RU2765545C1 - 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1h-pyrrol-3-carboxamide causing irregularities in the phases of the cell cycle and exhibiting cytotoxic activity against soft tissue sarcomas and gastrointestinal stromal tumours (gist) - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to an application of 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrol-3-carboxamide I by the formula below for producing an anti-tumour medicinal product.EFFECT: compound exhibits cytotoxic activity against soft tissue sarcomas and gastrointestinal stromal tumours.1 cl, 3 dwg, 2 tbl

Description

The invention relates to the field of medicine, derivatives of pyrrole, namely the new biologically active 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole- 3-carboxamide (2-ABK) I formula:

which can be used in medicine as an anticancer drug.

The closest structural analogue to the claimed compound are compounds of general formula II, exhibiting antitumor activity ethyl esters of 2-amino-1-benzoylamino-4-oxo-5-(2-oxo-2-aryl-ethylidene)-4,5-dihydro-1H -pyrrolidine-3-carboxylic acids [Patent No. 2607920 Russian Federation, IPC C07D 417/00, A61K 31/195. Ethyl esters of 2-amino-1-benzoylamino-4-oxo-5-(2-oxo-2-aryl-ethylidene)-4,5-dihydro-1H-pyrrolidine-3-carboxylic acids exhibiting antitumor activity, and their method receipt: No. 2015120527: dec. 05/29/2015: publ. 01/11/2017 / S.S. Zykova, N.M. Igidov, M.V. Kiselev, S.V. Boychuk, A.R. Galembikova]:

As a comparison standard, we took the anticancer drug - paclitaxel [Patent No. 2059631, IPC C07D 305/14, A61K 31/195. Taxol derivatives and a pharmaceutical composition having antitumor activity: No. 91 5010248: Appl. 11/29/1991: publ. 05/10/1996 / Valentina J. Stella, Abraham E. Matthew].

The claimed compound 2-ABPC in the acute toxicity test on mice when administered intraperitoneally in the form of a suspension in 2% starch mucus has a toxicity of more than 1000 mg/kg [Kolla V.E., Syropyatov B.Ya. Doses of drugs and chemical compounds for laboratory animals, Moscow: Medicine. - 1998. - 263 p.].

The aim of the present invention is the synthesis of a new, not previously described 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxamide ( 2-ABPC) (I) exhibiting cytotoxic activity against soft tissue sarcomas and gastrointestinal stromal tumors (GIST).

This goal is achieved by obtaining 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxamide (I) by the interaction of N-( 5-(tert-butyl)-2-oxofuran-3(2H)-ylidene)benzohydrazide (II) with cyanoacetamide in the presence of triethylamine in toluene medium according to the scheme:

Method for the preparation of 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxamide (I). A mixture of 1.36 g (0.005 mol) of N-(5-(tert-butyl)-2-oxofuran-3(2Н)-ylidene)benzohydrazide (II), 0.42 g (0.005 mol) of cyanoacetamide, and 0.5 g (0.005 mol) of triethylamine was boiled in 60 ml of benzene for 25 minutes. The precipitate formed on cooling was filtered off and recrystallized from ethanol. Received 0.99 g (yield 56%) of crystalline substance (I) with T _pl 230-231°C. C ₁₈ H ₂₀ N ₄ O ₄ . IR spectrum, v/cm ^-1 , liquid paraffin: 3408 (NH), 3318 (NH), 3204 (NH), 1679 (C=O), 1648 (C=O), 1591 (C=C). 1H NMR spectrum ⁽ DMSO-d ₆ ), δ, ppm: 1.02 s (9H, C(CH ₃ ) ₃ ), 6.42 s (1H, CH), 7.01 br. s (1H, NH), 7.34 br. s (1Н, NH), 7.65 m (5Н, arom.), 8.70 br. s (1Н, NH), 9.15 s (1H, NH), 10.86 s (1Н, NH).

13C NMR spectrum, δ, ppm, DMSO-d ₆ : ^25.98 , 87.47, 101.80, 128.02, 128.05, 131.16, 132.08, 141.20, 164.90, 165.52, 167.01, 179.99, 204.97.

Mass spectrum, m/z (I _rel ., %): 356 (74.7) [M]+., 300 (19.2) [M-2CO)]+, 299 (38.9) [M-t-BuCO]+, 282 (23.7) [М-2СО-Н ₂ O]+, 271 (12.0) [Mt-BuCO]+, 105 (100.0) [PhCO]+, 77 (20.9) [Ph]+, 271 (14.3) [t -Bu]+.

The claimed compound is a light yellow crystalline substance, soluble in DMSO, DMF, acetic acid, when heated - in ethyl alcohol, stable during storage.

The invention is supported by the following drawings, which show:

In FIG. 1 - photomicrographs of GIST cells (GIST T1-R) incubated with 2-ABD (10 μM) and paclitaxel (1 μM) for 24 hours.

In FIG. 2 - cleaved forms of caspase-3 and PARP, phosphorylated forms of histones 2AX and 3 in GIST T1-IM-R (left panel) and SK-LMS-1 (right panel) cell lines incubated for 48 hours with 2-ABD ( 5 and 10 μM), paclitaxel (1 μM) and vinblastine (0.01 μM). β-actin was used as a loading control.

In FIG. 3 - cell cycle flow cytometry histograms of the GIST T1-IM-R line incubated with 2-ABK (10 μM) and paclitaxel (1 μM) for 48 hours.

According to the results of microscopy of cell lines of soft tissue sarcomas, including leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma (SK-LMS-1, RD, HT1080, respectively), and GIST (GIST T1-R), the following morphological change was revealed - the transition of the cell shape to a rounded . This may indicate a delay of cells in the mitosis phase. Corresponding changes were found when cells were incubated with paclitaxel (comparator drug), which initiates cell death by apoptotic mechanism (Fig. 1).

Half inhibitory concentrations (IC50) for 2-ABD, determined using the MTS test against soft tissue sarcoma cell lines (SK-LMS-1, RD, HT1080), as well as naive, imatinib- and paclitaxel-resistant GIST cell lines ( GIST T1, GIST T1-R, GIST 430 and GIST Tx-R) and non-transformed human fibroblasts (BJ tert) are presented in Table 1. The high cytotoxic activity of the studied compound against a number of tumor cell lines, including paclitaxel-resistant GISTs, compared to non-transformed human fibroblasts, indicates a wide therapeutic window for 2-ABD, which makes it a potential candidate for the treatment of tumors with acquired resistance to paclitaxel.

Cell death by apoptosis was confirmed by immunoblotting, as evidenced by elevated levels of cleaved forms of caspase-3 and poly-ADP-ribose polymerase (PARP) in GIST T1-IM-R and SK-LMS-1 cell lines treated with 2- ABPK, paclitaxel and vinblastine (positive control). It is important to note that the severity of 2-ABD-induced apoptosis was significantly higher compared with paclitaxel and vinblastine. Apoptosis of cells incubated with 2-ABD can be caused either by the induction of DNA double-strand breaks, which is evidenced by an increased level of the phosphorylated form of histone 2AX (Fig. 2, left panel), or by the phenomenon of "mitotic catastrophe", a marker of which is an increase in the level of the phosphorylated form of histone 3. (Fig. 2, right panel).

Using flow cytometry (Flow Cytometer BD FACSCanto II), the cell cycle analysis of GIST T1-IM-R cells was performed. Thus, in samples incubated for 48 hours with 2-ABD and paclitaxel (positive control), an increased number of hypodiploid cells (subG1) was found, which indicates the pro-apoptotic activity of 2-ABD.

Representative flow cytometry histograms (FIG. 3) illustrate the cell cycle distribution of T1-IM-R GISTs incubated with 2-ABD (10 μM) and paclitaxel (1 μM) for 48 hours. The experiment was performed three times, in each case at least 150,000 cells were counted. Apoptosis of cells incubated in the presence of 2-ABD and paclitaxel is indicated by the subG1 peak (arrowed). The results of the quantitative assessment are shown in table 2.

Thus, the results of the studies performed indicate that 2-ABK exhibits cytotoxic and proapoptotic activity against soft tissue sarcomas and gastrointestinal stromal tumors (GISTs).

Claims (3)

Application of 2-amino-1-benzamido-5-(3,3-dimethyl-2-oxobutylidene)-4-oxo-4,5-dihydro-1H-pyrrole-3-carboxamide I formula

to obtain an anticancer drug.

Images