. The units of acceleration are m/s/s. N = normal cardiac function; A = abnormal heart function.

The rabbits were intravenously injected with 1 mg/kg doxorubicin (DOC) or Compounds (DOC), 2 times per week for 8 weeks (total cumulative dose of 16 mg/kg). Suitable age animals, employees pairwise power control group, which was administered doxorubicin, and group, which entered the Compound And were injected with only the media.

The group, which was administered doxorubicin, was significantly different from the groups that were administered Compound A, SH, or C (P<0,05; 2X2 and two-factor analysis of a correlation characteristics using Chi-squares).

Rabbits were injected with intravenous 1 mg/kg doxorubicin (DOC) or Compound A (DPXA) 2 times a week for 8 weeks (total cumulative dose of 16 mg/kg). Suitable age animals, employees pairwise power control group, which was administered doxorubicin (C), and groups, which were administered Compound A (CX), were injected with only the media.

N = normal cardiac function or histopathology; A= abnormal CE is, two of the three tests cardiotoxicity were abnormal. Abnormal shortening fraction was defined as the tendency or reasonable values are around twenty-five percentile or below. Anomalous acceleration of the aorta was determined values below 9,0 mists. Abnormal histopathology was defined as the presence of vacuoles, damage microfibril and inflammation caused by mononuclear cells (see discussion of the methods above).

The histopathology was considered normal, weak, medium or strong, as described above. In the group treated with doxorubicin was significantly more animals with abnormal total cardiac status, than in the groups treated with Compound a, C and CX (P<0,02; two-factor criterion accuracy Fischer) (see table 6).

Among the conclusions based on the study of cardiotoxicity were the conclusions that the Connection is not altered cardiac function in these conditions and showed only mild histopathological effects in 2/6 rabbits. On the other hand, doxorubicin altered cardiac function in 5/6 rabbits and all rabbits were observed abnormal histopathology in this model of cardiac toxicity after repeated administration in rabbits. Compared to doxorubicin is out And has no noticeable effect on Hematology and weight gain. In the existing scheme dosing Connection And causes lower cardiotoxicity in rabbits in comparison with doxorubicin.

In tests on subacute toxicity in mice has also been shown that the Connection And causes lower toxicity in the bone marrow than doxorubicin, as shown below (see table 7).

Drugs were administered intravenously on days 1, 5 and 9. The measurements were carried out at day 15. Values represent mean values ±SE. +=contrast media, p<0,05, #=contrast Compounds And p>0,05. Both doses are maximal sublethal doses.

The results of the above studies clearly show that Compound a is nocardioides form of doxorubicin. Because the Connection And saves a group of 14 IT is possible that the Connection And will find application in the treatment of sarcomas and carcinomas, in addition to leukemia. It is not expected of the existence of any disagreecause cardiotoxicity, because the Connection And does not form toxic 13-alcohol metabolite. Therefore, the Connection And unlike doxorubicin can be entered so long as is necessary to obtain remission and/or to t a major breakthrough in anthracycline chemotherapy of cancer.

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). These groups are regularly applied, respectively, doxorubicin, GPX 150, GPX-100 and a simple solvent-filler (0.9% NaCl) as 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 figure 14). 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 transmits is equal 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.

Scheme dosing

Optimal antitumor dose GPX-100 approximately twice the dose of doxorubicin, while the 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). Figure 17 shows that the control level RBC (upper graph - Weekly RBC levels) remained stable throughout yjetsa in all groups compared with control (figure 17). 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, growing at 7 weeks and returned to values corresponding to the control, in connection with the termination of treatment with doxorubicin at the seventh week. The impact of GPX-100 on the levels of RBC and hematocrit (STC) continues to decline throughout the study (see figure 18). 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 figure 16, 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 numbers of neutrophils, chemi used in these experiments doses CPX-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 takes an intermediate value between the same variable in doxorubicinol and control groups.

Dose GPX-100 and doxorubicin lead to less weight gain compared with the control rabbits. The effect of doxorubicin, resulting in slower weight gain compared with control, not Oblasty differences in feed consumption, 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, treats, the 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 in two, swelling (of the hind limbs and the chest wall). None of the rabbits treated GPX-100 or GPX-150, was not observed depletion of muscle, 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 figure 15) shows the program 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, none of the rabbits treated GPX-100 or GPX 150, at any time during the experiment did not have any 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 GPX-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 seconds PRP). Enhanced airway should be the result of an increase in reserves of calcium in sarcoplasmatic reticulum (SR), which is formed during intervals in the comfort on the breaks palpitations. As expected, doxorubicin significantly impairs contractility (dF/dt) palpitations rest period (see figure 20), but airway (dF/dt) of the Atria obtained from rabbits treated GPX-150, not deteriorated as compared with the control values. Airway (dF/dt) palpitations rest period of the Atria obtained from rabbits 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) compared 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 the power frequency, but the 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.

1) Summary of the experiment. Antitumor activity of GRX-100, GPX 150 and doxorubicin in vivo investigated 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 8-10). Dox shows the expected p is t, the higher the value of T/S from observed during the experiment (200% at 20% survival time, table.8).

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 subgroup 1. As a standard for comparison, choose the dose of dox 0.8 mg/kg

Dose GPX-100 2.4 and 3.2 mg/kg lead to retaliatory T/S, which is similar to the response of the T/S on the effect of dox (see tables 8-10). 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 will demonstrirujut 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 possess antitumor aktivnog scientific 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 exhibit antitumor activity equal to the activity of doxorubicin on the model R murine leukemia CD2F1 mice, injected 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 (4C). Doxorubicin (manufactured by GEM) was weighed for each dose daily and prepared in saline solution. The medicinal substance was stored in the refrigerator (4C).

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 animal

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

6) Test Protocol

On day 0 mice CD2F1 do intraperitoneal injection (ip) of 1.6 million freshly harvested tumor cells R. My 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 administered ip on days 1-9, n=10);

6) GPX-100 group (6.4 mg/kg administered ip on days 1-9, n=10);

7) GPX-150 group (0.8 mg/kg administered ip on days 1-9, 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 is ltati

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 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 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 8-10).

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 10)). However, the values of T/s for dox dose of 1.6 mg/kg was V 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 8-10). 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 8-10).

Dose GPX 150 6.4 mg/kg resulted in the largest values of T/C in all groups, subgroups 1 at 20% survival time (T/S=200%, table 8). However, the values of T/S from the action of 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% vs. 153%, respectively, according to tables 9 and 10).

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 8-10). 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) Discuss the th 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 was investigated on the model R murine leukemia.

The results show that the derivative anthracyclines like Connection And would be clinically more effective than their not-13-diethoxyanthracene counterparts because they can be entered in a higher effective doses and for longer periods of time, as they cause more low systemic toxicity and do not have disagreecause cumulative present. Derivatives of 13-diethoxyanthracene used in accordance with this invention in the treatment of patients suffering from cancers that are treated by doxorubicin and daunorubicin, are the possibility of introducing them in doses of at least 1.5 times the effective or equal activity cumulative rozpolozenie derivatives 13-diethoxyanthracene. Table 11 presents examples of derivatives of 13-diethoxyanthracene, which can be synthesized according to this invention. As discussed above, it is known that compounds such as those presented in table 11, have anticancer properties.

In contrast to the known processes, methods, corresponding to the present invention, are less temperaturesalinity. For example, the processes can be conducted at a temperature of from about 0With up to approximately 75C.

Preferably, when the process is conducted at a temperature of approximately 65With up to approximately 75C. More preferably, when the process is conducted at a temperature of from about 68With up to approximately 72C. Temperatures above approximately 72With usually lead to decomposition of the reactants and products.

The method that corresponds to this invention, includes a number of basic conditions. For example, the process is preferably carried out under acidic conditions. In other words, the pH value must be 6.5 or less. It was found that the known methods obtained the reactants and products. Reaction or any part thereof, for example only heated in a flask with reflux condenser, may be conducted at temperatures up to approximately 75In the absence of oxygen, in the absence of water and/or under nitrogen.

In addition, as the oxygen and water should be excluded from the reaction. Preferably, when the reaction is performed in a nitrogen atmosphere or an inert gas, using anhydrous solvents.

Ways, corresponding to the present invention, provide a much higher yield than the known methods of producing compounds. For example, it was shown that the known processes have access to approximately 30%. On the other hand, methods, corresponding to this invention, has been found to have the output from approximately 70% to approximately 80%.

According to the above, in the present invention are methods of making connections to the basic formula I, above.

The following formulas are examples of the transformation of the molecules in the reaction.

where R1, R2, R3, R4and R5defined as above.

Kedoya 13-deoxydoxorubicinol, which is a 13-diethoxyanthracene derived.

The following are examples of derivatives anthracyclines, the synthesis of which is described in this context.

In the compounds R5may be a modified version of analogues of various anthracyclines. In addition, the cycle D can be fluorinated.

Basically ways, corresponding to the present invention include the obtaining of a solution of 13-diethoxyanthracene with regenerating agent. The solution is gently heated in a flask with reflux condenser. Then the reaction mixture is cooled. According to one example, the reaction mixture is cooled to a temperature of from about 0With up to approximately 4C. Then the reaction mixture are added the base. The base can be cold. For example, the base has a temperature of from about 0With up to approximately 4C. One example of a reason is a saturated aqueous solution Panso3. In the reaction mixture may be added to the solvent, presents halogenate the ü simultaneously with the Foundation. Halogen-substituted hydrocarbon solvent can be cold. For example, a halogen-substituted hydrocarbon solvent may have a temperature from about 0With up to approximately 4C. Example halogen-substituted hydrocarbon solvent, which can be used is l3. Then the reaction mixture is filtered. Filtering takes place at low temperature. For example, the filtering may take place at a temperature of from about 4With up to approximately 15C.

The above addition of the base and halogensubstituted hydrocarbon solvent preferably initiates the deposition as a result of hydrolysis. Sediment presents the precipitate of inorganic salts, which can be filtered from the reaction mixture. After filtration, the filtrate may be acidified. The filtrate may be subjected to a column chromatography on silica gel. Hydrophobic impurities can be distinguished by elution of less polar solvents. Then 13-diethoxyanthracene products can be suirvey and the eluate may be further purified.

Preferably, when the methods, with the methanol with the addition of p-toluenesulfonic acid and laborgerate sodium. The solution is gently heated in a flask with reflux condenser under nitrogen and then cooled. Add saturated aqueous sodium bicarbonate solution and chloroform. Salt precipitated and was filtered and the filtrate is acidified hydrogen chloride in diethyl ether, and then allocate on a column of silica gel. Hydrophobic impurities resulting from the decomposition, elute mixed solution of chloroform and methanol. Products 13-diethoxyanthracene, elute with methanol. The methanol eluate is additionally purified preparative HPLC.

According to any of the above methods before or after allocation 13-diethoxyanthracene 13-diethoxyanthracene can be processed by one or more reducing and/or other agents that can restore 13-metastructure to a methylene group.

Next provide an example of a method of the invention.

Example

13-deoxydoxorubicinol hydrochloride

1 g of the hydrochloride 13-tailgatenation and 2.4 g of p-toluenesulfonic acid are dissolved in 50 ml of anhydrous methanol. To this solution was added 0.8 g lanbridge sodium. The resulting solution was heated to 68-72With and continue to gently heat in Kolb is tons to approximately 20 ml and cooled in the freezer to 0-4C. Add 2 ml of a saturated aqueous solution of sodium sulfate, and then 200 ml of chloroform. Add anhydrous sodium sulfate and filtered salt after shaking. The filtrate is acidified with hydrogen chloride in diethyl ether.

Then the solution is passed through a column of silica gel (2,55 cm). Then the column is washed with a mixture of chloroform/methanol (10/1) up until the eluate becomes colorless. The bound fraction containing the product, elute with methanol. The methanol eluate is evaporated and the residue is dissolved in 30% acetonitrile in ammonium formatnum buffer (pH 4.0, 0.5%) and allocate preparative HPLC. Use column containing phenyl groups, and separating the product from other impurities is achieved by using a gradient of acetonitrile/ammonium formate (from 27% to 30% acetonitrile over 30 minutes). Fraction, purified by HPLC, lyophilizers, receiving 13-deoxydoxorubicinol hydroformed in solid form, which is then dissolved in methanol containing hydrogen chloride. The solvent is evaporated, the product is precipitated with methanol in ethyl ether and receive 600 mg of 13-deoxydoxorubicinol hydrochloride. The yield is 80%.

UV spectrum:p>

1H-NMR (methanol d4): (see below)

of 1.30 (d, 3 H, 6’-H3),

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 (m, 3H, O-CH3),

of 4.25 (m, 1H, 4’-H),

of 4.95 (m, 1H, 3’-H),

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

to 7.50 (dd, 1H, 3-H) and

7,80 (m, 2H, 1 - and-2-N).

The invention also includes methods of treating a mammalian host in need of cancer treatment. The methods include the introduction of the hosts of effective anti-cancer amount of at least one of the compounds of formula I

Effective anti-cancer amount of compounds suitable for the invention may be introduced depending on the type of mammal body weight, age, individual condition, and also forms the introduction. Compounds corresponding to the present invention, can be introduced by conventional means, suitable for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be entered in the form of monotherapy, but mostly they are administered with a pharmaceutical carrier selected on the basis of hispanohablante from known factors, such as the pharmacodynamic characteristics of the particular agent and its appearance and method of administration, the age, condition and body weight of the recipient, the nature and extent of symptoms, kind of joint treatment, frequency of injections and the desired effect. It can be expected that the daily dosage of the active ingredient will be 0.001 to 1000 milligrams (mg)/kilogram (kg) of body weight with the preferred dose comprising from 0.1 mg/kg to about 30 mg/kg

Dosage forms (compositions suitable for use contain from about 1 mg to about 100 mg of active ingredient/unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5 to 95 wt.% by weight of the total composition.

The active ingredient can be administered orally in solid dosage forms such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms. The active ingredient may also be administered intranasal (nasal drops) or by inhalation. Other dosage forms are potentially possible, such as extremely durable, gradient and media in powder form, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., Similar fillers can be used for preparation of pressed pellets. As tablets and capsules can be in the form of products with the delayed output to ensure a continuous release of drug over a period of time, measured in hours. Molded tablets may be coated with sugar or shell from film to mask the unpleasant taste and protect the tablet from the weather, or have intersolubility membrane for selective destruction in the gastrointestinal tract.

Liquid dosage forms for oral administration may contain tint and flavor and fragrances to improve the attitude and cooperation of the patient.

In General, suitable carriers for parenteral solutions are water, suitable oil, saline, aqueous dextrose (glucose) and similar solutions of sugars and glycols, such as propylene glycol or polyethylene glycols. Solutions for parenteral administration preferably contain vodorastvorimuu salt of the active ingredient, nami are antioxidants, such as sodium bisulfite, sodium sulfite, or ascorbic acid, in the form of monocomponent or in combination. Also use citric acid and its salts and sodium salt of EDTA (ethylenediaminetetraacetic acid).

In addition, the dosage form for intravenous or intraperitoneal administration may contain lyophilized powder for recovery of sterile water or sterile saline for injection. Such solutions can contain preservatives like benzalkonium chloride, methyl - or propylparaben and chlorbutanol.

Acceptable pharmaceutical carriers are described in Handbook of pharmaceutical Sciences, Remington, Mack Publishing Company, a standard literary source referenced in this field.

Claims

1. A method of treating a mammal in need of cancer treatment, wherein the mammal impose no limit on the total cumulative dose of an effective anticancer amount of at least one compound represented by the formula I

where R1- H or HE; R2- N., HE, or OMe; R3- H or HE; R4- H or HE; R5is yset, at least 1.5 times the dose equal anticancer activity anthracycline containing 13-ketogroup.

3. The method according to p. 1, characterized in that the connection use 13-dezoksiguanosin.

4. The method according to p. 1, wherein the compound has the formula

5. The method of obtaining derivatives of 13-diethoxyanthracene formula I

where R1- H or HE; R2- N., HE, or OMe; R3- H or HE; R4- H or HE; R5- carbohydrate or substituted carbohydrate,

wherein the solution prepared 13-tailgatenation add reducing agent, the reaction mixture is heated in a flask under reflux, then cooled, add saturated aqueous solution of NaHCO3add to the reaction mixture, the solvent is represented halogen-substituted hydrocarbon, filter the reaction mixture, acidifying the resulting filtrate, chromatografic the filtrate and produce the target product.

6. The method according to p. 5, characterized in that by means of a reducing agent 13-metastructure reduced to a methylene group.

7. The method according to p. 5, characterized in that the solution 13-dailytribuneherald.com agent, heated in a flask with reflux condenser spend cautiously in the atmosphere of inert gas, particularly nitrogen, as solvent for the reaction mixture are added chloroform, acidification of the filtrate spend hydrogen chloride in diethyl ether chromatography of the filtrate is carried out on a column of silica gel, with separate salt, elute hydrophobic impurities formed in the decomposition of salts, a mixed solution of chloroform and methanol, 13-diethoxyanthracene products elute with methanol and the methanol eluate additionally purified by preparative HPLC.

8. The method according to p. 7, characterized in that the recovery is carried out at pH of 6.5 or less.

9. The method according to p. 7, characterized in that the heating is carried out at a temperature of approximately 65 - 75C.

10. The method according to p. 7, characterized in that the heating is carried out at a temperature of approximately 68 - 72C.

11. The method according to p. 7, characterized in that the heating is carried out at a temperature up to approximately 75C.

12. The method according to p. 7, characterized in that the heating is carried out in the absence of oxygen.

13. The method according to p. 7, is this heating is carried out in nitrogen atmosphere.

15. The method according to p. 7, characterized in that the specified product is obtained with a yield of approximately 70 - 80%.

16. The method according to p. 5, characterized in that the dissolved hydrochloride 13-tailgatenation and p-toluensulfonate in anhydrous methanol are added to a solution of Lamborghini sodium, the reaction mixture is heated to a temperature of approximately 68 - 72With, gently heat the resulting solution in the flask under reflux for about one hour in a nitrogen atmosphere, the reaction mixture was concentrated and cooled in a freezer to a temperature of approximately 0 - 4With add saturated aqueous sodium bicarbonate solution to the reaction mixture, add chloroform, anhydrous sodium sulfate, filtered salts formed by adding anhydrous sodium sulfate after shaking, the filtrate is acidified hydrogen chloride in diethyl ether, pass the solution through a column of silica gel, then wash the column with chloroform/methanol up until the eluate becomes colorless, elute with methanol fractions containing the product, evaporated methanol eluate, rastut the product of preparative HPLC using a column containing phenyl groups, separate the product from other impurities using a gradient of acetonitrile/ammonium formate and lyophilizers purified using HPLC fraction with getting 13-deoxydoxorubicinol hydrochloride defined General formula I, in which R1- H; R2- OMe; R3- HE; R4- HE; R5-



 

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