RU2847846C1 - 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidine-5-yl salts with cytotoxic activity - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to the salt of 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidine-5-ium of the general formula:

where X = S or N-CH₃ , A^- = I^- or ClO₄ ^- , provided that X is not S when A^- = ClO₄ ^-. Salts of 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidine-5-ium.

EFFECT: they have a pronounced cytotoxic effect on the tumour cell lines HeLa, PANC-1, MCF-7, A549 and HCT-116 and lower systemic toxicity compared to cisplatin.

1 cl, 1 tbl, 2 ex

Description

The invention relates to new chemical compounds, which are salts of condensed heterocyclic compounds containing a dimethylaminostyryl substituent at the 2,4-position and exhibiting cytotoxic activity. This property makes these compounds suitable for medical use.

Styryl dyes are a widely used class of compounds. Their interest is driven by the relative ease of their preparation and their unique physicochemical and optical properties. Currently, styryl dyes are widely used for detecting biomolecules. Due to the fact that the fluorescence of these compounds can increase hundreds of times upon interaction with DNA, and due to their ability to penetrate cells, styryl dyes are successfully used to visualize intracellular DNA (Akbay N., Losytskyy M.Y., Kovalska V.B., Balanda A.O., Yarmoluk S.M. J. Fluoresc. 18: 139–147 (2007)). Results published in 2022 in the journal Pharmaceuticals show that fused systems containing a styryl moiety (2-styrylchromones) are safe for human neutrophils and at the same time represent a basis with great potential for the development of new drugs with cytotoxic action (Lucas M., Freitas M., Zanchetta M., Silva AMS., Fernandes E. Pharmaceuticals. 15(3): 288 (2022)).

In recent decades, significant efforts have been made to study the therapeutic potential of guanine analogues and their nucleosides. Currently, a number of antiviral drugs based on them have been developed (US Patents Nos. 5,041,426 and 4,880,784). US Patents Nos. 5,994,321, 5,446,045, and EA013594B1 describe the synthesis and evaluation of biological activity of new compounds, 3,5-disubstituted and 3,5,7-trisubstituted 3H-oxazolo- and 3H-thiazolo[4,5-d]pyrimidin-2-ones. However, the antitumor activity of the above-described thiazolopyrimidines containing bisdimethylaminostyryl substituents in the main chain has not been previously studied. We have synthesized new heterocyclic systems containing bisdimethylaminostyryl substituents at positions 2 and 4.

The technical result of the present invention is the creation of new low-toxic chemical compounds based on bis-dimethylaminostyryl substituents in the azine fragment of the molecule at positions 2,4 of the condensed azoloazinium nucleus, containing a nodal quaternized nitrogen atom, possessing a pronounced cytotoxic effect, which can be used as active components in the creation of antitumor drugs.

This result is achieved by creating new compounds, namely 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidinium-5-ium salts, which have a pronounced cytotoxic effect on human tumor cell lines (HeLa, PANC-1, MCF-7, A549), which can be used as an active component in the creation of antitumor drugs.

where, X = S or N-CH ₃ ;

A ^- = I ^- or ClO ₄ ^- .

The claimed invention - 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidinium-5-ium salts with cytotoxic action are hybrid molecules containing a 2,4-bisdimethylaminostyryl substituent in a heterocyclic and aromatic condensed system containing a nodal quaternized nitrogen atom. The cytotoxic action of the proposed compound is due to the presence of a quaternized nitrogen atom in the molecule of the substance and a synergistic effect when binding to a DNA macromolecule. The claimed compound belongs to the class of DNA-tropic cytotoxic agents. In turn, the choice of another anion, not specified in the formula, will not affect the declared biological activity of compounds belonging to the general formula, and can only provide an increase or decrease in the solubility of the resulting salts.

The synthesis of the claimed 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidinium-5-ium salts was carried out according to the scheme:

The essence of the claimed invention is explained by examples.

Example 1.

A mixture of 5,7-dimethyl-3-(4-bromophenyl)-azolo[3,2-a]pyrimidin-4-ium salt (1 mmol) and 4-(dimethylamino)benzaldehyde (4 mmol) was heated at 150 °C with reflux and stirring for 2 hours in 1.5 ml of dimethylformamide. Complete conversion of the starting compounds was demonstrated by TLC analysis. The ternary system butan-1-ol/acetic acid/water (4:4:1) was used as the eluent. After cooling the reaction mixture, a precipitate formed, which was filtered off.

2,4-bis((E)-4-dimethylaminostyryl)benzo[4,5]thiazolo[3,2-a]pyrimidin-4-ium perchlorate (I)

Product I can be used without further purification. Yield: 398 mg, 69% yield. Dark blue powder, mp <300°C.

^1H NMR spectrum (DMSO _- d6 ), δ, ppm: 8.31 (m, 2H, Ar), 8.19 (d, J = 15.5 Hz, 1H, styryl), 8.06 (s, 1H, pyrimidine), 7.91 (d, J = 15.5 Hz, 1H, styryl), 7.83 – 7.70 (m, 4H, Ar), 7.64 (d, J = 8.9 Hz, 2H, Ar), 7.51 (d, J = 15.6 Hz, 1H, styryl), 7.15 (d, J = 15.5 Hz, 1H, styryl), 6.82 (d, J = 8.9 Hz, 2H, Ar), 6.75 (d, J = 9.0 Hz, 2H, Ar), 3.05 (s, 6H, N(CH ₃ ) ₂ ), 3.02 (s, 6H, N(CH ₃ ) ₂ ). ¹³ C NMR spectrum (DMSO- d ₆ ), δ, ppm: 164.1, 162.3, 153.1, 152.8, 146.2, 144.3, 136.1, 131.8, 131.5 (2H), 129.0, 128.6, 126.7, 124.9, 122.7, 122.4, 119.8, 117.5, 112.9, 112.6, 112.5, 112.5, 40.2, 40.13. MS (EI, 70 eV) m/z: 577 (M ⁺ ) (perchlorate- _free ). Found (%): C, 62.41; H, 6.77; N, 9.74. Calculated for _{C30H29N4SO4Cl} . C, _{62.39 ;} H, 6.76; N, 9.71.

2,4-bis((E)-4-(dimethylamino)styryl)benzo[4,5]thiazolo[3,2-a]pyrimidin-4-ium iodide (II)

Product II can be used without further purification. Yield: 458 mg, 76% yield. Dark burgundy powder, mp <300°C.

¹ H NMR spectrum (DMSO- d ₆ ), δ, ppm: 8.30 (m, 2H, Ar), 8.20 (d, J = 15.6 Hz, 1H, styryl), 8.11 (s, 1H, pyrimidine), 7.90 (d, J = 15.4 Hz, 1H, styryl), 7.85 – 7.71 (m, 4H, Ar), 7.66 (d, J = 9.0 Hz, 2H, Ar), 7.50 (d, J = 15.5 Hz, 1H, styryl), 7.11 (d, J = 15.6 Hz, 1H, styryl), 6.88 (d, J = 8.8 Hz, 2H, Ar), 6.75 (d, J = 9.0 Hz, 2H, Ar), 3.1 (s, 6H, N(CH ₃ ) ₂ ), 3.09 (s, 6H, N(CH ₃ ) ₂ ). MS (EI, 70 eV) m/z: 478.22 (M+) (without iodide). Found (%): C, 75.44; H, 6.12; N, 11.73. Calculated for C ₃₀ H ₂₉ N ₄ S: C, 76.34; H, 5.10; N, 11.72.

Example 2.

A mixture of 2,4,10-trimethylbenzo[4,5]imidazo[1,2-a]pyrimidin-10-ium salts (0.65 mmol) and 4-(dimethylamino)benzaldehyde (2.79 mmol) was heated at 150 °C with reflux and stirring for 20 min in 0.5 ml of dimethylformamide. Complete conversion of the starting compounds was demonstrated by TLC analysis. The ternary system butan-1-ol/acetic acid/water (4:4:1) was used as an eluent. After cooling the reaction mixture, a precipitate formed, which was filtered off under vacuum.

4-((E)-4-(dimethylamino)styryl)-2-((Z)-4-(dimethylamino)styryl)-10-methylbenzo[4,5]imidazo[1,2-a]pyrimidin-10-ium iodide (III)

Product III can be used without further purification. Yield: 223 mg, 63% yield. Dark blue powder, mp <300°C.

¹ H NMR spectrum (DMSO- d ₆ ), δ, ppm: 8.43 (d, J = 8.6 Hz, 1H, Ar), 8.21 (d, J = 15.6 Hz, 1H, styryl), 8.06 (d, J = 15.5 Hz, 1H, styryl), 8.03 – 8.00 (m, 2H, Ar, pyrimidine), 7.87 – 7.78 (m, 3H), 7.71 – 7.61 (m, 3H), 7.19 (d, J = 15.7 Hz, 1H, styryl), 6.84 (d, J = 8.9 Hz, 2H, Ar), 6.78 (m, 3H, Ar), 4.06 (s, 3H, CH ₃ ), 3.08 (s, 6H, N(CH ₃ ) ₂ ), 3.04 (s, 6H, N(CH ₃ ) ₂ ). ¹³ C NMR NMR spectrum (DMSO _-d6 ) , δ, ppm: 164.1, 162.3, 153.1, 152.8, 146.2, 144.3, 136.1, 131.8, 131.5 (2H), 129.0, 128.6, 126.7, 124.9, 122.7, 122.5, 119.8, 117.5, 112.9, 112.6, 112.5 (2H), 112.5 (4H), 40.2 (2H), 40.1 (2H). MS ( _EI , 70 eV) m/z: 474 (M ⁺ ) (without iodide). Found (%): C, 61.88; H, 5.37; N, 11.62. Calculated for _C31H32N5I. C, _61.90 ; H, 5.39; N, 11.65.

4-((E)-4-(dimethylamino)styryl)-2-((Z)-4-(dimethylamino)styryl)-10-methylbenzo[4,5]imidazo[1,2-a]pyrimidin-10-ium perchlorate (IV)

Product IV can be used without further purification. Yield: 398 mg, 69% yield. Dark green powder, mp <300°C.

¹ H NMR spectrum (DMSO- d ₆ ), δ, ppm: 8.43 (d, J = 8.6 Hz, 1H, Ar), 8.21 (d, J = 15.6 Hz, 1H, styryl), 8.06 (d, J = 15.5 Hz, 1H, styryl), 8.03 – 8.00 (m, 2H, Ar, pyrimidine), 7.87 – 7.78 (m, 3H), 7.71 – 7.61 (m, 3H), 7.19 (d, J = 15.7 Hz, 1H, styryl), 6.84 (d, J = 8.9 Hz, 2H, Ar), 6.78 (m, 3H, Ar), 4.06 (s, 3H, CH ₃ ), 3.08 (s, 6H, N(CH ₃ ) ₂ ), 3.04 (s, 6H, N(CH ₃ ) ₂ ). Found (%): C, 61.88; H, 5.37; N, 11.62. Calculated for C ₃₁ H ₃₁ N ₅ ClO ₄ : C, 64.97; H, 5.45; N, 12.22. Found: C, 64.90; H, 5.49; N, 11.43.

Experimental studies of cytotoxicity and general toxicity of synthesized salts of 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidinium-5-ium.

Experiment 1.

The study of the cytotoxicity of the claimed compounds and reference compounds with DNA-tropic action (doxorubicin) was carried out using the MTT test on HeLa, PANC-1, MCF-7, A549 and HCT-116 cell lines according to the method published in the work (Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays // Journal of Immunological Methods. - 1983. - Vol. 65, no. 1-2. - P. 55-63). The cells were cultured in a _CO2 incubator at +37 °C in a humidified atmosphere containing air and 5% _CO2 in DMEM-F12 nutrient medium containing 10% heat-inactivated fetal bovine serum, 1% L-glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin.

For the experiment, cells were seeded in a 96-well plate and incubated overnight in a _CO2 incubator to allow cell attachment to the well surface (5,000 cells in 200 µl of DMEM-F12 medium were added to each well). Cell numbers were counted using a Luna II cell viability analyzer (LogosBio, South Korea). DMEM-F12 medium containing the test compounds was then added to the wells. Cells were incubated in the plates for 48 hours in a _CO2 incubator at 37°C. At the end of the incubation period, the DMEM-F12 medium was removed by inverting the plate. Next, 100 μl of DMEM-F12 medium and 20 μl of MTT reagent (5 mg ml ^-1 ) were added to the wells and the plates with cells were incubated for 1 h in a CO ₂ incubator at +37 °C. After removing the supernatant, formazan crystals were dissolved for 15 min with stirring in 200 μl / well DMSO and the optical density was measured on an Allsheng AMR-100 plate photometer (Allsheng Instruments Co., Ltd, China) at wavelengths of 540 nm and 690 nm. As a result of the interaction of MTT with NAD (P) H-dependent cellular oxidoreductases, a colored product is formed - formazan, with a maximum absorption at a wavelength of 540 nm, the content of which is proportional to the number of viable cells. To correct for background, the optical density values at 690 nm for the corresponding wells were subtracted from the optical density values at 540 nm. Data were normalized as a percentage relative to cells incubated without test compounds.

The claimed 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidinium-5-ium salts exhibit cytotoxic activity against HeLa, PANC-1, MCF-7, A549, and HCT-116 cell lines, comparable to doxorubicin, which was selected as the reference compound. All synthesized compounds showed a significant difference in cytotoxicity compared to the reference drug against the PANC-1 cell line, and compound I also demonstrated this difference against the HeLa cell line (Table 1).

Table 1 – IC ₅₀ values (μM) for compounds (I-IV) and doxorubicin

Substance Name of cell line HeLa MCF-7 A549 HEK293 PANC-1 HCT-116 Compound (I) 0.19 7.1 12.9 0.50 0.90 3.6 Compound (II) 1.20 8.8 0.09 0.44 1.9 11.9 Compound (III) 0.7 7.2 0.06 0.52 1.3 3.3 Compound (IV) 1.33 8.5 0.1 0.47 2.1 12.8 Doxorubicin 1.5 1.3 0.07 0.9 46.3 0.50

Experiment 2.

Acute toxicity was assessed using female Wistar rats weighing 200–250 grams, obtained from the Rappolovo breeding farm. The synthesized compounds (I, II, III, IV) were administered intraperitoneally as a single solution at a dose of 8.5 mg/kg. The commercial drug Cisplatin-Teva was also administered intraperitoneally as a single solution at a dose of 8.5 mg/kg, which corresponded to the LD ₅₀ for this drug (the dose of a substance that causes death in 50% of animals in the test group; Jin P, Jiang J, Zhou L, Huang Z, Nice EC, Huang C, Fu L. J Hematol Oncol. 15(1):97 (2022)).

Five animals were used for each compound. The observation period was 14 days. During the studies, no weight loss, changes in behavior or appearance, or death were observed in the animal groups administered compounds (I, II, III, and IV). In the cisplatin group, mortality was 40%.

The obtained results indicate that the indicated compounds at a concentration of 8.5 mg/kg have lower toxicity compared to the commercial drug Cisplain-Teva, which is widely used in clinical practice.

Thus, the obtained 2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidinium-5-ium salts, when compared with the reference compound, have a more pronounced cytotoxic effect, as shown on tumor cell lines, and less systemic toxicity compared to cisplatin, which makes them promising agents for the creation of oncological drugs.

Claims (3)

2,4-bis(4-(dimethylamino)styryl)benzo[4,5]azolo[3,2-a]pyrimidin-5-ium salt of the general formula: where X = S or N-CH ₃ ; A ^- = I ^- or ClO ₄ ^- , provided that X is not S, when A ^- = ClO ₄ ^- .