PL247926B1 - 2-(3-Hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide, method of its production and its first medical application - Google Patents

Abstract

The subject of the application is a new hydrazinecarbothioamide derivative, of general formula 1 shown in the drawing, which is a simultaneous inhibitor of human topoisomerase II and tyrosinase, which is used in the treatment of melanoma. The application also covers a method for preparing the new hydrazinecarbothioamide derivative of general formula 1, wherein 3-hydroxy-4-nitrobenzoic acid hydrazide is reacted with 1-naphthylmethyl isothiocyanate in organic solvents. The reaction progress is monitored using chromatographic techniques. After the reaction is complete, the reaction mixture is cooled, the resulting precipitate is filtered off, dried, and then crystallized from polar solvents. Importantly, the reaction in organic solvents is carried out in boiling ethanol, preferably for 5-30 minutes, and the resulting reaction product is crystallized from ethanol.

Description

Description of the invention

The subject of the invention is a new hydrazinecarbothioamide derivative of general formula 1, shown in the drawing, characterized by simultaneous inhibition of the activity of human topoisomerase II and tyrosinase, exhibiting cytotoxic activity against melanoma cancer cell lines, a method of its preparation and its first medical application.

Hydrazinecarbothioamide derivatives, other than the one indicated in the invention, are known from the scientific literature to have anticancer activity (Biomolecules 2022, 12, 151; Molecules 2019, 24, 2065; Molecules 2020, 25, 2980; Anticancer Agents Med Chem 2018, 18 529-540).

In the case of single hydrazinecarbothioamide derivatives, it was found that their anticancer activity is the result of inhibition of topoisomerase II (Anticancer Agents Med Chem 2018, 18, 529-540). There are also single scientific reports indicating that some hydrazinecarbothioamide derivatives, but substituted only in position 2 and not having a substituted nitrogen atom in the carbothioamide group, are capable of inhibiting the tyrosinase enzyme (Bioorg Chem 2008, 16, 1096-1102; Sci Rep 2023, 13, 2578). For the hydrazinecarbothioamide derivatives described in the cited source texts, either no anticancer activity was determined, or, as in the case of one tested derivative described in Sci Rep 2023, 13, 2578, no significant inhibition of A375 cancer cell growth was observed up to a concentration of 32 gM. Importantly, to date, there are no reports in the scientific literature, including patents, regarding the existence of hydrazinecarbothioamide derivatives that demonstrate the ability to simultaneously inhibit both of the aforementioned enzymes—human topoisomerase II and tyrosinase. Furthermore, there are no scientific reports on the cytotoxic activity of such derivatives against melanoma cells or any other cancer cells.

Inhibition of both of these enzymes is beneficial in the treatment of melanoma, as inhibition of topoisomerase II activity results in limited proliferation of cancer cells, and inhibition of tyrosinase, the overexpression of which is observed in melanoma cells, increases the sensitivity of these cells to cytostatics and increases the activity of lymphocytes against cancer cells, thus increasing the effectiveness of the treatment (Int J Cancer 2009, 124, 1470-1477.

The number of cancer cases diagnosed globally has increased in recent years to over 23 million, while the number of cancer-related deaths has exceeded 10 million (JAMA Oncol 2022, 8 420-444). According to WHO data (Globocan 2020) on the incidence of melanoma, over 320,000 new cases of this type of cancer were recorded in 2020, with a mortality rate exceeding 60,000. Furthermore, statistics show that the number of newly diagnosed melanoma cases doubles every decade. These data demonstrate the importance of identifying and introducing into clinical practice new anticancer drugs that demonstrate efficacy against melanoma cells.

The Applicants conducted research to obtain hydrazinecarbothioamide derivatives capable of simultaneously inhibiting human topoisomerase II and tyrosinase, enzymes essential for effective anti-melanoma activity against melanoma cells. The results demonstrate the potent anti-melanoma activity of the hydrazinecarbothioamide derivative, which is the subject of the invention. This activity results from the simultaneous inhibition of topoisomerase II and tyrosinase activity in melanoma cells. Importantly, the hydrazinecarbothioamide derivative, which is the subject of the invention, demonstrated anti-melanoma activity against melanoma cells that was stronger than the clinically used anti-cancer drug, etoposide. The data presented suggest that the derivative, which is the subject of the invention, may be an attractive candidate for an anti-melanoma drug.

The essence of the invention is 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide, of the general formula 1 shown in the drawing.

The derivative of general formula 1 exhibits inhibitory activity against human topoisomerase II and tyrosinase, which leads to inhibition of melanoma cell growth and increases the sensitivity of these cells to cytostatics. It also increases the activity of lymphocytes against melanoma cells. The demonstrated dual activity of the derivative of the invention against these two enzymes contributes to its significant anticancer activity against melanoma cells.

The essence of the method for preparing the new hydrazinecarbothioamide derivative of general formula 1, according to the invention, is that 3-hydroxy-4-nitrobenzoic acid hydrazide (general formula 2) is reacted with 1-naphthylmethyl isothiocyanate (general formula 3), wherein the reaction is carried out in organic solvents, diethyl ether, methanol, preferably in boiling ethanol, for 5-30 minutes. The progress of the reaction is monitored by chromatographic techniques. After the reaction is complete, the reaction mixture is cooled, and the precipitate is filtered off, dried, and then crystallized from polar solvents, preferably ethanol.

The essence of the invention is also 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide for use in the treatment of melanoma.

The compound of the invention has a strong anti-tumor effect against melanoma cells and may therefore be used in the production of new drugs for the treatment of this cancer disease.

Example 1:

0.01 mol of 3-hydroxy-4-nitrobenzoic acid hydrazide was dissolved in 25 ml of anhydrous ethanol, and an equimolar amount of 1-naphthylmethyl isothiocyanate was added to the resulting solution. The mixture was heated to the boiling point of the solvent in a round-bottomed flask under a water condenser for 25 minutes, after which the reaction mixture was cooled, and the precipitate was filtered off, dried, and crystallized from ethanol.

2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide (of general formula 1) was obtained with a yield of 82%.

The chemical structure of the compound was determined based on the results of 1H-NMR and IR spectra. The reaction progress was monitored using thin-layer chromatography.

Physicochemical and spectral parameters of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide of general formula 1;

Reaction yield: 82%; melting point: 180-182°C; 1H-NMR (600 MHz): 3.70 (s, 2H, CH2), 7.00-8.12 (m, 10H, ArH), 9.32 (s, 1H, NH), 9.52 (s, 1H, OH), 10.21 (s, 1H, NH), 10.85 (s, 1H, NH); IR (γ, cm-1): 3341 (NH), 1670 (C=O), 1336 (C=S).

The anticancer properties of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide were determined using the MTT assay. The following melanoma cancer cell lines were used in the studies: A375 and G361. Cell culture media dedicated to each cell line were used for cell culture.

Increasing concentrations of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide were added to 96-well plates containing a given cell type at a density of ^1x105 cells/ml, followed by 24-hour incubation, after which antitumor activity was determined based on the degree of inhibition of the metabolic activity of melanoma cells using the MTT assay. Etoposide, a clinically used anticancer drug, was used as a positive control. Example 2:

Melanoma cell lines (A375 and G361), purchased from the American Type Culture Collection (ATCC), were cultured in recommended media supplemented with 10% FBS and the antibiotics penicillin (100 U/ml) and streptomycin (100 μg/ml). Culture was performed in an incubator maintained at 5% CO2 and 37°C. The melanoma cell suspension was transferred to a 96-well plate (in a volume of 100 μl/well) at a suspension density of 1 x ¹⁰⁵ cells/ml. After 24 hours, required for complete cell attachment to the bottom of the wells, solutions containing increasing concentrations of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide or etoposide (a positive control in the MTT assay) were added to the plates and incubated for another 24 hours, after which MIT reagent solution was added. After a 3-hour incubation, 10% SDS was added to the wells to dissolve the formed formazan crystals. After overnight incubation, the absorbance of the solutions in the wells was read at 570 nm using a microplate reader (Epoch, BioTek). Results are expressed as IC50 values. The results are presented in Table 1.

PL 247926 Β1

Table 1. Antitumor activity of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide against melanoma cell lines G361 and A375.

IC50 ± SD [pg/ml] G361 A375 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide 4.13 ±0.17 5.71 ± 0.10 Etoposide 8.25 ±0.39 14.17 ±0.42

The ability of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide to inhibit human topoisomerase II and tyrosinase activity was determined using enzymatic activity assay kits: Topoisomerase II Drug Screening Kit (manufacturer: TopoGEN) and Tyrosinase Inhibitor Screening Kit (manufacturer: Sigma-Aldrich).

Example 3:

A mixture containing DNA, topoisomerase II enzyme, an appropriate buffer, and 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide at concentrations of 10, 25, 50, and 100 μg/ml was incubated for 30 minutes at 37°C. After the addition of 10% SDS, proteinase K, and loading buffer, electrophoresis was performed on a 1% agarose gel. After electrophoretic separation, DNA fragments were stained with ethidium bromide and analyzed densitometrically using the Syngene G.BOX gel documentation system. Results were expressed as IC50 values, defining the concentration at which the enzyme activity is inhibited by 50% of the initial activity. The level of inhibition of topoisomerase II activity by 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide is presented in Table 2.

Example 4:

The assay for the ability of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide to inhibit tyrosinase activity was performed according to the protocol provided by the manufacturer of the Tyrosinase Inhibitor Screening Kit. A 20 μI solution containing increasing concentrations of 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide from 10 to 100 μg/ml was added to 50 μI tyrosinase solution (containing 48 μI MAK257A buffer and 2 μI tyrosinase). The mixture was incubated for 10 minutes at room temperature, then 30 μl of the solution containing the tyrosinase substrate was added and the mixture was mixed thoroughly. Enzyme activity was then assessed spectrophotometrically at 510 nm every 5 minutes over a 30-60 minute period. Results were expressed as IC50 values. The level of tyrosinase inhibition by 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)methyl]hydrazinecarbothioamide is presented in Table 2.

Table 2. Inhibitory capacity of topoisomerase II and tyrosinase enzymes by 2-(3-hydroxy-4-nitrobenzoyl)-N-[(naphth-1-yl)]hydrazinecarbothioamide

Enzyme IC50 ± SD [pg/ml] topoisomerase II 73.53 ±2.27 tyrosinase 54.04 ±1.77

Claims (5)

1. A new hydrazinecarbothioamide derivative, with the general formula 1 shown in the figure, which is a simultaneous inhibitor of human topoisomerase II and tyrosinase. 2. A method for producing a new hydrazinecarbothioamide derivative of the general formula 1, shown in the drawing, characterized in that 3-hydroxy-4-nitrobenzoic acid hydrazide is reacted with 1-naphthylmethyl isothiocyanate, carrying out the reaction in organic solvents, where the progress of the reaction is monitored using chromatographic techniques, and after the reaction is completed, the reaction mixture is cooled, the resulting precipitate is filtered off, dried and then crystallized from polar solvents. 3. The method according to claim 2, characterized in that the reaction in organic solvents is carried out in boiling ethanol, preferably for 5-30 minutes. 4. The method according to claim 2, characterized in that the obtained reaction product is crystallized from ethanol. 5. A new hydrazinecarbothioamide derivative of general formula 1 shown in the drawing, which is a simultaneous inhibitor of human topoisomerase II and tyrosinase, of general formula 1, for use in the treatment of melanoma.