RU2647588C1 - Method of obtaining diethrye salt 2,4-di(1-metoxyethyl)deuteroporphyrin-ix (dimegin) - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to pharmacology, in particular to method of preparation of disodium salt of 2,4-di(1-methoxyethyl)deuteroporphyrin-IX (DIMEGIN). Method includes carrying out of alkaline hydrolysis of tetramethyl ether of hematoporphyrin-IX of general formula (3)

obtained by mixing protogemin of general formula (4)

with acetic acid with obtaining of batch, in which methanol is added and then sodium acetate is added in water to give tetramethyl ester of hematoporphyrin-IX (3). Hydrolysis is carried out in preporation of dioxane-water at 70–80 °C for 1 hour, followed by the addition of isopropyl alcohol to the batch, excrete of the desired disodium salt of 2,4-di(1-methoxyethyl)deuteroporphyrin-IX (1).

EFFECT: invention allows to increase output of the end product and simplify the technology of its production.

1 cl, 3 dwg, 2 ex

Description

The invention relates to pharmacology, namely to the preparation of a biologically active compound of the porphyrin series -, disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX (DIMEGINA) of the general formula (1) in freeze-dried form, which can be used in as a highly effective photosensitizer (PS) for photodynamic therapy (PDT) of some forms of cancer and other neoplasms of various genesis, numerous inflammatory and purulent processes, in cosmetology for skin photorejuvenation, as well as for fluorescence diagnostics Ostik cancer cells.

Hematoporphyrin-IX (GP) derivatives of the general formula (2) are used in the majority of modern photosensitizers (PS), the so-called first generation, for PDT of cancer and other diseases.

Some of these derivatives have found a real embodiment in world medical practice and are widely used at present as approved drugs for PDT cancer and other neoplasms. The most famous among these PSs are PHOTOFRIN II (USA) and PHOTOGEM (Russia), which are a complex mixture of condensation products of hematoporphyrin-IX molecules in the form of sodium salts without an exact determination of the molecular structure and molecular weight of this mixture, which mainly contains isomeric dimers containing ether and ester bonds, as well as, probably, C — C bound polymers. (Pushpan SK, Venkatraman S., Anand V., Sankar J. et al. (2002) Curr. Med. Chem., Anti-Cancer Agents, 187-207; Pat. Russia 2128993, 04.20.1999, Mironov A.F. .; Nokel A.Y.)

The main disadvantages of the above FS are the following:

1) the oligomeric structure of PS requires very careful chromatographic separation of certain fractions from a complex mixture of hematoporphyrin-IX condensation products to obtain a standardized dosage form of the drug, which significantly increases the cost of the entire process;

2) the polymer structure of the PS determines a significant variability of the electronic spectra depending on the pH of the solution and the ratio of the components in the composition of the PS;

3) the presence of close-lying tetrapyrrole rings in the PS composition of macrocycles leads to a hypochromic effect and a decrease in fluorescence due to self-quenching of photoexcited hematoporphyrin molecules and, accordingly, a decrease in the generation of singlet oxygen, which affects the effectiveness of PHOTOFRIN II or PHOTOGEM as a photosensitizer.

Recently, a new dimeric porphyrin, SINOPORPHIN, has been synthesized, an analog of PHOTOFRIN, the structure of which is a mixture of three isomeric dimers, the porphyrin units of which are connected by an ether bond. But its synthesis presents significant difficulties, in comparison with the already known FS of a similar structure. (Photochem Photobiol Sci. 2015 Apr, No. 14 (4), p. 815-32. Doi: 10.1039 / c4pp00463a.Lin N1, Li C, Wang Z, Zhang J, Ye X, Gao W, Wang A, Jin H, Wei J.)

A known method of obtaining DIMEGIN disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX (1) (Auth. Certificate USSR 1160710 from 04/28/84). The method consists in the fact that 1 g of 2,4-di- (α-methoxyethyl) deuteroporphyrin-IX dimethyl ether is maintained at room temperature in 100 µk of a 25% hydrochloric acid solution, after 12 hours it is neutralized with a saturated sodium acetate solution, the precipitate formed filtered off, washed repeatedly with hot distilled water and dried at 40-50 ° C. The dry residue is dissolved in a mixture of chloroform-methanol (5: 1), filtered, hexane is added until complete precipitation, the precipitate is filtered off, dried in air and 0.82 g (85%) of 2,4-di- (α-methoxyethyl) deuteroporphyrin is obtained -IX.

To a solution of 1.0 g (1.6 mmol) of porphyrin in a mixture of methanol and chloroform (40 ml, 1: 9), 73.6 mg (3.2 mmol) of sodium metal in 10 ml of methanol are added. After 30 minutes, the solvent was removed in vacuo, the residue was dissolved in a minimum amount of methanol (20 ml) and precipitated by adding hexane-ether (1: 1) gradually, the precipitate was filtered off, dried in a vacuum desiccator over NaOH and 0.9 g was obtained ( 84%) of the disodium salt of 2,4-di- (α-methoxyethyl) deuteroporphyrin-IX.

The main disadvantage of this method is the fact that during the acid hydrolysis of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX dimethyl ether], partial removal of methanol molecules from the methoxyethyl groups of deuteroporphyrin-IX necessarily occurs with the appearance of porphyrins containing vinyl substituents, which leads to to reduce the output of DIMEGIN.

The closest in technical essence and selected as a prototype is patent EP 0149995 B1, 03/07/1990, which consists in obtaining DIMEGIN by hydrolysis of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX dimethyl ether in a solution of pyridine with sodium hydroxide in methanol at stirring at 90 ° C for 2 hours, after which a crystalline precipitate of the target product is isolated with a yield of ~ 10%.

A technical problem is the creation of PS with higher consumer properties for PDT than the well-known drugs PHOTOFRIN II PHOTOGEM or SINOPORFIN, i.e.:

1) a high degree of purity and homogeneity of the dosage form of the drug FS;

2) sufficient solubility in water to create the optimal concentration when creating convenient injection solutions, as well as the ability to dissolve in gel and ointment compositions in the form of monomolecular structures for external use,

3) the best ability to generate singlet oxygen when irradiated with laser light with different wavelengths (from the region of 390-410 nm, up to 600-625 nm),

4) as well as the simplicity and convenience of technological operations upon receipt of the dosage form and its stability during long-term storage.

The technical result from the use of the invention in comparison with the prototype is to increase the yield of the target product to 95%, many times simplifying the technology for obtaining, based on the starting material, “hematoporphyrin-IX tetramethyl ester obtained from available starting material - protogemin.

In our present invention, we have developed conditions for alkaline saponification of ester residues of propionic acid esters in the tetramethyl ether molecule of hematoporphyrin-IX. The TMG compound of the general formula (3)

obtained in turn from natural protogemin of the general formula (4) immediately before the formation of the crystalline disodium salt of 2,4 (1-methoxyethyl) deuteroporphyrin-IX (DIMEGINA) of the general formula (1) with a high yield and a content of at least 99 in the sample of the main product %, which is confirmed by the data of elemental analysis, electronic and PMR spectra, as well as HPLC data.

In FIG. Figures 1a and 1b show the electronic spectra of DIMEGIN in water and ethanol, from which it can be seen that this porphyrin in both aqueous and organic media is in monomeric form, which contributes to the maximum generation of singlet oxygen and its effective use as a photosensitizer for PDT. From FIG. 1a and 1b it is seen that the region of the Soret band (380-410 nm) in integrated intensity in both water and alcohol significantly exceeds absorption in the visible part of the spectrum. Therefore, the use of laser or LED sources to excite DIMEGIN molecules in the "violet" spectral region "occurs with maximum efficiency, in contrast to standard methods for irradiating related known photosensitizers such as PHOTOFRIN, PHOTOFRIN II or PHOTOGEM, for which a laser source of radiation at ~ 630 nm is usually used .

In FIG. 1a shows the electronic spectrum (general view) of DIMEGIN in water (blue color) and alcohol (red color).

In FIG. 1b shows the electronic spectrum of DIMEGIN in the visible part of the spectrum (450 nm - 650 nm).

The method for producing disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX (DIMEGINA) by alkaline hydrolysis is characterized in that hematoporphyrin-IX tetramethyl ether is used as the starting component for hydrolysis. (TMH) (3) obtained by mixing protogemin (4) with acetic acid to give a reaction mixture to which methanol is added and then sodium acetate in water is added to obtain hematoporphyrin-IX tetramethyl ester. (TMH) (3), which is used as the starting material for hydrolysis in a solution of dioxane-water at 70-80 ° C for 1 hour, followed by the addition of isopropyl alcohol to the reaction mixture, isolation of the target product of 2,4-di disodium salt (1-methoxyethyl) deuteroporphyrin-IX (1).

PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION

Example 1

30 g of protogemin (4) are stirred on a rocking chair in 300 ml of acetic acid saturated with HBr (d = 1.45) in a flask with a tightly closed stopper at room temperature for 24-48 hours until the precipitate is completely dissolved, the reaction mixture is poured slowly with stirring for 15 minutes to 2400 ml of methanol, after 12 hours, a solution of 1 kg of sodium acetate in 1 l of water is added to the reaction mixture with stirring so that the temperature does not exceed 18-20 ° C, after 24 hours the precipitated crystalline precipitate is filtered off, washed with hot (60 ° C) distilled water, air dried 29 g (96.2%) of technical “tetramethyl ether” hematoporphyrin IX (3) are obtained, which are dissolved in 700 ml of chloroform, chromatographed on a column of aluminum oxide (III stage of activity) in chloroform, the main bright red fraction is separated, evaporated to dryness, the residue dissolved in ethanol, kept at 8-10 ° C for 24 hours, the precipitated coarse-grained precipitate was filtered off, dried in air and 21.9 g (72.7%) of tetramethyl ether of hematoporphyrin-IX were obtained. (TMG) (3). Electronic spectrum in chloroform λ max, nm (ε): 400 (166000), 500 (12800), 534 (8100), 569 (5900), 623 (3900). TMG in CDC13 (δ): 10.55, 10.51, 10.13, 10.08 all s, meso-H, 4H; 6.05 k, CH-CH3; 2H; 4.42 t and 3.30 t 2хСН2СН2СОО, 4Н and 4Н; 3.71, 3.68, 3.66, 3.65, 3.61, all s, 24H, 4xCH3 rings, 2xCH-CH3, 2xCOOCH3; NH-3.92, s, 2H.

Example 2

To a solution of 21.9 g of TMG in 130 ml of freshly distilled dioxane at 70 ° C, add a solution of 2.85 g of sodium hydroxide in 38 ml of water, stir for 1 hour at 70-80 ° C, and 250 ml of isopropyl alcohol are poured slowly (over 20 minutes) with stirring, cooled to 50 ° C, poured with stirring in 10 minutes 700 ml of acetone, the precipitated precipitate is filtered off, washed with a filter of 300 ml of acetone, dried in air at 50-55 ° C, until there are no organic solvents in the sample by GLC and 21.6 g are obtained ( 96.6%) of the disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX, counting at ref projectile loader "tetramethyl ester" hematoporphyrin-IX. PMR spectrum in DMSO-d6 (δ) 10.67, 10.56, 10.46, 10.18 all s, meso-H, 4H; 6.16, 6.15 two k, C H- OMe, 2H; 4.33 t, 4.31 t, 2xC H 2CH2COONa, 4H; 3.71, 3.69, 3.57, 3.56, 3.54, 3.51 all s, 3Hx4 rings and 2xCH- OMe ; 2.92 t, 2.90 t, 2xCH2C H 2COONa, 4H; 2.20, 2.19, 2.15, 2.14 two d, CH- Me , 6H; -3.92, s, 2H. NH shown in FIG. 2 “The PMR spectrum of disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX” indicates that the sample contains no impurities of accompanying porphyrins, and from the HPLC data it follows that the content of the main substance is 99.2%.

Below are the photosensitizing properties of DIMEGIN.

The high effectiveness of Dimegin as a photosensitizer was established by a comparative study of its photophysical properties with the properties of the photosensitizers Photodithazine and Radachlorin. First of all, Dimegin has high photo stability (22 times higher), intense luminescence (2 times higher), efficient generation of singlet oxygen (2.5 times higher) .. An additional advantage of Dimegin is its low toxicity (at / in the introduction of 240 mg / kg) in comparison with "Photoditazine" (with the on / in the introduction of 86 mg / kg). (A.V. Dadeko, T.D. Muravyova, A.M. Starodubtsev, S.I. Gorelov, M.V. Dobrun, T.K. Krisko, I.V. Bagrov, I.M. Belousova, G .V. Ponomarev. Optics and Spectroscopy. 2015, Volume 119, No. 4, pp. 617-622).

Earlier, the accumulation of a photosensitizer in a cell, its intracellular localization, and cell survival after photodynamic treatment with Dimegin were studied using the example of human cell lines obtained from the skin. (SCL1 and SCL2, carcinoma cells and fibroblasts.) As noted, "Dimegin" mainly accumulates in the mitochondria of cells. The high ability to generate singlet oxygen upon irradiation of cells treated with Dimegin induces oxidative stress, which leads to the release of caspases and the subsequent mitochondrial development of apoptosis, which ultimately leads to cell death.

, O.S. Wolfbeis, M. Landthaler // Photodermatology, Photoimmunology & Photomedicine, V. 14, №3-4, P. 125-131, 1998.)Irradiation of the cellular material treated with a 15 mM Dimegin solution with an incoherent light source (580-740 nm) with a dose of 24 J / cm ² in all cases led to a significant phototoxic effect. (S. Fickweiler, R.-M. Szeimies, C. Abels, GV Ponomarev, F.

, OS Wolfbeis, M. Landthaler // Photodermatology, Photoimmunology & Photomedicine, V. 14, No. 3-4, P. 125-131, 1998.)

The photodynamic properties of DIMEGINA were evaluated by the example of its antimicrobial action, which showed that LED irradiation (λ = 400 nm) with a power density of 0.05 W / cm ^{2 of} microbial cells S. aureus, E. coli, C. Albicans, etc. ., pre-treated with a 0.35% Dimegin solution, led to a bactericidal effect comparable to that of standard antiseptics - chlorhexidine and dioxidine.

For work, we used the Dimegin solution prepared the day before the experiment, which was stored after preparation at room temperature.

It was established that the effectiveness of "Dimegin" is not inferior to the effectiveness of "Photoditazine" in this wavelength range, however, given the simplicity and cheapness of its manufacture, it may turn out to be quite competitive in mass production (V.M. Bondarenko, Yu.V. Alekseev, O V.V. Mislavsky, G.V. Ponomarev, Biomedical Chemistry, 2014, Volume 60, Issue 3, pp. 338-347).

Claims (4)

A method of producing a disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX (DIMEGINA) by alkaline hydrolysis, characterized in that hematoporphyrin-IX tetramethyl ether (TMG) of the general formula is used as the starting material for hydrolysis

obtained by mixing protogemin of the General formula

with acetic acid to obtain a reaction mixture to which methanol is added and then sodium acetate in water is obtained to obtain hematoporphyrin-IX tetramethyl ester (TMH) (3), which is used for hydrolysis in dioxane-water solution at 70-80 ° C for 1 hour, followed by the addition of isopropyl alcohol to the reaction mixture, isolation of the target product of the disodium salt of 2,4-di (1-methoxyethyl) deuteroporphyrin-IX (1).

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