Esters octa-4,5-carboxylic acid phthalocyanine cobalt, and their inclusion complexes with propylene glycol ether- cyclodextrin and method of suppressing tumor growth

 

(57) Abstract:

The invention relates to organic chemistry, and medicine, and it applies to substances used in combination with ascorbic acid for the treatment of malignant neoplasms (binary catalytic "dark" therapy of malignant tumors) and method of suppressing tumor growth. Describes esters OCTA-4,5-carboxy phthalocyanine cobalt General formula I, where R is CH3O[CH2CH2O]nor HO[CH2CH2O]nn = 3 - 7, and their inclusion complexes with propylene glycol ether-cyclodextrin, and a method of suppressing tumor growth using these compounds and biogenic reducing agent ascorbic acid. Application of the proposed connections provides persistent biologically significant inhibition of tumor growth and a significant increase in the life expectancy of mice with transplantable malignant tumors of various origins. 3 C. p. F.-ly, 4 PL.

The present invention relates to medicine and organic chemistry, as it relates to the suppression of tumor growth by chemotherapy and moreover, using chemical compounds.

The new strategy in what is "binary" therapy of tumors includes two components needed to obtain a therapeutic effect, each of which separately shows antineoplastic activity. This kind of treatment is photodynamic therapy, which combines the use of a photosensitizer with a local laser irradiation. This method of cancer therapy is based on the formation by the interaction of a photosensitizer with laser radiation of reactive oxygen species (ROS) and other free radical particles of different chemical structure, which are cytotoxic agents. The treatment is clinically tested worldwide [Chiss C. I., Sokolov, C. C., E. Filonenko Century and other FACES, 1998, No. 4, S. 4-12; L. W. Ma, J. Moan, Grahn, M. F., V. Iani Proc SPIE, 1996, v. 2924, p. 219-223; Raymond Bonnett. Chemical Society Reviews, 1995, v. 24, p. 19-33].

The disadvantage of this method of treatment of malignant tumors is the need for activation of the photosensitizer by light to achieve a clinical effect, which significantly limits the use of this method, not allowing the use of PDT for the treatment of deep common malignant tumors.

During clinical studies were also identified undesirable pharmacological Italia and its connective tissue layer (collagenosis, sclerotic changes) and due to the fact that the drug has long been preserved in the skin and in conditions of natural illumination can move to the active state. It is also possible lesions of the conjunctiva and retina.

Another example of a "binary" therapy would be the use of a catalytic system consisting of a metal complex Co or Fe with substituted phthalocyanines and biogenic reducing agent [RF Patent 2106146, borisenkova S. A., Girenko E. G., Potassium O. L. Russian journal of chemistry, vol XXLII, N 5, 1998; wolpin M. E., kraineva N. Y., Levitin I. I and other Russian chemical journal, volume XXLII, N 5, 1998; Syrkin, A. B., Zhukova O. S., ' C. B. and other Russian chemical journal, volume XXLII, N 5, 1998]. This kind of "binary" therapy, in contrast, PDT, called "dark" or catalytic therapy of malignant tumors. Today revealed the complex - sodium salt - Oct - 4,5 - carboxyfullerene cobalt

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which is the most active of all the tested complexes of Co and Fe with substituted phthalocyanines, which, in combination with nutrient reducing agent is ascorbic acid (AA) has antitumor activity. This metal complex of cobalt was named "Terephthal" (Thobani in the composition of the binary catalytic system. Binary catalytic system "TF + AK has a marked antitumor activity in vitro and in vivo [Syrkin, A. B., Zhukova O. S.,' C. B. and other Russian chemical journal, volume XXLII, N 5, 1998].

The task of the invention was the development of new metal complexes of cobalt with substituted phthalocyanines, which in combination with ascorbic acid have a marked antitumor activity, effectively destroying tumor cells.

To solve this problem were synthesized esters OCTA-4,5-carboxylic acid phthalocyanine cobalt formula 1

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where R is CH3[CH2CH2O]nor HO[CH2CH2O]n;

n = 3-7.

The synthesis of these esters is carried out by interaction of Oct-4,5-carboxyfullerene cobalt Co with methylpredisolone or tetraethylene glycol.

For further creation of new compounds that can be used to suppress tumor growth, were synthesized complexes include esters of formula 1 (ETF or TATP) with propylene glycol ether-cyclodextrin (CDOP), hereinafter referred to as (ETF-or TATP-K).

These inclusion complexes obtained by dissolution of the esters of formula 1 in water, and.

There is a method of suppressing tumor growth using salt OCTA-4,5-carboxyfullerene Co and ascorbic acid [RF patent 2106146]. This method allows to achieve inhibition of proliferation of human tumor cells in vitro, and inhibition of tumor growth and increase the life expectancy of animals with transplantable tumors in vivo.

The task of the invention was to provide such a method of suppressing tumor growth, which would provide a more stable biologically significant inhibition of the tumor and a significant increase in the life expectancy of animals with transplantable tumors. To solve this problem there has been proposed a method of suppressing tumor growth with the use of a complex of cobalt with substituted phthalocyanines and ascorbic acid, in which the complex use of the above compounds of formula 1 or their inclusion complexes with propylene glycol ether-cyclodextrin.

The antitumor effectiveness of the proposed method was evaluated both in vitro and in vivo. Methods for the assessment of antitumor efficiency are presented below.

Methodology 1.

Zitat the key epidermoid carcinoma of the hypopharynx man (cell culture Hep-2).

Cells were cultured in the medium Needle-MEM with the addition of 2 mm L-glutamine and 8% fetal calf serum, at 37oC in humidified atmosphere containing 5% carbon dioxide.

Cells were dissipated in wells of 96-well microplate, 100 μl at a concentration of 70-80 thousand cells/ml To evaluate the effectiveness of the cytotoxic effects of catalytic pair after 24 h (early logarithmic phase of growth in the wells was made by serial dilution of the investigated esters and ascorbic acid.

As Comparators used traditional cytotoxic drug "Platinum".

After 24 h incubation of the cells with drugs was assessed by the number of viable cells by colorimetric method using the MTT-test [Mossmann, T. R. Rapid colorimetric for cellular growth and survival: application to proliferation and cytotoxity assays/ Journal of immunological methods, 65, 1983, p. 55 to 63] .

The level of inhibition of culture growth was calculated by the formula:

ER (%)=[(Nto-Pabout)/Pto]100%,

where IL is the inhibition of culture growth, in percent;

Paboutand Ptothe number of viable cells, expressed in units of optical density, respectively, in the experimental (with drugs) and control (without drugs) samples.

aktivnosti in vitro study drug was calculated using the integral indicator of the cytotoxic activity IR50representing the concentration of a cytotoxic agent, in which there is inhibition of culture growth by 50%.

Method 2.

Antitumor activity in vivo was assessed in mice with transplantable tumors. Used the following transplantable malignant tumors of mice - the ascitic Ehrlich carcinoma (EAC), mammary adenocarcinoma Ca-755 (Ca - 755) and lymphocytic leukemia P-388 (solid variant of a tumor).

AKE instilled outbred white mice females administered intraperitoneally in 0.1 ml of ascitic fluid obtained from a mouse donor on day 7 of tumor growth and diluted 1:1 with 0.9% sodium chloride solution.

Ca-755 vaccinated mice females line C57B1/6 subcutaneously with 60 mg of tumor tissue in 0.3 ml of medium 199, taken from mouse donor on the 10 - 12th day of tumor growth.

P-388 vaccinated mice hybrids F1 females subcutaneously with 0.1 ml of ascitic fluid obtained from a mouse donor on day 7 of tumor growth and diluted 1:100, 9% solution of sodium chloride.

Treatment of animals with EAC and P-338 started within 24 to 48 h, and animals with Ca-755 after 48 - 72 h after inoculation of the tumor.

The control animals with transplantable tumors were administered placebo (0.9% saline Nadia" (cisdichlorodiaminoplatina, The MDC).

Doses and modes of introduction of the investigated drugs are detailed in the examples.

The antitumor effect was evaluated by the average life expectancy (ALE), the increase in life expectancy (UPI), calculated by the formula:

UPG-[(ALEexperience- ALEcontrol)/ ALEcontrol]100%

and inhibition of tumor growth, calculated by the formula:

SRW=[(POcontrolRHOexperience)/RHOcontrol]100%.

Significant biological effect is believed to increase life span of animals by 25% and the inhibition of tumor growth by 50%. [Experimental evaluation of anticancer drugs in the USSR and the USA / edited by H. P. Sofianou, A. B. Syrkin, A. Goldin, A. Klein, M., "Medicine", 1980].

The invention is illustrated by the following examples.

Example 1.

Synthesis metilprednisolona ether.

In the autoclave load 280 g dried over aluminum oxide and relocated methylcarbazole and 0.4 g of epirate boron TRIFLUORIDE, heated to a temperature of 100oC and gradually add 410 g of ethylene oxide. Then the autoclave is cooled to room temperature and the reaction mixture is discharged. Dolgopolov.

Synthesis metilprednisolona ether OCTA-4,5-carboxylic acid phthalocyanine cobalt (ETF). In a flask with a capacity of 50 ml equipped with a stirrer, a thermometer and a nozzle Dean-stark placed 3.8 g OCTA-4,5-carboxy phthalocyanine cobalt, 16.6 g of metropolitanareas and 0.07 g of orthophosphoric acid. The reaction mass is heated with stirring to a temperature of 160 -165oC, then take a sample and check its solubility in water. If a positive test result samples of the reaction mass is transferred to a beaker, add 200 ml of toluene and stirred. The resulting oil is separated from the solution by decantation and washed with a small amount of hexane. The residue is dissolved in water, the solution is filtered, distilled water and dried in vacuum. Get 7.9g ETF representing a viscous homogeneous mass of dark blue color, slowly soluble in water.

IR spectrum:c=o1710 cm-1.

Electronic spectrum (phosphate buffer pH 7,4): band 1 - 3252 nm, band 2 - 6672 nm.

Example 2.

In the conditions of example 1 from 3,5 g OCTA-4,5-carboxyfullerene cobalt and 16.0 g metilprednisolona ether so Kip. 160 - 200oC/3-5 mm RT. senior get of 7.1 g ETF.

IR spectrum:c=o1710 cm is EP 3.

In the conditions of example 1 from 3.8 g of OCTA-4,5-carboxyfullerene cobalt and 16.0 g of tetraethyleneglycol obtain 6.3 g tetraethyleneglycol ether OCTA-4,5-carboxyfullerene cobalt (TETF).

IR spectrum:c=o1710 cm-1.

Electronic spectrum (phosphate buffer pH 7,4): band 1 - 3252 nm, band 2 - 6682 nm.

Example 4.

Synthesis of the complex include ETF with propylene glycol ether-cyclodextrin (CDOP)*- substance ETF-K.

Substance ETF from example 1 in the amount of 7.9 g dissolved in warmed up to 50-60oC distilled water, the aqueous solution is filtered, separating the undissolved residue. To the filtrate add 31.6 CDOP and stirred at a temperature of 60-70oC, then the water is distilled off on the rotor, the residue is dried to constant weight in a drying Cabinet. Get of 37.8 g of the complex ETF, representing the blue crystalline powder, soluble in water. Output 95,8%.

IR spectrum:c=o1710 cm-1.

Electronic spectrum (phosphate buffer pH 7,4): band 1 - 3262 nm, band 2 - 6702 nm.

*- CDOP synthesized in accordance with the method described in U.S. patent N 3459731.

Example 5.

In the conditions of example 4 from 7,P>-1.

Electronic spectrum (phosphate buffer pH 7,4): band 1 - 3262 nm, band 2 - 6702 nm.

Example 6.

In the conditions of example 4 of 6.3 g of the substance TATT from example 3 25.2 g CDOP get to 29.5 g of the complex TATF-K.

IR spectrum:c=o1710 cm-1< / BR>
Electronic spectrum (phosphate buffer pH 7,4): band 1 - 3262 nm, band 2 - 6722 nm.

Example 7.

The cytotoxic activity of different substances ether of octacarbonyldicobalt cobalt WAS (ETF, ETF and TATP-K), taken both individually and in combination with ascorbic acid, compared to the cytotoxic activity of the drug "Platinum and binary catalytic system "TF + AK" in relation to cell epidermoid carcinoma of the hypopharynx man (cell culture Hep-2).

WAS in the culture medium were made at concentrations from 10 to 10-5M to 3 10-6M, and the drug "Platinum" - from 2 to 10-5M to 2,610-6M

Ascorbic acid was introduced into the culture medium after WAS with the interval from 0 to 5 min at concentrations ranging from 110-3M to 610-4M, using the molar ratio WAS:AK - 1:20.

The results are presented in table 1.

As can be seen from the data presented in the rye catalytic system "ETF + AK", "ETF-K+AK", "TETT + AK" and "TATF-K+AK effectively destroy tumor cells. Cytotoxic activity of the above binary catalytic systems comparable to those for traditional anticancer drug "Platinum and binary catalytic system "TF+AK" (PL.1).

Example 8.

The effect WAS different (ETF, ETF), taken both individually and in combination with ascorbic acid preparation "Platinum and binary catalytic system "TF+AK" to increase the life expectancy of mice with ascitic Ehrlich carcinoma.

WAS injected once intraperitoneally at a dose of active substance) 250 mg/kg of Ascorbic acid was administered intraperitoneally immediately after the introduction WAS at a dose of 443 mg/kg (molar ratio of substance:AK - 1:30). Binary catalytic system was introduced as follows: TF (40 mg/kg) 1 h + AK (88 mg/kg), the molar ratio of HF:AK -1:10.

The results are presented in table 2.

As can be seen from the data presented in table 2, individually ETF, ETF-K and AK does not have any impact on the life expectancy of mice-carriers of tumour. Binary catalytic system "ETF+AK" and "ETF-K+AK" have proty is velichanii life expectancy (UPI was 110 - 114). The use of binary catalytic system "TF+AK increases the life expectancy of mice with EAC by 42%.

Example 9 N.

The effect WAS different (ETF and ETF-K), taken both individually and in combination with ascorbic acid preparation "Platinum and binary catalytic system "TF+AK" on the dynamics of tumor growth in mice with mammary adenocarcinoma Ca-755.

WAS injected once intraperitoneally at a dose of active substance) 250 mg/kg of Ascorbic acid was administered intraperitoneally 1 h after injection WAS at a dose of 443 mg/kg (molar ratio of substance:AK - 1:30). Binary catalytic system was introduced as follows: TF (40 mg/kg) 1 h + AK (88 mg/kg), the molar ratio of HF:AK - 1:10.

The results are presented in table 3.

As can be seen from the data presented in table 3, individually ETF, ETF is To not have any influence on the dynamics of tumor growth and survival rate of mice - carriers of tumour.

The use of binary catalytic systems "ETF+AK" and "ETF-K+AK" leads to a stable (for 18 days) biologically significant inhibition of tumor growth, where TRO on day 10 was 82-85% and 18 day - 72-74%. In Torola tumors only 55% (minimal biologically significant anti-tumor effect).

Example N 10.

The effect WAS different (ETF and ETF-K), taken both individually and in combination with ascorbic acid preparation "Platinum and binary catalytic system "TF+AK" on the dynamics of tumor growth in mice with lymphocytic leukemia P-388.

WAS injected once intraperitoneally at a dose of active substance) 250 mg/kg of Ascorbic acid was administered intraperitoneally 1 h after injection WAS at a dose of 443 mg/kg (molar ratio of substance:AK - 1:30). Binary catalytic system was introduced as follows: TF (40 mg/kg) 1 h + AK (88 mg/kg), the molar ratio of HF:AK - 1:10.

The results are presented in table 4.

As can be seen from the data presented in table 4, individually ETF, ETF is To not have any influence on the dynamics of tumor growth and survival rate of mice - carriers of tumour.

The use of binary catalytic systems "ETF+AK" and "ETF-K+AK" leads to a stable (within 9 days) biologically significant inhibition of tumor growth, where TRO on day 5 was 70-73%, and on day 9 - 58-61%. Intravenous binary catalytic system "TF+AK" animals with a solid form of leukemia P-388 causes biologically significant inhibition is atom, declared esters and their inclusion complexes with propylene glycol ether-cyclodextrin in combination with ascorbic acid have a marked cytotoxic activity against human tumor cells (culture of cells Hep-2) in vitro, comparable to those for binary catalytic systems on the basis of similar drug "Terephthal" and traditional anticancer drug "Platinum", and in vivo in mice with transplantable malignant tumors of various origins provide significant persistent inhibition of tumor growth and increase the life expectancy of animals - carriers of tumour.

1. Esters OCTA-4,5-carboxylic acid phthalocyanine cobalt General formula 1

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where R is CH3O[CH2CH2O]nor HO[CH2CH2O]n;

n = 3-7.

2. The complex include esters of formula 1 with propylene glycol ether-cyclodextrin.

3. The method of suppressing tumor growth with the use of a complex of cobalt with substituted phthalocyanines and ascorbic acid, characterized in that as complex use connections under item 1 or 2.

 

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