New 1,3,5-triazine derivative and its application

Abstract

A new derivative of tryptamine-1,3,5-triazine, which is N2-(2-(1H-indol-3-yl)ethyl)-6-morpholino-N4-phenethyl-1,3,5-triazine-2,4-diamine hydrochloride of formula 1 for use in the treatment of colorectal cancer.

Description

[0001] NEW 1,3,5-TRIAZINE DERIVATIVE AND ITS APPLICATION

[0002] TECHNICAL FIELD

[0003] The subject of the invention is a new tryptamine-l,3,5-triazine derivative of formula 1 and its use in the treatment of colorectal cancer.

[0004] BACKGROUND ART

[0005] The new tryptamine-l,3,5-triazine derivative in the form of a hydrochloride salt of the general formula 1, which is the subject of the invention, is a compound hitherto unknown in the literature. The closest solution in terms of chemical structure to the described new compound is a series of 1,3,5-triazine derivatives published by Kutag D. et al. in a series of three articles: "Aminotriazines with indole motif as novel, 5-HT7 receptor ligands with atypical binding mode" Bioorg. Chem. 2020, 104, 104254, "Design and synthesis of new potent 5-HT7 receptor ligands as a candidate for the treatment of central nervous system diseases" Eur. J. Med. Chem. 2022, 227, 113931 and "Design, Synthesis and Biological Evaluation of Novel 1,3,5-Triazines: Effect of Aromatic Ring Decoration on Affinity to 5-HT7 Receptor" Int. J. Mol. Sci. 2022, 23(21), 13308.

[0006] Colorectal cancer (CRC) is one of the most lethal types of malignancy. It is estimated that over 1.9 million new cases were diagnosed in 2020. Modern oncological therapy mainly involves the resection of the tumor with a wide margin of healthy tissue, while chemotherapy is used as a supportive method. In the case of chemotherapy, several drugs are known (5-FU, folinic acid, oxaliplatin and capecitabine) which, in addition to destroying tumor cells, negatively affect healthy tissues, causing a number of side effects. Currently, the FDA (U.S. Food and Drug Administration) has approved three compounds for the treatment of colorectal cancer - cetuximab, panitumumab and bevacizumab. Despite their effectiveness, they have side effects and not every patient can be qualified for their use due to potential mutations of the EGF receptor or its low expression. In the case of small-molecules, only regorafenib has been approved by the FDA, among others for the treatment of colorectal cancer, but due to similar drawbacks, therapy with this drug is limited.

[0007] One of the important molecular targets that can be used in the treatment of CRC is the FOXM1 (Forkhead Box M) protein - an oncogenic transcription factor, the overexpression of which is associated with tumor growth, as well as with the angiogenesis process and the mechanism of metastasis formation, and plays a key role in the mechanism of resistance to typical anticancer drugs.

[0008] The compound STL427944 is known to have cytotoxic effects on colorectal cancer cells. The result of its use is the inhibition of FOXM1 protein production, described by Comacho C. and Gartel A. in Cell Death & Disease (2021) 12:704 "Novel FOXM1 inhibitor identified via gene network analysis induces autophagic FOXM1 degradation to overcome chemoresistance of human cancer cells" . This compound, described by formula 2, is a representative of hydrazones, comprising a 1,3,5-triazine motif connected by a hydrazone moiety to a phenyl ring, which in turn is substituted with a 2-furancarboxylic acid ester in the third position.

[0009] For the SW480 cell line (human primary colorectal cancer cells), it showed weak FOXM1 inhibition at a concentration of 25 pM, and significant inhibition at a concentration of -50pM. According to the authors, this compound also showed activity on other types of cancer cells including LNCaP (prostate cancer), PC3 (prostate cancer) and A549 (non-small cell lung cancer). DISCLOSURE OF THE INVENTION

[0010] The essence of the invention is a new tryptamine-l,3,5-triazine derivative, which is N2-(2-(lH-indol-3-yl)ethyl)-6-morpholino-N4-phenethyl-l,3,5-triazine- 2,4-diamine hydrochloride of formula 1, for use in the treatment of colorectal cancer, as a substance that inhibits the production of FOXM1 protein.

[0011] The new compound, N2-(2-(lH-indol-3-yl)ethyl)-6-morpholino-N4- phenethyl-l,3,5-triazine-2,4-diamine hydrochloride of formula 1, is obtained in the sequence of four reactions:

[0012] 1. tryptamine of formula 3 is alkylated with cyanuric chloride of formula 4 to obtain an intermediate of formula 5;

[0013] 2. the intermediate of formula 5 is subjected to a condensation reaction with morpholine of formula 6 to obtain an intermediate product of formula 7;

[0014] 3. the intermediate product of formula 7 is reacted with 2-phenylethylamine of formula 8, which produces the second intermediate, a base of formula la;

[0015] 4. the second intermediate, the base of formula la, is converted into the final hydrochloride compound of formula 1 using HCI 4M solution in dioxane.

[0016] It turned out unexpectedly that, despite the close structural similarity to known serotonin 5-HT7receptor ligands, the described compound being N2-(2- (lH-indol-3-yl)ethyl)-6-morpholino-N4-phenethyl-l,3,5-triazine-2,4-diamine hydrochloride of formula 1 has antiproliferative effects on SW480 and SW620 cell lines (human colorectal cancer cells) by blocking the production of the FOXM1 protein, while the line of normal colorectal epithelial cells CCD841 is treated as a control.

[0017] An exemplary method of obtaining a new tryptamine-l,3,5-triazine derivative of formula 1 is presented below:

[0018] In the first step, 1.0 g (5.369 mmol) of cyanuric chloride (formula 4) was dissolved in 20 mL of anhydrous tetra hydrofuran (THF) and then cooled to 0°C. 0.94 mL (5.369 mmol) of diisopropylethylamine (DIPEA) was added dropwise to the prepared mixture, followed by 0.78 g (4.881 mmol) of tryptamine (formula 3), dissolved in 15 mL of anhydrous tetra hydrofuran (THF). The addition was carried out at a rate of 2 drops / s, so that the temperature did not exceed 3°C. After the addition, the temperature of the reaction mixture was raised to 20°C and, maintaining this temperature, stirring was carried out using a magnetic stirrer for another 10 minutes at a speed of 700 rpm. After this time, 25 mL of the solvent was evaporated using a low-speed evaporator (temperature 40°C and pressure 300 mbar), and the residue was transferred to a separatory funnel and 50 mL of methylene chloride was added. The resulting mixture was washed three times with 50 mL of 0.1 M hydrochloric acid solution and then separated. The organic layer obtained in this way was dried over anhydrous magnesium sulfate (VI), filtered, and then the filtrate was evaporated using a low-speed evaporator (temperature 40°C and pressure 300 mbar) during 10 minutes. The dark beige solid residue obtained in this way was macerated in 15 mL of cold methanol and then filtered, obtaining a light beige precipitate constituting an intermediate of formula 5 in an amount of 1.45 g. The course of the reaction is shown in Scheme I.

[0019] Formula 3 Formula 4 Formula 5

[0020] Scheme I

[0021] In the second step, 1.45 g (4.705 mmol) of the intermediate of formula 5 was dissolved in 40 mL of anhydrous tetra hydrofuran (THF) and then cooled to temperature of 0°C. To the mixture prepared in this way, 0.82 mL (4.705 mmol) of diisopropylethylamine (DIPEA) was added dropwise, followed by 0.41 mL (4.705 mmol) of morpholine of formula 6. The addition was carried out at a rate of 2 drops / s, so that the temperature did not exceed 3°C. After the addition, the temperature of the reaction mixture was raised to 20°C, and maintaining this temperature, it was stirred using a magnetic stirrer for 15 hours at a speed of 700 rpm. After the reaction was completed, 30 mL of the solvent was evaporated using a low-speed evaporator (temperature 40°C and pressure 300 mbar), and the liquid residue was transferred to a separatory funnel and 50 mL of methylene chloride was added. The resulting mixture was washed three times with 50 mL of IM hydrochloric acid solution and separated. The organic layer obtained in this way was dried over anhydrous magnesium sulfate (VI), filtered, and then the filtrate was evaporated using a low-speed evaporator (temperature 40°C and pressure 300 mbar) during 10 minutes, obtaining a white intermediate product of formula 7 in an amount of 1.58 g. The course of the reaction is shown in Scheme II.

[0022] Formula 5 Formula 5 Formula 7

[0023] Scheme II

[0024] In the third step, 250 mg (0.697 mmol) of intermediate product 7 was ground in a porcelain mortar with 0.29 g (2.090 mmol) of potassium carbonate (K2CO3) and 0.02 g (0.070 mol) of tetrabutylammonium bromide (TBAB). The whole was transferred to a glass reaction vessel, 0.22mL (1.743 mol) of 2-phenylethylamine of formula 8 and 0.5 mL of N,N-dimethylformamide (DMF) was added. The vessel was placed in the reaction chamber of the microwave reactor. The synthesis was carried out for 2.5 minutes at a power of P = 50 W. After the reaction, the mixture was diluted with 10 mL of methylene chloride and washed three times with 20 mL of 0.1 M hydrochloric acid solution, then separated. The organic layer obtained in this way was dried over anhydrous magnesium sulfate (VI), filtered, and the filtrate was evaporated using a low-speed evaporator (temperature 40°C and pressure 300 mbar). The brown, thick residue was purified using a chromatographic column, using a mixture of chloroform and methanol as the eluent in the following volume ratios: 99:1, 98:2, 97:3, and ending with 96:4. The collected fractions with the purified compound of formula la were combined, evaporated using a low- speed evaporator (temperature 40°C and pressure 300 mbar) obtaining the second intermediate in the form of a transparent oil in the amount of 221 mg. The course of the reaction is shown in Scheme III.

[0025] Scheme III

[0026] In the last step, 221 mg (0.499 mmol) of the second intermediate of formula la was dissolved in 5 mL of acetone and then acidified with 4M HCI in dioxane to pH 2. The white precipitate formed was precipitated by adding 30 mL of cold diethyl ether and filtered, obtaining in this method 243 mg of the final product in the form of N2-(2-(lH-indol-3-yl)ethyl)-6-morpholino-N4-phenethyl- l,3,5-triazine-2,4-diamine hydrochloride of formula 1. The course of the reaction is shown in Scheme IV.

[0027] Scheme IV

[0028] Results of analyzes confirming the chemical structure

[0029] The compound of formula 1 was characterized by: nuclear magnetic resonance spectroscopy (proton -1H NMR and carbon -13C NMR), chromatographic analysis combined with mass spectroscopy (HPLC-MS), thin- layer chromatography (TLC) and melting point measurement.

[0030] Nuclear magnetic resonance

[0031] The proton nuclear magnetic resonance spectrum is shown in Fig. 1.

[0032] XH NMR (600 MHz, MeOD) 6 7.56 (s, 1H), 7.39 - 7.21 (m, 6H), 7.11 (d, J = 7.0 Hz, 2H), 7.02 (s, 1H), 3.84 - 3.61 (m, 12H), 3.06 (s, 2H), 2.90 (s, 2H).

[0033] The carbon nuclear magnetic resonance spectrum is shown in Fig 2.

[0034] 13C NMR (151 MHz, MeOD) 6 161.69, 154.70, 138.59, 136.86, 128.51, 128.24, 127.36, 126.22, 122.44, 121.07, 118.29, 117.68, 111.31, 111.01, 44.14, 41.76, 41.21, 34.81, 24.74, 14.05.

[0035] HPLC - MS analysis (chromatographic analysis with mass spectroscopy) - chromatogram and mass spectrum are shown in Fig. 3.

[0036] HPLC: purity = 100 %, retention time = 4,639 min, m / z = 444,5 [M+H]

[0037] Thin layer chromatography - TLC TLC: Rf = 0,44 (eluent system chloroform: methanol 9:1 v / v)

[0038] Melting point (determined on a Boetius apparatus)

[0039] Tt = 213-216°C

[0040] Molar mass of salt: 480,01 g / mol

[0041] General formula of salt: C25H30CIN7O

[0042] The analyzes performed allow for unambiguous confirmation that a new product was obtained in the form of N2-(2-(lH-indol-3-yl)ethyl)-6-morpholino-N4- phenethyl-l,3,5-triazine-2,4-diamine hydrochloride described by formula 1, which is a new derivative of tryptamine-l,3,5-triazine.

[0043] Cultivation of cell lines

[0044] The cell lines SW480 and SW620 and CCD841 purchased from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendations were used in the cultivation. The cultivation for each cell line and their passage twice a week, with subcultivation in a 1:3 ratio, was carried out according to standard schemes presented in the book Davis, J. M. (2011). "Basic techniques and media, the maintenance of cell lines, and safety. Animal cell culture. Chichester: Wiley-Blackwell, 91-125." Lines SW480 and SW620 were cultured, according to standards, in DMEM (Dulbecco's Modified Eagle Medium) with gentamicin (50 pM / mL) in 75 cm2flasks, and lines CCD841 in EMEM (Eagle's Minimum Essential Medium) with gentamicin (50 pM / mL ) in culture flasks having a surface of 75 cm2. The lines were grown at a temperature of 37°C in an atmosphere of 5% CO2.

[0045] Assessment of antiproliferative potential

[0046] The assessment of the antiproliferative potential of the new tryptamine- 1,3,5-triazine derivative of formula 1 against colorectal cancer cells was carried out using the MTS test on SW480 and SW620 cell lines. The normal colorectal epithelial cell line CCD841 was used as a reference sample. Additionally, the ability to inhibit FOXM1 production was determined in the ELISA test. For the MTS proliferation test and ELISA test, a dose of 5.8 mg of a new tryptamine-l,3,5-triazine derivative of formula 1 was prepared and dissolved in 120.8 pL of DMSO (dimethyl sulfoxide) to obtain a concentration of 0.1 M / L (starting solution). Then, using DMEM culture medium with 10% (v / v) addition of FBS - fetal bovine serum (1 mL of FBS was added to 9 mL of DMEM), a series of concentrations of a new tryptamine-l,3,5-triazine derivative of the formula 1 was prepared: 200 pM / L, 100 pM / L, 50 pM / L, 25 pM / L, 12.5 pM / L, 6.25 pM / L, which is shown below in Table 1.

[0047] Table 1

[0048] MTS proliferation test

[0049] The MTS test is a colorimetric method for determining the number of viable cells in proliferation and cytotoxicity tests. In this test, the reagent contains the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole], which is reduced by living cells into colored formazan, soluble in the cell culture medium. The level of cell proliferation is directly proportional to the amount of formazan produced, and thus to the color change assessed colorimetrically at a wavelength of 490 nm.

[0050] Performing the MTS proliferation test using a test available on the market (here: Cel ITiter 96® AQueous One Solution Cell Proliferation Assay, Promega). On a flat-bottomed plate with a working volume of 200 pL wells, 104cells were seeded in a volume of 50 pL of culture medium per well, in 24 wells for each line, and the plate was placed in an incubator for 1 hour. Then, 50 pL of a solution of the new tryptamine-l,3,5-triazine derivative of formula 1 was added to the wells, with concentrations consistent with Table 1. Each concentration was added to 3 wells for the tested cell line. For the SW480 and CCD841 lines, concentrations of 100 pM / L, 50 pM / L, 25 pM / L, 12.5 pM / L, 6.25 pM / L were used, thus obtaining the concentrations of the new tryptamine-l,3,5-triazine derivative of formula 1 in the wells: 50 pM / L, 25 pM / L, 12.5 pM / L, 6.25 pM / L and 3.125 pM / L, and for the SW620 line concentrations of 200 pM / L, 100 pM / L , 50 pM / L, 25 pM / L, 12.5 pM / L, obtaining concentrations of a new tryptamine-l,3,5-triazine derivative of formula 1 in the wells: 100 pM / L, 50 pM / L, 25 pM / L , 12.5 pM / L, 6.25 pM / L.

[0051] Additionally, for each line, 50 pL of DMEM with addition of 0.2% DMSO was added to the remaining 9 wells, 50 pL of DMEM with 10% addition of FBS to the next 3 wells, and 50 pL of DMEM with 0.1% addition of triton-X (compound that kills all cells, control) into the remaining 3 wells. Such prepared culture plates were placed in an incubator for 24 hours. After 24 hours, in accordance with the manufacturer's recommendations, 20 pL of CellTiter 96® reagent was added to each well, and after 4 hours, spectrophotometric absorbance measurement was performed at a wavelength of 490 nm.

[0052] Cells incubated with DMEM with addition of 10% FBS were considered "100% viable". The number of viable cells from the remaining groups was normalized to the control and presented as % of control. For analysis, the results from 3 wells in which a given concentration was tested were averaged. The experiment was performed 4 times. IC50 was calculated, it is a measure of cytotoxic activity, i.e. the concentration that inhibits the growth of the tested cell population by 50%. GraphPad Prims 8 software was used to calculate IC50.

[0053] % cell proliferation for a given concentration was calculated according to the formula:

[0054] % cell proliferation

[0055] For the SW480 line, an IC50 of 8.596 pM / L was obtained, for SW620 it was 12.862 pM / L, and for the CCD841 line it was approximately 913.3 pM / L. The result obtained for the CCD841 line shows that the new tryptamine-l,3,5-triazine derivative of formula 1 does not affect the proliferation of normal cells, constituting the control group.

[0056] The results of the MTS proliferation test are presented in Fig. 4, where the dependence of % cell proliferation on the concentration of the new tryptamine- 1,3,5-triazine derivative of formula 1 is presented in the graphs.

[0057] Determination of the ability to inhibit FOXM1 production for the SW480 line in the ELISA test

[0058] In the ELISA test, antibodies that specifically detect the antigen (in this case FOXM1) are immobilized on the surface of the plate. The FOXM1 protein present in the material combines with the above-mentioned antibodies. Enzymatically labeled antibodies are then added, again directed against FOXM1. The amount of enzyme-labeled antibody attached corresponds to the amount of FOXM1 protein detected and is measured by enzymatic activity using a substrate that changes color when modified by the enzyme used for labelling the antibodies. The light absorption of the product formed after adding the substrate is measured spectrophotometrically and converted into numerical values.

[0059] On a flat-bottomed plate with a 1 mL working volume of wells, 0.5xl06cells of the SW480 line were seeded to 6 wells in a volume of 250 pL of culture medium for each well, and the plate was placed in an incubator for 1 hour. Then, 250 pL of the new tryptamine-l,3,5-triazine derivative of formula 1 with a concentration of 25 pM / L was added to 3 wells, thus obtaining a final concentration of 12.5 pM / L in the wells. The concentration of 12.5 pM / L of the new tryptamine-l,3,5-triazine derivative of formula 1 was determined based on the above-described results of proliferation of SW480 line cells in the presence of the test compound, for which the IC50 was 8.596 pM / L. 250 pL of culture medium was added to 3 subsequent wells. Culture plates prepared in this way were placed in an incubator for 24 hours. After 24 hours, the cultures were assessed using an inverted microscope, then the supernatant was collected from all cultures, the cells were washed with ImL of PBS (Phosphate-buffered saline), 0.5mL of trypsin was added and the plate was placed in an incubator for 10 minutes. After assessing the degree of cell detachment from the vessel surface using an inverted microscope, the trypsinization reaction was stopped by adding 10 pL of FBS. The cells were collected in an Eppendorf tube and centrifuged for 5 minutes at 300xg. The supernatant was collected and the cell pellet was suspended in 250 pL of PBS. Samples were frozen at -20°C. In order to obtain the minimum number of replicates (n=3) for statistical analysis, cell isolation cultures were repeated 3 times.

[0060] Before performing the ELISA test, cell lysis was performed using a sonicator, 3 times for 1 minute each. The ELISA test was performed using Forkhead FOX Protein Ml (FOXM1) kit (Abbexa, United Kingdom) according to the manufacturer's protocol. On the plate provided in the kit, 100 pL of standards were pipetted into 3 subsequent wells and cell lysate after culturing with the tested active substance, tryptamine-l,3,5-triazine derivative of formula 1, into 3 subsequent wells, and as a control, cell lysate after culturing in the culture medium also into 3 subsequent wells. The plate was incubated for 1 hour at 37°C. 100 pL of Detection Reagent A was added to each well. It was incubated for 1 hour at 37°C.

[0061] The plate was rinsed 3 times with the Wash Buffer included in the kit. 100 pL of Detection Reagent B was added to each well. It was incubated for 1.5 hours at 37°C. The plate was rinsed 5 times with Wash Buffer. 90 pL of TMB substrate solution was added. It was incubated for 15 minutes at 37°C. O.D. - optical density was measured at a wavelength of 450 nm. The average was taken from three repetitions.

[0062] The average results of the ELISA test are shown in Fig. 5, where A is the production of FOXM1 protein for the SW480 cell line and B for the SW480 cell line with the addition of the new tryptamine-l,3,5-triazine derivative of formula l. This confirms that the compound of formula 1 has a strong potential to inhibit the production of FOXM1 protein.

[0063] As confirmed by the performed research, the compound of formula 1 has antiproliferative properties for the SW480 and SW620 colorectal cancer lines, while remaining practically indifferent to the proliferation of the healthy colorectal epithelial cell line CCD841. Additionally, this compound has a strong FOXM1 inhibitory potential.

Claims

Zastrzezenia patentowe1. A new derivative of tryptamine-l,3,5-triazine, which is N2-(2-(lH-i ndol-3- yl)ethyl)-6-morpholino-N4-phenethyl-l,3,5-triazine-2,4-diamine hydrochloride of formula 1.

2. A new tryptamine-l,3,5-triazine derivative according to claim 1 for use in the treatment of colorectal cancer.

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