RU2607920C2 - Ethyl ethers of 2-amino-1-benzoylamino-4-oxo-5-(2-oxo-2-aryl-ethylidene)-4,5-dihydro-1h-pyrrolidine-3-carboxylic acids, having anti-tumour activity and method for production thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of general formula Ia-d, where R=CH₃O (Ia), C₂H₅O (Ib), Br (Ic), Cl (Id), which have anticancer activity. Also disclosed is a method of producing compounds of formula Ia-d and their application for producing drug preparations for treating oncological diseases.

EFFECT: compounds exhibit low toxicity.

3 cl, 4 dwg, 1 tbl, 6 ex

Description

The invention relates to the field of medicine and organic chemistry, namely to drugs for the treatment of tumor diseases.

The closest structural analogues of the claimed compounds are known, obtained by the interaction of 5-aryl-3-arylimino-3H-furan-2-ones with ethyl cyanoacetic acid in the presence of triethylamine [Kharitonova SS, Igidov NM, Zakhmatov AV , Rubtsov A.E. Chemistry of iminofurans VIII *. Recyclization of 5-aryl-3-arylimino-3H-furan-2-ones under the action of cyanoacetic acid derivatives // Zh. Organ. chemistry. 2013.V. 49. - Issue. 2. - S. 252-261]. The synthesis of the nearest structural analogues is carried out according to the following scheme:

The antitumor activity of the nearest structural analogues has not been previously studied.

The synthesis of the starting precursors of the new compounds Ia-3-benzoylhydrazones 5-aryl-2,3-dihydro-2-furandiones (III) was carried out by a known method [Tyuneva A.V., Igidov N.M., Koryagina N.N., Borodin A.Yu., Zakhmatov A.V., Makarov A.S., Toksarova Yu.S., Rubtsov A.E. Chemistry of iminofurans. VII *. Intramolecular cyclization of 2-N-aryl substituted derivatives of 4-aryl-4-oxobut-2-enoic and 2-amino-5,5-dimethyl-4-oxohex-2-enoic acids // Zh. Organ. chemistry. 2011.V. 46. Issue. 2. S. 258-264].

Derivatives of Taxol

(RF patent No. 2059631, IPC C07D 305/14, A61K 31/195, publ. 05/10/1996).

Derivatives of amidoximes of propencarboxylic acids of the formula

compound of formula Ia

a compound representing an oxadiazoline derivative of the formula Ib

a method for producing an amidoxime derivative of a propencarboxylic acid of formula Ia, the method comprising reacting an oxadiazoline derivative of formula Ib with an aqueous alkali solution; if necessary, the resulting compound of formula Ia is reacted with an inorganic or organic acid to obtain a pharmaceutically acceptable acid addition salt, or the free base is isolated from its acid addition salt (Patent No. 2264387, IPC C07D 207/325, C07D 271/06, C07D 273/02, C07D 413/06, C07C 251/40, A61K 31/40, A61K 31/535, A61K 31/16, A61P 25/00, published on November 20, 2005), which are closest in technical essence and adopted as prototypes .

The objective of the invention is to obtain new compounds Ia-g, which can be used in medicine to treat or prevent the growth of tumor cells, as well as the development of a method for the synthesis of previously undescribed ethyl esters of 2-amino-1-benzoylamino-4-oxo-5- (2- oxo-2-arylethylidene) -4,5-dihydro-1H-pyrrolidine-3-carboxylic acids (Ia-g), which exhibit antitumor activity and have lower toxicity than the antitumor drug taxol.

The technical result is an increase in the antitumor activity of the compound, as well as a decrease in toxicity.

The invention is a derivative of pyrrole, namely, new ethyl esters of 2-amino-1-benzoylamino-4-oxo-5- (2-oxo-2-arylethylidene) -4,5-dihydro-1H-pyrrolidine-3-carboxylic acids formulas (I), as well as their pharmaceutically acceptable salts, which include non-toxic organic or inorganic salts, in particular hydrochlorides, sulfates, hydrosulfates, nitrates, phosphates, lactates, acetates, citrates, benzoates, succinates, tartrates and the like:

showing antitumor activity.

The problem is achieved by the interaction of 3-benzoylhydrazones 5-aryl-2,3-dihydro-2-furandiones (III) with cyanacetic ether in the presence of triethylamine in toluene medium according to the scheme:

The technical result is achieved with ethyl esters of 2-amino-1-benzoylamino-4-oxo-5- (2-oxo-2-arylethylidene) -4,5-dihydro-1H-pyrrolidine-3-carboxylic acids of the general formula Ia-g:

namely 2-amino-1-benzoylamino-4-oxo-5- [2- (4-methoxyphenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid ethyl ester (Ia), 2-amino-1-benzoylamino-4-oxo-5- [2- (4-ethoxyphenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid ethyl ester (Ib), ethyl ether 2-amino-1-benzoylamino-4-oxo-5- [2- (4-bromophenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid (Ic) and 2- ethyl ether amino-1-benzoylamino-4-oxo-5- [2- (4-chlorophenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid (Ig), which exhibits antitumor activity.

The technical result is also achieved by the method of producing ethyl esters of 2-amino-1-benzoylamino-4-oxo-5- (2-oxo-2-arylethylidene) -4,5-dihydro-1H-pyrrolidine-3-carboxylic acids of the general formula Ia- g:

consisting in the fact that 3-benzoylhydrazones of 5-aryl-2,3-dihydro-2-furandiones (III) are reacted with cyanacetic ether in the presence of triethylamine in toluene for 20 minutes and the precipitate formed is filtered off and recrystallized from a mixture of solvents benzene: ethanol (2: 1).

The technical result is also achieved by the use of compounds for the production of drugs for the treatment of cancer.

The invention is illustrated by figure 1, which shows a graph of the antitumor activity of compounds Ia-g and comparison drugs taxol and etoposide in relation to human osteosarcoma cells U2 OS (48 hours incubation with drugs) (the ordinate shows the number of viable cells (in%) from control “K.” On the ordinate axis is the concentration (in μM) of the studied compounds and preparations comparing taxol and etoposide).

Examples of a preferred embodiment of the invention.

Example 1. Synthesis of the inventive ethyl ester of 2-amino-1-benzoylamino-4-oxo-5- [2- (4-methoxyphenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid Ia

A mixture of 1.61 g (0.005 mol) of 3- benzoylhydrazone 5- (4-methoxyphenyl) -2,3-dihydro-2-furandione (III), 0.57 g (0.005 mol) of ethyl cyanoacetic acid and 0.5 g (0.005 mol) of triethylamine in 60 ml of toluene for 20 minutes. After a day, the precipitate formed was filtered off and recrystallized from a benzene-ethanol mixture (2: 1). Received 1.44 g (66%) of crystalline substance Ia with T _PL 259-261 ° C. C ₂₃ H ₂₁ N ₃ O ₆ . IR spectrum, ν / cm ^-1 , liquid paraffin: 3252 (NH), 1705 ( СО ОС ₂ Н ₅ ), 1656.1606 (С = O). ¹ H NMR spectrum (DMSO-d ₆ ), δ, ppm: 1.27 t (3Н, СН ₂ СН3 ), 3.77 s (3Н, СН ₃ О), 4.13 q (2Н, СН2 СН3), 6.53 s ( 1H, CH), 6.86 d, 7.23-7.66 m (9H, C ₆ H ₅ , C ₆ H ₄ ), 8.30 br. s (1H, NH), 9.03 br. s (1H, NH). 10.80 ears. s (1H, NH).

Example 2. Synthesis of the inventive ethyl ester of 2-amino-1-benzoylamino-4-oxo-5- [2- (4-ethoxyphenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid Ib

A mixture of 1.68 g (0.005 mol) of 3- benzoylhydrazone 5- (4-ethoxyphenyl) -2,3-dihydro-2-furandione (III), 0.57 g (0.005 mol) of ethyl cyanoacetic acid ester and 0.5 g (0.005 mol) of triethylamine in 60 ml of toluene for 20 minutes. After a day, the precipitate formed was filtered off and recrystallized from a benzene-ethanol mixture (2: 1). Received 1.68 g (75%) of crystalline substance IB with T _PL 250-252 ° C. C ₂₄ H ₂₃ N ₃ O ₆ . IR spectrum, ν / cm ^-1 , liquid paraffin: 3250 (NH), 1699 ( СО OS ₂ Н ₅ ), 1658.1600 (С = O). ¹ H NMR spectrum (DMSO-d ₆ ), δ, ppm: 1.29 t (3H, CH ₂ CH3 ), 1-36 t (3H, OCH ₂ CH3 ), 4.09 q (2H, OCH 2 CH ₃ ), 4.18 q (2H, CH2 CH ₃ ), 6.60 s (1H, CH), 6.86-7.88 m (9H, C ₆ H ₅ , C ₆ H ₄ ,), 8.41 s (1H, NH), 9.17 s (1H, NH). 10.95 s (1H, NH).

Example 3. Synthesis of the inventive ethyl ester of 2-amino-1-benzoylamino-4-oxo-5- [2- (4-bromophenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid Ic

A mixture of 1.85 g (0.005 mol) of 3- benzoylhydrazone 5- (4-bromophenyl) -2,3-dihydro-2-furandione (III), 0.57 g (0.005 mol) of ethyl cyanoacetic acid ester and 0.5 g (0.005 mol) of triethylamine in 60 ml of toluene for 20 minutes. After a day, the precipitate formed was filtered off and recrystallized from a benzene-ethanol mixture (2: 1). Received 1.79 g (74%) of crystalline substance Ib with T _PL 282-284 ° C. C ₂₂ H ₁₈ BrN ₃ O ₅ . IR spectrum, ν / cm ^-1 , liquid paraffin: 3267 (NH), 1707 ( СО ОС ₂ Н ₅ ), 1661.1593 (С = O). ¹ H NMR spectrum (DMSO-d ₆ ), δ, ppm: 1.23 t (3Н, СН ₂ СН3 ), 4.11 kv (2Н, СН2 СН ₃ ), 6.54 s (1Н, СН), 7.09-7.65 m (9H, C ₆ H ₅ , C ₆ H ₄ ), 8.38 br. s (1H, NH), 9.09 broad. s (1H, NH). 10.93 ears s (1H, NH)

Example 4. Synthesis of the inventive ethyl ester of 2-amino-1-benzoylamino-4-oxo-5- [2- (4-chlorophenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid Ig

A mixture of 1.63 g (0.005 mol) of 3- benzoylhydrazone 5- (4-chlorophenyl) -2,3-dihydro-2-furandione (III), 0.57 g (0.005 mol) of ethyl cyanoacetic acid ester and 0.5 g (0.005 mol) of triethylamine in 60 ml of toluene for 20 minutes. After a day, the precipitate formed was filtered off and recrystallized from a benzene-ethanol mixture (2: 1). Received 1.41 g (64%) of crystalline substance 1 g with a _{mp of} 249-251 ° C. C ₂₂ H ₁₈ ClN ₃ O ₅ . IR spectrum, ν / cm ^-1 liquid paraffin: 3269 (NH), 1704 ( СО ОС ₂ Н ₅ ), 1660.1591 (С = O). ¹ H NMR spectrum (DMSO-d ₆ ), 5, ppm: 1.24 t (3Н, СН ₂ СН3 ), 4.12 kv (2Н, СН2 СН ₃ ), 6.59 s (1Н, СН), 7.26-7.67 m (9H, C ₆ H ₅ , C ₆ H ₄ ), 8.39 s, (1H, NH), 9.13 s (1H, NH). 10.92c (1H, NH).

The obtained compounds Ia-d are yellow crystalline substances soluble in DMSO, dimethylformamide, sparingly soluble in alcohol, toluene and insoluble in water.

Example 5. Determination of acute toxicity of compounds Ia-g. Acute toxicity with intraperitoneal administration was determined on white nonlinear male mice weighing 18-22 g. The test substance and the reference drug were injected into the abdominal cavity as a solution in 2% starch mucus at the rate of 0.1 ml per 10 g of animal weight in increasing doses. All animals were in standard vivarium conditions. The results were processed according to Prozorovsky with the calculation of the average lethal dose (LD ₅₀ ) at p <0.05 (Table 1).

Example 6. Antitumor activity of compounds Ia-g.

A pronounced dose-dependent cytotoxic effect of these compounds is shown (Fig. 1), at doses of 10 and 20 μM, significantly exceeding the effect of the reference drug (paclitaxel), as well as the well-known antitumor drug etoposide, which is an inhibitor of type II topoisomerase. The effect was found on tumor cell lines of gastrointestinal stromal tumors GISO-882 and T-1, osteosarcoma U2-OS, lung cancer H1299, cervical cancer HeLa S3.

One of the mechanisms of action of these compounds is the induction of DNA damage, namely double-strand breaks, as evidenced by the accumulation of the phosphorylated form of histone 2A (γ-H2AX) in the GISO-T1 cell tumor line (Fig. 2). The effect of DNA damage was comparable to the chemotherapy drug doxorubicin and the targeted drug imatinib. Similar results indicating the ability of compounds 1a-d to induce DNA damage were obtained using all of the above cell tumor lines. The result of DNA damage in tumor cells was the activation of repair mechanisms, manifested in an increase in the expression level of phosphorylated forms of ATM kinase and p53 protein (pATM and pP53, respectively) (Fig. 2), as well as activation of restriction points leading to selective accumulation of cells in G2 / M phases of the cell cycle (Fig. 3). Subsequently, tumor cells died by the apoptosis mechanism, as evidenced by an increase in the expression level of the cleaved form of poly-ADP (ribose) polymerase (PARP), which is a consequence of caspase-3 activation (Fig. 2), as well as light microscopy data (Fig. 4 )

The synthesized compounds Ia-g are classified as low-toxic substances and are significantly less toxic in comparison with taxol [Koll V.E., Syropyatov B.Ya. Doses of drugs and chemical compounds for laboratory animals, M .: Medicine, 1998, 263 S.]

Claims (9)

1. Ethyl esters of 2-amino-1-benzoylamino-4-oxo-5- (2-oxo-2-arylethylidene) -4,5-dihydro-1H-pyrrolidine-3-carboxylic acids of the general formula Ia-g:

where R = CH ₃ O (Ia), C ₂ H ₅ O (Ib), Br (Ib), Cl (Ig), namely 2-amino-1-benzoylamino-4-oxo-5- [2- (4-methoxyphenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid ethyl ester (Ia), 2-amino-1-benzoylamino-4-oxo-5- [2- (4-ethoxyphenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid ethyl ester (IB), ethyl ether 2-amino-1-benzoylamino-4-oxo-5- [2- (4-bromophenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid (Ic) and 2- ethyl ester amino-1-benzoylamino-4-oxo-5- [2- (4-chlorophenyl) -2-oxoethylidene] -4,5-dihydro-1H-pyrrole-3-carboxylic acid (Id) exhibiting antitumor activity. 2. A method of obtaining ethyl esters of 2-amino-1-benzoylamino-4-oxo-5- (2-oxo-2-arylethylidene) -4,5-dihydro-1H-pyrrolidine-3-carboxylic acids of the general formula Ia-g:

where R = CH ₃ O (Ia), C ₂ H ₅ O (Ib), Br (Ib), Cl (Ig), consisting in the fact that 3-benzoylhydrazones of 5-aryl-2,3-dihydro-2-furandiones (III) are reacted with cyanoacetate in the presence of triethylamine in toluene for 20 minutes and the precipitate formed is filtered off and recrystallized from a mixture of solvents benzene: ethanol (2: 1). 3. The use of compounds according to any one of paragraphs. 1-2 to obtain drugs for the treatment of cancer.

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