RU2476218C1 - Photosensitisers for photodynamic therapy - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quaternary ammonium salts of meso-tetra[1-(4'-bromobutyl)-3-pyridyl]bacteriocholine of general formula

where

EFFECT: compounds possess high photoinduced activity and may be used for the photodynamic therapy of malignant growths as a photosensitiser.

Description

The invention relates to medicine, namely to photosensitizers for photodynamic therapy (PDT) of malignant neoplasms and a number of other pathological conditions.

The PDT method is based on the use of natural or synthetic photosensitizers (PS), which are capable of selective accumulation (tropism) in tumor tissue. When irradiated with light of a certain wavelength, the PS transitions to the activated state, which initiates the formation of cytotoxic agents - singlet oxygen ( ¹ O ₂ ) and free radicals, which cause the destruction of the structural elements of the tumor tissue.

The successful application of the PDT method for the treatment of malignant neoplasms stimulates the search for new FS with improved properties. The most promising for PDT are FS with a maximum absorption in the red and near infrared ranges (650-800 nm), the so-called “therapeutic window”, where the intrinsic absorption of biological tissue is minimal, which allows deeper penetration of radiation into the tissue and, as a result, high efficacy of therapy (Bonnett RJ Heterocyclic Chem. 2002 V.39. P.455-470).

Among them, FS based on chlorins (dihydroporphyrins), characterized by an intense long-wavelength band in the region of 660 nm, for example, water-soluble mono-L-aspartylchlorin e ₆ and other various forms of chlorin e ₆ , in particular, domestic drugs Photoditazine, Radachlorin and Belarusian Photolon, should be noted. (Chan Thi Hai Yen, G.V. Ramenskaya, N.A. Oborotova. Ross. Biotherapist. J. 2009. Issue 4. P. 99-104), as well as synthetic chlorins - 5,10,15,20- tetrakis (m-hydroxyphenyl) chlorin (Temoporfin, m-THPC, Foscan) and benzoporphyrin derivatives (Benzoporphyrin monoacids a, ring A).

Prospective FS are phthalocyanine derivatives. Thus, the Photosens preparation based on sulfonated aluminum phthalocyanine has a long-wavelength absorption maximum at λ _max = 675 nm with a high molar extinction coefficient ε (over 100,000) and a high fluorescence quantum yield (RF Patent No. 2220722, A61K 31/409, 2004). However, Photosens does not have a sufficiently high selectivity for accumulation in tumor cells, long-term preservation in tissues, which leads to skin phototoxicity.

Promising PS for PDT are bacteriochlorins (tetrahydroporphyrins), absorbing in the most optimal range of the “therapeutic window” - 740-820 nm depending on the structure. The problem here is the search for storage-stable derivatives of bacteriochlorin, since the hydrogenation of the porphyrin ring simultaneously with spectral shift decreases the stability of the molecule in oxidation reactions. One way to solve this problem is to use metal complexes. So, tucad, a palladium derivative of bacteriochlorin (Tookad, Israel-Germany-France) with a maximum absorption at 760 nm, is allowed for the treatment of the prostate.

The objective of the invention is the synthesis of PS, characterized by absorption in the near infrared range of the spectrum, high tropism for tumor tissue and possessing high photoinduced activity.

To solve this problem, PS was synthesized, which is positively charged bacteriochlorins - quaternary ammonium salts based on mesotetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin, mesotetra octabromides [1- (4'-pyridiniobutyl ) -3-pyridyl] bacteriochlorin (3-Py ₄ BC (BuPu) ₄ Br ₈ , compound 1), meso-tetra [1- (4 '- (dimethylethanolammoniobutyl) -3-pyridyl] bacteriochlorin (3-Py ₄ BC ( BuDMAE) ₄ Br ₈ , compound 2), General formula:

,

Where

,

Octabromides 1 and 2 were synthesized by the interaction of meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide with an excess of dry pyridine or dimethylaminoethanol, respectively, in boiling methanol for 4.5 hours in an inert atmosphere. The starting meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide was obtained by boiling meso-tetra (3-pyridyl) bacteriochlorin with an excess of 1,4-dibromobutane in nitromethane in an inert atmosphere.

Figure 1 shows the ¹ H NMR spectrum of compound 1 in CD ₃ OD in the region of aromatic protons.

The singlet of β-pyrrole protons is located at 8.27 m. The signals of the protons of the pyridinium rings are observed at 8.16 ppm (H-3 ¹ ), 8.53 ppm (H-5), 8.62 ppm (H-4 ¹ ), 9.17 ppm (H-6, H-2 ¹ ), 9.42 ppm (H-4), 9.84 ppm (H-2).

These photosensitizers (1 and 2) are readily soluble in water and water-salt solutions at room temperature. They are stable in aqueous multicomponent solutions for 1 month with varying concentrations from 2 to 45 μM under dark conditions (in the absorption spectra λ _max 763 nm for compound 1, 762 nm for compound 2 and in the fluorescence spectra λ _max 771 nm). It should be noted that the dyes in distilled water were less stable (a decrease in the absorption intensity at a maximum of 15–20% during the observation period was noted). Figure 2 shows the absorption spectra of a solution of compound 1 (14.2 μM) in a 0.9% NaCl solution and distilled water immediately after dissolution (black line - the spectra coincide) and after a month of storage of the solution at + 4 ° C in 0.9 % NaCl solution (blue line) and in distilled water (red line).

Photosensitizers have the ability to generate singlet oxygen in a water-salt solution with a quantum yield of φ ( ¹ O ₂ ) = 0.30 ÷ 0.34. In a cell-free medium, dyes are prone to fading, which was accompanied by a decrease in the fluorescence intensity at a maximum without changing the shape of the spectrum.

To evaluate the photosensitizing properties, we studied the ability of compound 1 to photoinduced generation of singlet oxygen in solution. It was established that the studied compound in the monomeric state has an average quantum yield of singlet oxygen generation φ ( ¹ O ₂ ) = 0.32 ± 0.02. In confirmation of the generation of precisely ¹ O _2, it was shown that the decolorization reaction of 4-nitroso-N, N-dimethylaniline in the presence of histidine, which underlies the definition of ¹ O ₂ , is completely suppressed by sodium azide, a known quencher of ¹ O ₂ .

Dyes had in vitro phototoxicity against human tumor cells of various epithelial origin: epidermoid larynx pharyngeal carcinoma (HEP2), lung adenocarcinoma (A549) and colon carcinoma (HT29) with varying dye solution concentrations from 0.15 to 40 μM, incubation time to light exposure to 0.5-6 hours and an energy density of 2-10 J / cm ² .

The present invention is characterized by the following examples:

Example 1

Meso-Tetra [1- (4'-pyridiniobutyl) -3-pyridyl] bacteriochlorin octabromide (1). Meso-Tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide (0.09 g, 0.06 mmol) is dissolved in 4 ml of methanol and 1 ml of dry pyridine is added. The reaction mass is stirred while boiling for 4.5 hours in an inert atmosphere. After cooling to room temperature, the reaction mixture was evaporated to dryness in vacuo. The residue was dissolved in 5 ml of methanol, 50 ml of benzene was added, the precipitate formed was filtered off and washed with benzene. 0.093 g (85.3%) of compound 1 is obtained. ESP, λ _max , nm (log ε), methanol: 761 (5.04), 516 (4.70), 421 (4.11), 374 (4, 93), 349 (4.95). ESP, λ _max , nm (log ε), water: 763 (5.02), 518 (4.71), 419 (4.34), 374 (4.95), 351 (4.97). ESP, λ _max , nm (log ε), PBS (pH 7.4): 760 (4.96), 694 (3.89), 515 (4.61), 416 (4.35), 372 (4 , 91), 348 (4.93). ¹ H NMR spectrum (CD ₃ OD), δ, ppm: 2.34-2.42 (m, 16H, CH ₂ ), 4.09-4.38 (m, 8H, β-H), 5.02-5.04 (m, 8H, CH ₂ ), 8.14-8.18 (m, 8H, m-H (Ru)), 8.27 (s, 4H, β-H), 8 50-8.56 (m, 4H, H-5 (Ru)), 8.60-8.64 (m, 4H, n-H (Ru)), 9.16-9.18 (m, 12H , H-6 (Ru), o-H (Ru)), 9.42 (d, 4H, H-4 (Ru)), 9.83-9.85 (m, 4H, H-2 (Ru) )

Example 2

Meso-Tetra [1- (4'-dimethylethanolammoniobutyl) -3-pyridyl] bacteriochlorin octabromide (2). Tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide (0.09 g, 0.06 mmol) was dissolved in 4 ml of methanol and 1 ml of dimethylethanolamine was added. The reaction mass is stirred while boiling for 4.5 hours in an inert atmosphere. The isolation of compound 2 is carried out according to the procedure described for compound 1. Get 0.09 g (80.9%) of compound 2. ESP, λ _max , nm (log ε), methanol: 763 (4.93), 516 (4, 64), 422 (4.44), 373 (4.87), 350 (4.89). ESP, λ _max , nm (log ε), water: 763 (4.90), 518 (4.55), 419 (4.41), 374 (4.83), 351 (4.85). Spectrum ¹ H NMR (CD ₃ OD), δ, ppm: 2.09-2.13 (m, 8H, CH ₂ ), 2.34-2.37 (m, 8H, CH ₂ ), 3 24 (s, 24H, CH ₃ ), 3.53-3.59 (m, 8H, CH ₂ ), 3.63-3.66 (m, 8H, CH ₂ ), 3.99-4.04 (m, 8H, CH ₂ ), 4.10-4.38 (m, 12H, β-H, OH), 5.03-5.06 (m, 8H, CH ₂ ), 8.27 (s, 4H, β-H), 9.50-9.53 (m, 4H, H-5 (Ru)), 9.18-9.23 (m, 4H, H-6 (Ru)), 9.44 (d, 4H, H-4 (Ru)), 9.86 (s, 4H, H-2 (Ru)).

Example 3

Determination of the photostability of compound 1.

The photostability of compound 1 was studied in an air-saturated aqueous solution under uniform irradiation with a continuous Nd ³⁺ -YAG laser (generation wavelength 532 nm, power 0.82 mW) for 30 min. Photodegradation 1 was recorded every 10 min by a decrease in absorption at the maximum of the long-wave Q band. The quantum yield of photobleaching (ϕ _f ) was determined by the slope of the graph of the dependence of the number of photobleached molecules in a solution N _mol , determined by formula (1), and the number of absorbed quanta N _square , determined by formula (2).

,

,

,

,

where D ₀ and D are the absorption at the maximum of Q are the photosensitizer bands before and after irradiation. C ₀ is the concentration of the photosensitizer in solution before irradiation; N _A is the Avogadro number, I is the power on the sample, λ is the wavelength of the irradiating light, D _Δ is the optical density of the irradiated solution at the laser wavelength, t is the irradiation time in seconds, s is the speed of light, h is the Planck constant, V is volume of solution in the cell.

The measured linear dependence of the number of photobleached molecules on the number of absorbed quanta is characterized by a slope of 9.5 × 10 ^-4 , which is numerically equal to the quantum yield of photobleaching ϕ. According to the ϕ value, photobleaching of photosensitizer 1 occurs after absorption of about 1000 light quanta per molecule, i.e. in terms of photostability, it is comparable with other derivatives of bacteriochlorin - meso-tetra (1-methyl-3-pyridyl) bacteriochlorin tetratosylate and meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide, for which ϕ are respectively 18 , 5 × 10 ^-4 and 8.7 × 10 ^-4 .

Example 4

Accumulation and intracellular localization of compound 1 in human lung carcinoma cells (A549 cell culture).

The study of intracellular localization of the dye in cells was carried out using the method of confocal microspectroscopy and reconstruction of spectral images (KOMIRSI). The concentration of the photosensitizer in the medium ranged from 0.5 to 10 μM, the incubation time was 1-6 hours. Fluorescence was excited by an Nd ³⁺ -YAG laser with a generation wavelength of 532 nm. It was revealed that the dye penetrates into cells and accumulates in the cytoplasm (diffuse distribution, as well as concentration in granules), but does not penetrate into the nucleus. When studying intracellular kinetics, it was shown that the maximum accumulation of compound 1 in the cytoplasm of A549 cells is observed after 4 hours of incubation (Figure 3).

An analysis of the intracellular spectra showed that in the cytoplasm of cells, compound 1 is present in various states described by spectra with fluorescence maxima of 771, 773, 776 and 780 nm, different from the spectrum in PBS or in water. In order to clarify the causes of these changes, a study was made of the effect of the microenvironment on the fluorescence of compound 1.

A spectrum with a maximum of 771 nm is closest to spectrum 1 in complexes with BSA and HSA, a spectrum with a maximum of 773 is closest to spectrum 1 in complexes with globulins, a spectrum with a maximum of 776 nm is in complexes with nucleic acids.

The kinetics of accumulation of compound 1 in A549 cells was studied. It was found that in cells Compound 1 accumulates rather slowly: saturation of intracellular accumulation is observed only after 4 h of incubation.

Example 5

The photoinduced activity of compound 1 in relation to human lung carcinoma cells (A549 cell culture). The photosensitizer was introduced into the culture medium at a concentration of 0.3 to 30 μM. Incubation times ranged from 30 minutes to 6 hours. Irradiation was carried out as in example 3, the level of inhibition of culture growth was calculated by the formula:

IR (%) = [(P _to -P _o ) / P _to ] × 100%,

where IR - inhibition of culture growth, in percent;

P _about and P _to - the number of viable cells, expressed in units of optical density, respectively, in experimental (with dye) and control (without dye) samples. Inhibition of culture growth by 50% (IC ₅₀ ) was considered a biologically significant effect.

Compound 1 was shown to have high phototoxicity to A549 culture cells. The maximum photoinduced activity was observed at 4 hours of incubation (IR ₅₀ was 1.5 ± 0.07 μM), with an increase in the time interval, the IC ₅₀ value changed slightly. Compound 1 did not have dark toxicity during the day of observation (IR ₅₀ >> 20 μg / ml).

In vitro testing of photosensitizers 1 and 2 showed that they also exhibit high phototoxicity against human tumor cells HEP2. The IC ₅₀ value was 0.67 ± 0.08 μg / ml and 0.9 ± 0.1 μg / ml, respectively. There was no dark toxicity.

Thus, the proposed FS has all the properties that will subsequently allow their effective use for PDT of malignant neoplasms.

Claims (1)

Quaternary ammonium salts of meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin of the general formula

Where

,

as photosensitizers for photodynamic therapy.

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