PL229106B1 - Bromides of butanamide 2-cyano-3-oxy-N-substituted derivatives and method for producing them - Google Patents

Abstract

The subject of the invention are bromides of 2-cyano-3-oxy-N-substituted butanamide derivatives of the general formula 1, where X is an aliphatic substituent, respectively ethyl, butyl, hexyl, octyl, dodecyl. Compounds of the general formula 1 are obtained in the acylation reaction of the appropriate 4-[(2-triphenylphosphino)alkyloxy]benzylamine bromides with ethyl 2-cyanoacetoacetate, the reaction is carried out in a polar solvent environment at room temperature for 4 - 6 hours, using the molar proportions 1: 1. After completion of the reaction, the precipitate is filtered off and purified by crystallization from a mixture of methanol-propan-2-ol. Derivatives of the general formula 1, where X is an aliphatic substituent, respectively ethyl, butyl, hexyl, octyl, dodecyl bromide for use in the treatment of neoplastic diseases.

Description

The subject of the invention are bromides of 2-cyano-3-oxy-N-substituted butanamide derivatives of the general formula 1, where X is an aliphatic substituent, respectively ethyl, butyl, hexyl, octyl, dodecyl, and a method for their preparation and use.

The known art describes a pharmaceutical agent called Leflunomide, which belongs to the group of drug precursors. It is only in vivo that it is converted to the therapeutically active metabolite of Leflunomide - Triflunomide, which has immunomodulatory, anti-proliferative, T-lymphocyte and anti-inflammatory properties. Triflunomide contains a 2-cyano-3-oxybutanamide moiety in its molecule. [patent EP 2004149 A1; Dieter Ruchatz, April 13, 2007].

The compounds obtained according to the invention were subjected to cytotoxicity assessment against selected cancer cell lines: myeloma (RPMI8226), acute myeloblastic leukemia (Kasumi 1), lung squamous cell carcinoma (NCIH2170) and gastric cancer (MKN74) as well as normal cells: hepatocytes and cardiomyocytes (H2C9). One of the strategies aimed at facilitating the delivery of drugs to the mitochondrion is the addition of a triphenylphosphine moiety [Fulda S., Galluzzi L., Kroemer G.: Targeting mitochondrial for cancer therapy. Nat Rev Drug Discov. 2010; 9: 447-464]. Chemical compounds with triphenylphosphine in their structure and forming lipophilic cations easily penetrate the mitochondrial membrane, made of a double layer of phospholipids. Hydrophilic cations are difficult to pass through biological membranes because in this process they use large amounts of energy needed to transfer ions from the hydrophilic environment to the hydrophobic interior of the lipid membrane. In the case of lipophilic cations, the activation energy is lower and the process is easier [Murphy M.P.: Targeting lipophilic cations to mitochondria. Biochemica et Biophysica Acta 2008; 1777: 1028-1031].

The new compounds of the invention are 2-cyano-3-oxy-N{4-[(triphenylphosphino)alkyloxy]benzyl}butanamide bromides of the general formula I, where X is an aliphatic substituent, respectively ethyl, butyl, hexyl, octyl, dodecyl.

Compounds of general formula 1, where X is an aliphatic substituent, respectively ethyl, butyl, hexyl, octyl, dodecyl, are obtained by acylation of the corresponding 4-[(2-triphenylphosphino)alkyloxy]benzylamine bromides of general formula 2,2-cyanoacetoacetate with ethyl Formula 3, the reaction is carried out in a polar solvent environment, preferably ethyl, methyl, propyl alcohol, at room temperature for 4-6 hours, using a 1:1 molar ratio. After completion of the reaction, the precipitate is filtered off and purified preferably by crystallization from the polar solvent propan-2-ol of a mixture of methanol-propan-2-ol in a ratio of 1:3.

The essence of the invention is also the use of a derivative of Leflunomide's metabolite - Triflunomide (A771726), containing in its structure triphenylphosphine of the formula 1 in the treatment of cancer, in particular myeloma and lung squamous cell carcinoma, acute myeloblastic leukemia.

Results of pharmacological tests on the example of derivatives: 2-cyano-3-oxy-N{4-[(triphenylphosphino)hexyloxy]benzyl}butanamide bromide, 2-cyano-3-oxy-N{4-[(triphenylphosphino)octyloxy] bromide benzyl}butanamide, 2-cyano-3-oxy-N{4-[(triphenylphosphino)dodecyloxy]benzyl}butanamide bromide are presented in Table No. 2.

Example 1: In a 100 cm ³ conical flask, 1.81 g (4 mmol) of a solution of 4-[(2-triphenylphosphino)ethoxy]benzylamine bromide in 30 cm ³ of ethyl alcohol was placed, to which an ethanolic solution of 2-cyanoacetoacetate was added in portions ethyl alcohol, in the amount of 0.62 g (4 mmol) dissolved in 30 cm ³ of ethyl alcohol. The contents of the flask were stirred using a magnetic stirrer at room temperature for 5-6 hours. After completion of the reaction, the precipitate of 2-cyano-3-oxy-N{4-[(triphenylphosphino)ethoxy]benzyl}butanamide bromide formula 1 was filtered off and purified by crystallization from a methanol-propan-2-ol mixture (in the ratio of 1: 3).

0.98 g (40% yield) of solid with m.p. 166-168°C

Elemental analysis for formula C32H30BrN3O3P (MW 601.48) calculated:

marked:

%C = 63.90 %C = 63.88 %H = 5.02 %H = 5.11 %N = 4.65 %N = 4.52 %Br = 13.28 %Br = 13.19

^1H NMR spectrum (DMSO-d6, δ, ppm)

9.31 (s, 1H, NH), 7.01-7.51 (m, 19H, H-Ar), 4.84 (s, 1H, CH), 3.63 (s, 2H, CH2benzyl), 1.96 (s, 3H, CH3), 1.48 -1.70 (m, 4H, CH2),

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The physicochemical data of the other bromides of 2-cyano-3-oxy-N-substituted butanamide derivatives are presented in Table No. 1.

Compounds: 2-cyano-3-oxy-N{4-[(triphenylphosphino)hexyloxy]benzyl}butanamide bromide, 2-cyano-3-oxy-N{4-[(triphenylphosphino)octyloxy]benzyl}butanamide bromide, bromide 2 -cyano-3-oxy-N{4-[(triphenylphosphino)dodecyloxy]benzyl}butanamide was tested for cytotoxicity on myeloma cancer cell lines (RPMI 8226), acute myeloblastic leukemia (Kasumi 1), lung squamous cell carcinoma (NCIH2170) and gastric cancer (MKN74) and normal cells: hepatocytes and cardiomyocytes (H2C9) (results are presented in Table No. 2). A series of concentrations were tested: 25, 50, 100 and 200 pM of the test compounds. Cytotoxicity was assessed on the basis of the number of dead cells in the microscopic image and the MTT test, on the basis of which EC50 (half maximal effective concentration) concentrations were determined.

2-Cyano-3-oxy-N{4-[(triphenylphosphino)hexyloxy]benzyl}butanamide bromide showed toxic effects on myeloma and lung squamous cell carcinoma cells with no effect on normal cells - cardiomyocytes.

2-Cyano-3-oxy-N{4-[(triphenylphosphino)octyloxy]benzyl}butanamide bromide has been shown to be cytotoxic to myeloma cells and hepatocytes, as well as to acute myeloblastic leukemia.

2-Cyano-3-oxy-N{4-[(triphenylphosphino)dodecyloxy]benzyl}butanamide bromide has a strong cytotoxic effect on myeloma cells, lung cancer cells and hepatocytes.

PL229 106 B1

Table No. 1

Physicochemical data of the obtained bromides of 2-cyano-3-oxy-N-substituted butanamide derivatives.

L-P x Sum formula. molecular weight ed. [%] mp [ °C] Calc/Identification Analysis 1HNMR spectra (δ, ppm, DMSO- di) c h n Bro 1. (CH2) _{4 C34H34 BrN20JP 629.53} 33 170-174 64.86 64.93 5.44 5.30 4.45 4.56 12.69 12.73 9.65 (s, 1H, NH), 6.91-7.45 (m, 19H, H-Ar), 4.62 (s, 1H, CH), 3.46 (s, 2H, CH2benzyl) > 1.81 (S, 3H, _CII3 ), 1.39-1.65 (m, 8H, CH2 ₎ 2. ₍ CH2) ₆ C ₃₆ H _3s BrN ₂ O ₃ P 657.5S 47 186-188 65.67 65.61 5.82 5.90 4.26 4.34 12.15 12.22 9.22 (s, 1H, NH), 7.11-7.86 (m, 19H, H-Ar), 4.78 (s, 1H, CH), 3.55 (s, 2H, CH2nzyl), 2.06 (s, 3H, CHj), 1.62- 1.79(m, 12H, Cl-h) 3. (CH2 _{) p C3aH42BrN2OjP 685.63 _} 53 195-197 66.56 66.49 6.17 6.11 4.08 4.05 11.65 11.60 9.88 (s, 1H, NH), 7.26-7.59 (m. 19H, H-Ar), 4.74 (s, 1H, CH), 3.49 (s, 2H, _CH2bcnzyl ), 1.90 (S, _3H , CH3 ), 1.64-1.80 (m, 16H, CH2 ₎ 4. (CH2 ₎ , _approx C ₄ oH ₄₄ BrN ₂ 0 ₃ P 713.68 1 38 201-203 67.31 67.38 6.21 6.18 3.92 3.86 11.19 11.29 9.17 (s, 1H, NH), 8.01-7.65(1′, 19H, H-Ar), 4.89 (s, 1H, CH), 3.68 (s, 211, _CH2bcnzyl )j 1.88 (S, 3H, CH ₃ ), 1.52-1.70 (m, 20H, CH2 ₎

PL229 106 B1

Table No. 2

Results of pharmacological studies of 2-cyano-3-oxy-N{4-[(triphenylphosphino)alkyloxy]benzyl}butanamide derivatives.

LINE CELLULAR N6 [µM] N8 [µM] N10 [µM] Myeloma (RPMI8226) Image microscopic (dead cells) 5 * 25 25 * 10 50 ΐ* 50 * * * 25 * ** 100 ** 100 * * * 200 *** 200 * ** Cytotoxicity (MTT) % of viable cells 5 83.5 25 103.2 25 109.12 10 67.5 50 92.8 50 27.76 25 61.8 100 73.25 100 8.69 200 36.69 200 9.56 EC50 [µM] - 165.6 40 Acute myeloblastic leukemia (Kasumi 1) Image microscopic (dead cells) 5 * 25 * 25 ** 10 * 50 * 50 ** 25 * 100 * 100 200 ** 200 Cytotoxicity (MTT) % of viable cells 5 86.58 25 90.13 25 93.67 10 87.08 50 90.29 50 113.08 25 91.21 100 72.99 100 74.43 200 56.29 200 63.71 EC50 [µMJ - - - Cancer flat- epithelial lungs (NCIH2170) Image microscopic (dead cells) 5 - 25 * 25 * 10 * 50 ** 50 25 100 ** 100 ** 200 ** 200 ** Cytotoxicity (MTT) % of viable cells 5 93.84 25 81.29 25 83.61 10:82.59 50 80.69 50 58.05 25 63.33 100 74.72 100 55.32 200 74.03 200 48.47 EC50 [µM] - - 178

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Stomach Cancer (MKN74) Image microscopic (dead cells) 5 - 25 - 25 - 10 - 50 - 50 - 25 - 100 * 100 * 200 * 200 * Cytotoxicity (MTT) % of viable cells 5 91.48 25 92.05 25 87.42 10 98.28 50 62.42 50 89.14 25 87.99 100 67.15 100 82.28 200 70.84 200 56.34 EC50 [µM] - - - Hepatocytes (THLE3) Image 5 * 25 ** 25 * microscopic (dead cells) 10 * 50 ** 50 ** 25 ** 100 m 100 * * * 200 200 Cytotoxicity (MTT) % of viable cells 5 90.90909 25 61.36 25 64.54 10 81.81818 50 52.27 50 56.82 25 45.45455 100 46.82 100 49.54 200 200 EC50 [µMl 25 50 100 Cardiomyocytes (H2C9) Image microscopic (dead cells) 5 - 25 *! 25 - 10 - 50 * 50 * 25 * 100 * 100 ** 200 * 200 *** Cytotoxicity (MTT) % of viable cells 5 130.00 25 83.00 25 86.00 10 137.00 50 97.00 50 70.00 25 120.00 100 77.00 100 58.00 200 82.00 200 68.00 EC50 [µM] - - -

Legend:

* more than 50% dead cells ** 25-50% dead cells *** 10-25% dead cells

Compound #6: 2-cyano-3-oxy-N{4-[(triphenylphosphino)hexyloxy]benzyl}butanamide bromide Compound #8: 2-cyano-3-oxy-N{4-[(triphenylphosphino)octyloxy] bromide benzyl}butanamide Compound #10: 2-cyano-3-oxy-N{4-[(triphenylphosphino)dodecyloxy]benzyl}butanamide bromide

Claims (5)

1. Bromides of 2-cyano-3-oxy-N-substituted butanamide derivatives of the general formula I, where X is the aliphatic substituent ethyl, butyl, hexyl, octyl, dodecyl, respectively. 2. A process for the preparation of 2-cyano-3-oxy-N-substituted butanamide derivatives bromides of the general formula 1, where X is as defined in claim 1, characterized in that the corresponding 4 - [(2-triphenylphosphino) alkyloxy] benzylamine bromides of the general formula 2 is acylated with ethyl 2-cyanoacetylacetate of the formula 3 in a polar solvent, preferably ethyl, methyl, propyl alcohol at room temperature for 4-6 hours, using a molar ratio of 1: 1, after completion of the reaction, a precipitate formed are filtered off and purified preferably by crystallization. PL229 106 Β1 3. The method according to p. A method according to claim 2, characterized in that the separated precipitate is purified by crystallization from a polar solvent of propan-2-ol or a mixture of methanol: propan-2-ol in the ratio 1: 3. 4. Bromides of 2-cyano-3-oxy-N-substituted butanamide derivatives of general formula I, where X is an aliphatic substituent ethyl, butyl, hexyl, octyl, dodecyl, respectively, for use in the treatment of neoplastic diseases. 5. Bromides according to claim 1 4 for use in the treatment of myeloma, lung squamous carcinoma and acute myeloblastic leukemia.