CZ202322A3 - A heterocyclic compound for the inhibition of growth of malign tumours - Google Patents

Abstract

The presented solution provides derivatives of 1,1-dioxidobenzo[b]thiophene, which are growth inhibitors of malignant cell lines. It also describes the method of their preparation and their therapeutic use.

Description

A heterocyclic compound for inhibiting the growth of malignant tumors

Field of technology

The invention relates to a covalent inhibitor of the protein STAT3 (Signal Transducer and Activator Of Transcription 3), its use in inhibiting the growth of malignant cell lines and its therapeutic use.

Current state of the art

The protein STAT3 (Signal Transducer and Activator Of Transcription 3) is a transcription factor that transmits extracellular signals to the cell nucleus, and then the transcription of target genes is activated. The STAT3 protein has been identified as a possible molecular target due to its role in the initiation and growth of tumor tissue (Levy D. E. et al. Nat Rev Mol Cell Biol. 2002;3:651).

For this reason, efforts have been made to find inhibitors of the STAT3 protein, thereby preventing the growth of malignant cells. Compounds affecting STAT3 can be divided into non-covalent and covalent inhibitors.

Non-covalent inhibitors are usually targeted at inhibiting the SH2 domain, inhibiting the DNA binding domain of STAT3, or have been identified as other non-covalent inhibitors. Non-covalent STAT3 inhibitors aimed at inhibiting the SH2 domain can be classified into several structural types, i.e. phosphate inhibitors, salicylic acid analogues, 4-naphthoquinone derivatives, phenylsulfonamides, aminotetrazole analogues, quinoline analogues, carboxylic acid ester analogues or carboxylic acid analogues. Non-covalent inhibitors of STAT3 aimed at inhibiting the DNA binding domain were derived, for example, from niclosamide or the compound InS3-54. Other non-covalently acting inhibitors are benzyloxyphenyl analogues.

Covalent inhibitors of STAT3 are targeted at reactive cysteine residues of the protein, with which it forms an irreversible bond. In 2006, the molecule Stattic was identified, which was able to inhibit the function of the SH2 domain of STAT3 (Schust J. et al. Chem Biol. 2006;13:1235). Using mass spectrometry, the binding of Stattic to the cysteine residues of STAT3 was verified, and thus its mechanism of action was determined (Heidelberg S. et al. Bioorg Med Chem Lett. 2013;23:4719).

Static

In 2013, Chen et al. reported derivatives of Stattic, where the nitro group was replaced by a carboxamide, with the ability to inhibit the growth of human malignant lines. Some carboxamides have shown activity against the human cell line MDA-MB-231 (breast adenocarcinoma), but their biological activity has not been directly compared with the model molecule Stattic (Chen et al. Eur J Med Chem. 2013;62:498).

OH

Br^ .N

CN

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In 2014, Chen et al. extended these findings to structurally modified carboxamides structurally derived from 5-chloro-2-hydroxybenzoic acid and prepared their 2-alkoxy analogues. Some compounds showed activity comparable to the model molecule Stattic on the human cell line MDA-MB-231 (Chen et al. Eur J Med Chem. 2014; 82:195).

Cl

In 2017, Zhang et al. published Stattic derivatives based on modification with a secondary amine at position 6 or 7. Some compounds showed comparable or slightly improved antiproliferative activity compared to the model molecule Stattic on the human cell line MDA-MB-231 (Zhang W. et al. Eur J Med Chem. 2017;125:538).

The essence of the invention

The subject of this invention are inhibitors of the proliferative activity of malignant cells.

This invention relates to 6-((4-methoxybenzyl)amino)benzo[ b ]thiophene-1,1-dioxide, its pharmaceutically acceptable acid addition salts.

Further, the subject of the invention is 6-((4-methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide, its pharmaceutically acceptable acid addition salt for use as a medicine, especially for the treatment of malignant tumors. The substance according to the invention is an inhibitor of the proliferative activity of malignant cells.

The subject of the invention is also a pharmaceutical preparation containing 6-((4methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide, its pharmaceutically acceptable acid addition salt, and at least one pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include, in particular, fillers, binders, slip agents, solvents, disintegrants. Particularly suitable pharmaceutically acceptable excipients are fillers such as carbohydrates, starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidine, alginic acid and its salts, solvents, binders.

Clarification of the drawing

fig. 1 and 2 show the results of determining the ability of glioblastoma lines to form colonies for the compound K2071 (a compound according to the invention) and for the comparison substance Stattic. fig. 1 is a picture of culture dishes, Fig. 2 is a graph of the calculated area of a colony of cells after treatment with the tested compound and the comparator.

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Examples of the implementation of the invention

The invention is described in the following examples, which do not limit its scope in any way. The starting raw materials are available from commercial sources.

Example 1: Preparation of the compound according to the invention

Preparation procedure:

To anhydrous methanol (8.3 mL) were added 4 Å molecular sieves corresponding to the aldehyde (0.91 mmol) and anhydrous CH3COOH (0.91 mmol) under N2 atmosphere. The reaction mixture was stirred for 30 min at room temperature, then 6-amino-1,1-dioxobenzo[b]thiophene (0.83 mmol) was added and the mixture was heated to 50 °C for 4 h. After subsequent cooling to room temperature, NaBH^CN (2.06 mmol) was added to the mixture, after which it was stirred at room temperature for 19.5 hours. The mixture was filtered through a frit to remove molecular sieves, the solvent was evaporated under reduced pressure and the residue was quantitatively transferred to a separatory funnel using dichloromethane (60 ml) and distilled water (60 ml). After shaking, the organic phase was separated and the aqueous phase was extracted with three portions of dichloromethane (40 ml each). The combined organic phases were washed with saturated aqueous NHCO3 (100 mL), then saturated aqueous NaCl (100 mL), dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified using a flash chromatography system using a mobile phase consisting of a mixture of heptane/ethyl acetate and a proportion of secondary components in the range of 30 to 80%. The final molecule was obtained by evaporation of the solvents.

6-((4-Methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide (K2071)

Appearance: yellow solid, yield: 61%, mp: 125 to 127 °C. 1H NMR (500 MHz, CDCb) δ: 3.81 (3H, s, CH3O), 4.30 (2H, s, CH2), 4.58 (1H, bs, NH), 6.43 (1H, d , Jh,h = 6.7 Hz, Ar), 6.61 (1H, dd, Jh,h = 8.2 Hz, Jh,h = 2.3 Hz, Ar), 6.87 and 6.91 ( 2H, m, Ar), 6.93 az to 6.97 (1H, m, Ar), 7.09 (1H, d, Jh,h = 8.2 Hz, Ar), 7.09 (1H, dd, Jh,h = 6.8 Hz, Jh,h = 0.9 Hz, Ar), 7.23 and 7.26 (2H, m, Ar). ¹³ C NMR (126 MHz, CDCb) δ: 47.5 (CH 2 ), 55.5 (CH 3 O), 106.1, 114.4, 115.4, 119.5, 126.1, 126.5, 128 .9, 129.7, 133.4, 138.9, 150.5, 159.3. HRMS (ESI) calcd for C16H16NO3S+ [M+H]+ 302.0851, found 302.0846.

Example 2: Testing compounds for inhibition of the MDA-MB-231 cell line

The prepared strip and the Stattic reference strip were tested for growth inhibition of the human breast adenocarcinoma-derived cell line MDA-MB-231 (purchased from the American Type Culture Collection, ATCC). The cell line was propagated in DMEM cell culture medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum, 4 mM GlutaMAX™ and penicillin/streptomycin (Thermo Fisher Scientific, MA, USA) in a 5% CO2 humidified atmosphere at 37 °C in a monolayer. Before the viability test, cells were harvested, counted and seeded in 384-well plates (Corning Inc., NY, USA) at 1000 cells/well in 20 μl of total medium volume. Test compounds were diluted in DMSO and were transferred to the respective wells using non-contact acoustic transfer using an ECHO 655 (Labcyte, Inc., USA) integrated in a fully automated robotic HTS station (HighRes® Biosolutions, USA). Compounds were tested in a concentration range of 80 to 0.045 μΜ, in triplicate. Cells were incubated with the compounds for 72 hours. Cell viability was assessed by determining the level of intracellular ATP using the CellTiter-Glo® luminescence test (Promega, USA). The luminescence signal was measured on an Envision multimode plate reader (Perkin Elmer, USA). The data were normalized and processed using the ScreenX LIMS system. The resulting relative inhibition of the growth of the malignant cell line MDA-MB-231 is shown in Table 1.

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Table 1: Relative growth inhibition of the malignant cell line MDA-MB-231 in the presence of Stattic derivatives.

Compound number R ED50 (μΜ) Static (cf.) NO2 18.74 K2071 (4-Methoxybenzyl)amino 2.41

Example 3: Determination of the ability of glioblastoma lines to form colonies

Human glioblastoma cell lines A172, T98 and U87 were seeded at the same number of cells (12,500 cells per cm ² ). 24 hours after seeding, cells were exposed to a 5 μΜ concentration of either compound K2071 (a compound of the invention) or the comparator Stattic for 24 hours.

Cell cultures were then harvested and 10,000 (A172) or 4,000 cells (T98 and U87) were seeded per 20 cm ² dish. Seeding was performed in triplicate. Grown colonies were detected after 14 days of growth using crystal violet solution (0.05% crystal violet; 1x PBS buffer; 1% formaldehyde; 1% methanol; incubation for 20 minutes at room temperature, followed by 5 ^Z water rinses). Plates with colonies were scanned and colony area was calculated in the FiJi program. The results are shown in Fig. 1 (culture dishes after staining with crystal violet) and Fig. 2 (total colony area). The values used are from two biological replicates.

Claims (5)

1. 6-((4-Methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide or a pharmaceutically acceptable acid addition salt thereof. 5 2. 6-((4-Methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide according to claim 1 or a pharmaceutically acceptable acid addition salt thereof for use as a medicament. 3. 6-((4-Methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide according to claim 1 or a pharmaceutically acceptable acid addition salt thereof for use in the treatment of malignant tumors. 4. 6-((4-Methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide or a pharmaceutically acceptable derivative thereof 10 acid addition salt for use according to claim 2 for inhibiting the proliferative activity of malignant cells. 5. Pharmaceutical preparation, characterized in that it contains 6-((4-methoxybenzyl)amino)benzo[b]thiophene-1,1-dioxide according to claim 1 or its pharmaceutically acceptable acid addition salt, and at least one pharmaceutically acceptable auxiliary bar.