RU2278119C1 - Tetraazachlorines as photosensibilizators for photodynamic therapy - Google Patents

Abstract

FIELD: organic chemistry, medicine, oncology.

SUBSTANCE: invention relates to photosensitizing agents used in photodynamic therapy of neoplasm and some other diseases. Invention describes tetraazachlorines of the general formula:

wherein R₁R₂ mean -C₆H₄; R₃ = R₄ = R₅ mean -CH₃; M means HH (H₂TBTAC); R₁R₂ means 1,2-C₁₀H₆; R₃ = R₄ = R₅ mean -CH₃; M means Zn(Zn-1,2-TNTAC); R₁ = R₂ mean -C₆H₅; R₃ = R₄ = R₅ mean -CH₃; M means HH (H₂TACPh₆); R₁ = R₃ mean hydrogen atom (H); R₂ =R₄ mean -C₆H₅; R₅R₅ mean -CH₂N(CH₃)CH_2; M means HH (H₂PyrTACPh₄); R₁R₂ mean 4-C(CH₃)₃C₆H₃; R₃ = R₄ mean hydrogen atom (H); R₅R₅ mean -CH₂N(CH₃)CH₂; M means HH (H₂TB^tTACPyr^N-Me) used as photosensitizing agents in the near IR-region of spectrum for carrying out the photodynamic therapy. These photosensitizing agents possess the intensive absorption in spectral region 710-750 nm and can be used in treatment of deep tumor tissues by the photodynamic therapy method.

EFFECT: valuable medicinal properties of compounds.

1 cl, 4 ex

Description

The present invention relates to medicine, and more specifically to photosensitizers for photodynamic therapy (PDT) of neoplasms and a number of other diseases.

The PDT method is based on the use of drugs - photosensitizers, which, when introduced into the body, accumulate mainly in the tumor. Subsequent light, for example laser, irradiation of the pathological site of the photosensitizer molecule catalyzes the formation of cytotoxic agents, in particular singlet oxygen, which destroy tumor cells.

A drawback of clinical photosensitizers based on hematoporphyrin derivatives, for example, HPD (hematoporphyrin derivative), photofrin-2, is the low absorption intensity in the photoexcitation band (625-640 nm). Significant intrinsic absorption of biological tissue in this spectral region causes a small depth of radiation penetration and complicates the treatment of large tumors (R. Bonnett. Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy. Chem. Soc. Rev. 24 (1), 19- 33, 1995). In this regard, chlorine-based photosensitizers (dihydroporphyrins) have an advantage, the more intense long-wavelength absorption band of which is shifted to the red region compared to porphyrins. Among them, it should be noted water-soluble mono-L-aspartylchlorin e _6u (preparations NPe6, MACE) and other various forms of chlorin e ₆ , in particular, domestic preparations Photoditazine, Radachlorin, as well as synthetic chlorins - 5,10,15,20 -tetrakis (m-hydroxyphenyl) chlorin (temoporfin, m-THPC, foscan) and benzoporphyrin derivatives (benzoporphyrin monoacid, ring A) with a maximum absorption at 690 nm. However, these compounds are very difficult to access, in addition, their extinction coefficient, as a rule, is low.

Very promising photosensitizers are derivatives of phthalocyanine, tetrabenzotetrase - an analog of porphyrins. Thus, the photosensitizer "Photosens" based on sulfonated phthalocyanine hydroxyaluminium, absorbing at 675 nm with an extinction coefficient exceeding 10 ⁵ L × mol ^-1 · cm ^-1 (EA Lukyanets. New sensitizers for photodynamic therapy. Ross. Chemical journal, 42 (5 ), 9-16, 1998), is approved for clinical use in Russia for the treatment of a number of oncological diseases of various localizations. However, even in the Photosens absorption region, the intrinsic absorption of unsensitized tissues remains significant. It is known that it is minimal only at wavelengths exceeding 700 nm in the region of the so-called “therapeutic window”. In addition, the disadvantage of "Photosense" is the long retention in the skin, which requires careful observance by patients of the light regime. "Photosens" is the closest analogue of the photosensitizers proposed in this application.

In the present invention, the task is to create photosensitizers for PDT with intense absorption in the spectral region of 710-750 nm. This problem is solved by the fact that as a photosensitizer for PDT, new compounds are proposed - derivatives of tetraazachlorin - of the general formula

where R ₁ R ₂ = C ₆ H ₄ , R ₃ = R ₄ = R ₅ = CH ₃ , M = HH (H ₂ TBTAC);

R ₁ R ₂ = 1,2-C ₁₀ H ₆ ; R ₃ = R ₄ = R ₅ = CH ₃ , M = Zn (Zn-1,2-TNTAC);

R ₁ = R ₂ = C ₆ H ₅ ; R ₃ = R ₄ = R ₅ = CH ₃ , M = HH (H ₂ TACPh ₆ );

R ₁ = R ₃ = H, R ₂ = R ₄ = C ₆ H ₅ , R ₅ R ₅ = CH ₂ N (CH ₃ ) CH ₂ , M = HH (H ₂ PyrTACPh ₄ );

R ₁ R ₂ = 4-С (СН ₃ ) ₃ С ₆ Н ₃ , R ₃ = R ₄ = Н, R ₅ R ₅ = СН ₂ N (СН ₃ ) СН ₂ , М = НН (H ₂ TB ^t TACPyrB ^N-Me ).

for example, tetramethyltribenzotetraazachlorin (H ₂ TBTAC), tetramethyltri-1,2-naphtho-tetraazachlorin zinc (Zn-1,2-TNTAC), tetramethylhexaphenyltetraazachlorin (H ₂ TACPh ₆ ), N-methylpyrrolidino- [3,4-tetra tetrafluoro H ₂ PyrTACPh ₄ ), N-methylpyrrolidino- [3,4-b] tri- (4'-tert.-butyl) benzotetraazachlorin (H ₂ TB ^t TACPyr ^N-Me ).

The proposed tetraazachlorin derivatives are synthesized according to the methods developed by us. Tetramethyltribenzotetraazachlorin (H ₂ TBTAC) and hexaphenyltetraazachlorin (H ₂ TACPh ₆ ) were obtained by condensation of a mixture of tetramethylsuccinonitrile with the corresponding 1,2-dinitriles in dimethylaminoethanol in the presence of lithium dimethylaminoethylate and tri-1,2-nitro-nitro-nitrin - similar condensation in high boiling organic solvents in the presence of zinc chloride [RF Patent No. 2188200, cl. C 07 D 487/22 (2002). BI No. 24 (2002)]. N-Methylpyrrolidino [3,4-b] tetraphenyltetraazachlorin (H ₂ PyrTACPh ₄ ) and tri- (4'-tert.-butyl) benzotetraazachlorin (H ₂ TB ^t ACPyr ^N-Me ) were obtained by analogy with tetraarylporphyrins (Silva AMG, Tome AC., Neves GPMS, Silva AMS, Cavaleiro JAS, II J. Chem. Soc. Chem. Commun., 1999, p. 1767) by the reaction of 1,3-cycloaddition of the corresponding tetraazaporphins with azomethinilide generated in situ from N-methylglycine and paraform. All synthesized tetraazachlorins are insoluble in water and were used as their emulsions in aqueous 4% solutions of Cremophor EL and Proxanol 268 (table 1, drawing).

The present invention is illustrated by the following examples.

EXAMPLE 1. Synthesis of N-methylpyrrolidino [3,4-b] tetraphenyltetraazachlorin (H ₂ PyrTACPh ₄ ). A solution of 50 mg (0.08 mmol) of tetraphenyltetraazaporphine in 5 ml of o-dichlorobenzene is heated to 100 ° C under argon atmosphere with stirring, and 14 mg (0.16 mmol) of sarcosine and 12 mg (0.4 mmol) of paraform are added. Next, the reaction mixture is heated to 150 ° C and maintained at this temperature for 1.5 hours. Then, the cooled reaction mass is chromatographed on silica gel with chloroform. The second fraction (a mixture of chlorin and isobacteriochlorin) is rechromatographed on alumina with toluene. The raspberry fraction is collected and after removal of the solvent, 15 mg (27.5%) of H ₂ PyrTACPh _{4 is} obtained. Mass spectrum, m / z: 675 (M ⁺ ). ESP, λmax. (Intensity ratio), chloroform: 711 (1.0), 656 pl. (0.39), 547 (0.49), 358 (1.02), 333 (0.99).

Similarly, from tri- (4'-tert.-butyl) benzotetraazaporphine N-methylpyrrolidino- [3,4-b] tri- (4'-tert.-butyl) benzotetraazachlorin (H ₂ TB ^t TACPyr ^N-Me ) was obtained.

EXAMPLE 2. Synthesis of tetramethylhexaphenyltetraazachlorin (H ₂ TACPh ₆ ). Dissolve 0.01 g (14 mmol) of lithium by heating in 20 ml of dimethylaminoethanol, cool to room temperature. A mixture of 0.82 g (3.6 mmol) of diphenylmaleonitrile and 0.49 g (3.6 mmol) of tetramethylsuccinonitrile is added, it is slowly heated to boiling with stirring and boiling is continued for 4 hours. Then the reaction mixture is cooled, diluted with 100 ml of water, the precipitate formed is filtered off, washed successively with hot water, 50% aqueous ethanol, and dried in air. The residue was triturated, extracted with ethanol to remove impurities, then with chlorobenzene. The chlorobenzene extract was evaporated in vacuo and chromatographed on silica gel with chloroform, and the raspberry fraction was collected. Obtain 0.025 g (3.8%) of H ₂ TACPh ₆ . Found,%: C 78.45; H 5.66; N, 12.76. C ₅₆ H ₄₄ N ₈ H ₂ O. Calculated,%: C 79.41; H 5.47; N 13.23. Mass spectrum, m / z: 829.2 (M ⁺ ). Electronic absorption spectrum. λ _max , nm (log ε), chloroform: 727 (4.63), 691pl. (3.85), 662pl. (3.69), 557 (4.10), 488 (3.57), 371 (4.32), 336 (4.33).

EXAMPLE 3. In vitro determination of phototoxicity of N-methylpyrrolidino [3,4-b] tetraphenyltetraazachlorin metastane (H ₂ PyrTACPh ₄ ).

Testing was performed on a cell culture of human epidermoid carcinoma of the larynopharynx HEP2. Cells were scattered at a concentration of 40 × 10 ³ cells / ml in the wells of a 96-well plate (Costar, USA) in Igla-MEM medium containing 7% fetal calf serum. After 24 hours (the beginning of the logarithmic phase of culture growth), the substance was added in successive dilutions, the cells were incubated for 2-5 hours, and a 500 W halogen lamp was irradiated using a KS-13 filter (λ≥640 nm). The power density was 13.6-15.0 mW / cm ² . The calculated light dose in all experiments was 10 J / cm ² . The control was: 1) cells that were not treated with dye, solvent and irradiation, 2) cells that were irradiated without prior incubation with dye, 3) cells incubated with dye without subsequent irradiation, 4) cells treated with 4% Cremophor followed by irradiation .

The results of the determination of phototoxicity are shown in table 2.

EXAMPLE 4. Determination of the photodynamic activity of aqueous emulsions of tetraazachlorins in vivo.

The solid form of Ehrlich ascites carcinoma (OE) was grafted subcutaneously onto the outer surface of the lower extremity of CBA female mice. Diluted ascitic fluid (~ 2.2 × 10 ⁶ tumor cells) taken from a donor mouse on the 7th day of tumor growth was used for implantation.

Samples of tetraazachlorins in 4% Cremophor EL or Proxanol 268 were filtered through a 0.22 μm membrane filter and administered to animals intravenously (in the tail vein) once on the 6th day of tumor growth 48, 24 or 0.5 hours before PDT.

A sensitized tumor was irradiated using an ATO-1 therapeutic oncological apparatus for photodynamic therapy and KS-18 filters with a maximum transmission at a wavelength of 700 nm and SZS for filtering the ultraviolet part of the spectrum. The radiation power density is 250 mW / cm ² , the dose density is 300 J / cm ² . Before irradiation, the hair over the tumor was removed. The duration of observation of animals was 25 days after treatment.

The effectiveness of the impact was evaluated by the change in tumor volume (V _op ) and the values of inhibition of tumor growth (SRW), calculated by the formulas (2) and (3):

where D ₁ , D ₂ and D ₃ are three mutually perpendicular diameters of the tumor.

In the study of the in vivo photodynamic activity of aqueous emulsions of H ₂ PyrTACPh ₄ in 4% Cremophor in mice with a P-388 tumor, irradiation was performed 30 minutes after administration using the ATO-1 apparatus with KS-10 and SZS filters. The radiation power density is 300 mW / cm ² , the light dose is 270 J / cm ² .

The results of the determination of photodynamic activity are shown in tables 3-5.

The emulsion of H ₂ PyrTACPh ₄ in 4% Cremophor (H ₂ PyrTACPh ₄ -CrEL) has photo-induced activity on a culture of human tumor cells (table 2). Under standard screening conditions, the IR ₅₀ during irradiation averages 1-33 μM (HEP2 culture) and 1.63 μM (A549 culture); there is no "dark" toxicity in this concentration range. Varying the incubation time and irradiation conditions allows us to conclude that a near-maximum effective concentration of H ₂ PyrTACPh ₄ in cells is reached by 2 hours and phototoxicity for this period is mainly due to the intracellular photosensitizer. A similar Zn-1,2-TNTAC emulsion has a relatively low photo-induced activity: the IC ₅₀ value for it turned out to be 31.0 μM (HEP2 culture).

The in vivo photodynamic activity of aqueous emulsions of H ₂ PyrTACPh _{4 was} studied in mice with a solid form of Ehrlich ascites carcinoma (OE). H ₂ PyrTACPh ₄ in an aqueous solution of 4% Cremophor EL (at doses of 0.1-6.0 mg / kg) and 4% Proxanol 268 (at doses of 1.0-7.0 mg / kg) did not inhibit tumor growth in the absence of light exposure, i.e. did not have “dark” cytotoxicity (table 3). When PDT was performed 48 and 24 hours after intravenous administration of H ₂ PyrTACPh ₄ at doses of 1.0, 3.0 and 6.0 mg / kg, a pronounced antitumor effect was observed throughout the observation period (TPO = 52.2-92.3%). When conducting an irradiation session 0.5 hours after administration of the photosensitizer at a dose of 1.0 mg / kg, the therapeutic effect was recorded during 25 days of observation (TPO = 61.9-65.1), and at a dose of 0.5 mg / kg, only on the 15th day after PDT (61.8 %).

A solution of H ₂ PyrTACPh ₄ in 4% Proxanol 268 showed high photodynamic activity against experimental OE when administered at doses of 3.5 and 7.0 mg / kg 24 hours before the PDT session - inhibition of tumor growth reached 91.8-100% (table 3). At lower doses (1.0 and 0.5 mg / kg), biologically significant inhibition of tumor growth was observed upon irradiation 0.5 hours after administration and amounted to 52.6-70.0% on the 15-25th day of observation. Comparison of the photodynamic activity of H ₂ PyrTACPh ₄ in Cremophor EL and Proxanol 268 did not reveal significant differences (table 3).

Aqueous emulsions of H ₂ TACPh ₆ , H ₂ TBTAC, and H ₂ TB ^t TACPyr ^N-Me in 4% Proxanol 268 under similar conditions also did not possess “dark” toxicity and showed pronounced activity during the PDT session 2 hours after administration (table 4 ) It should be noted the high activity of H ₂ TWTAS, which provides high TPO values at low doses (up to 0.35 mg / kg).

The in vivo photodynamic activity of aqueous emulsions of H ₂ PyrTACPh ₄ in 4% Cremophor in mice with P-388 tumor was also studied. PDT with H ₂ PyrTACPh ₄ -CrEL led to the development of characteristic tissue reactions - edema, followed by the formation of a necrotic scab in all animals 8-9 days after irradiation. PDT with H ₂ PyrTACPh ₄ -CrEL led to a marked inhibition of tumor growth of P-388 for all periods of observation and to increase the life expectancy of animals (table 4). The therapeutic effect of PDT is higher at a dose of 1.0 mg / kg photosensitizer than at a dose of 0.5 mg / kg: SRW exceeded 80% at a dose of 1.0 mg / kg and 70% at a dose of 0.5 mg / kg; UPJ at a dose of 1.0 mg / kg was 42.9%, at a dose of 0.5 mg / kg - 24.9%.

Thus, the intensively absorbed tetraazachlorin derivatives of the present invention in the spectral region of 710-750 nm represent a new class of effective near-infrared photosensitizers that can be used for PDT treatment of deep tumor tissues.

Claims (1)

Tetraazachlorins of the general formula

where R ₁ R ₂ = C ₆ H ₄ , R ₃ = R ₄ = R ₅ = CH ₃ , M = HH (H ₂ TBTAC); R ₁ R ₂ = 1,2-C ₁₀ H ₆ ; R ₃ = R ₄ = R ₅ = CH ₃ , M = Zn (Zn-1,2-TNTAC); R ₁ = R ₂ = C ₆ H ₅ ; R ₃ = R ₄ = R ₅ = CH ₃ , M = HH (H ₂ TACPh ₆ ); R ₁ = R ₃ = H, R ₂ = R ₄ = C ₆ H ₅ , R ₅ R ₅ = CH ₂ N (CH ₃ ) CH ₂ , M = HH (H ₂ PyrTACPh ₄ ); R ₁ R ₂ = 4-C (CH ₃ ) ₃ C ₆ H ₃ , R ₃ = R ₄ = Н, R ₅ R ₅ = CH ₂ N (CH ₃ ) CH ₂ , М = НН (H ₂ TB ^t TACPyr ^N-Me ). as photosensitizers for photodynamic therapy.