RU2642426C1 - 1-ethyl-6-fluoro-4-oxo-7-(8-ethoxy-2-oxo-2h-chromen-3-yl)-1,4-dihydroquinoline-3-carboxylic acid with anti-tubercular activity - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to a fluoroquinolone carboxylic acid derivative, namely 1-ethyl-6-fluoro-4-oxo-7-(8-ethoxy-2-oxo-2H-chromen-3-yl)-1,4-dihydroquinoline-3 carboxylic acid of formula

.

EFFECT: high antitubercular activity, including that with respect to strains of mycobacteria with multiple drug resistance.

2 tbl, 1 ex

Description

The invention relates to the field of medical chemistry and relates to a new derivative of fluoroquinolone carboxylic acid, 1-ethyl-6-fluoro-4-oxo-7- (8-ethoxy-2-oxo-2 H -chromen-3-yl) -1,4- dihydroquinoline-3-carboxylic acid, which has anti-tuberculosis activity and may find application in medicine and veterinary medicine.

Bacterial infections occupy an important place in the structure of socially significant diseases, and tuberculosis is one of the 10 leading causes of death. In 2015, 10.4 million new cases of tuberculosis infection were recorded in the world, and the mortality rate was 1.8 million. In Russia, in 2015, 130,904 cases of tuberculosis were registered. The Russian Federation is included in the list of countries with a high burden of tuberculosis, since the incidence and mortality rate of the population from tuberculosis is 5-8 times higher than in Europe (http://www.who.int/tb/en) (2009); Global Tuberculosis Control: Surveillance, Planning, Financing; WHO Report 2009; World Health Organization: Geneva, 2009).

http://www.who.int/mediacentre/factsheets/fs104/en/. These data indicate how great the socio-economic importance of tuberculosis is. Unfortunately, the fight against tuberculosis with the methods of modern chemotherapy does not have the desired effect.

The main problems of treating tuberculosis include the limited arsenal of anti-TB drugs and the development of multidrug resistance of the pathogen (MDR), when mycobacterium tuberculosis is resistant to at least two main anti-TB drugs - isoniazid and rifampicin. Mycobacterium tuberculosis (MBT) is hypermutable - when it enters a cell, its numerous mutations begin, leading to its resistance, increased growth rate and other unpleasant qualities of this pathogen. In 2015, there were about 480,000 new cases of multidrug-resistant tuberculosis (TB) with MDR-TB in the world, including 42,000 cases in Russia. In addition, more than 100,000 patients with rifampicin-resistant forms of tuberculosis (PP-TB) were diagnosed. India, China and the Russian Federation accounted for 45% of the total number of new MDR-TB and RR-TB cases - 261,000 cases.

Fluoroquinolone drugs are highly active against Mycobacterium tuberculosis (MBT) and are of significant interest as drugs for combined chemotherapy for drug-resistant tuberculosis [Caminero J., Sotgiu G., Zumla A. et al. Best drug treatment for multidrug-resistant and extensively drugresistant tuberculosis // Lancet Infect Dis. 2010. No. 10. P. 621-629]. According to the WHO guidelines (2011), in the treatment of MDR-TB, levofloxacin is preferred; moxifloxacin is recommended only with proven broad drug resistance.

The mechanism of action of fluoroquinolones on a mycobacterial cell is fundamentally different from the mechanism of action of known anti-TB drugs, which ensures the bactericidal effect and activity of fluoroquinolones in relation to strains that are sensitive and resistant to chemotherapy. The search for new drugs, the mechanism of action of which on the mycobacterial cell is fundamentally different from the mechanism of action of known anti-TB drugs, is an urgent task [V. Villemagne, C. Crauste, M. Flipo et al. Eur. J. Med. Chem . 2012 , 51, 1-16].

Fluoroquinolones have an inhibitory effect on the microbial cell, both during dormancy and during growth. The mechanism of action is the inhibition of gyrase DNA, which leads to disruption of transcription and replication processes. As a result, DNA synthesis is disrupted, which ultimately disrupts cell division.

Bacterial DNA gyrase is a 400 kDa protein tetramer consisting of two A subunits (GyrA) and two B subunits (GyrB). DNA binding and cleavage is associated with GyrA, while ATP binds and hydrolyzes GyrB protein. GyrB consists of an amino-terminal domain that has ATPase activity and a carboxy-terminal domain that interacts with GyrA and DNA. Ideally, antibiotics, the mechanism of action of which is based on the inhibition of bacterial DNA gyrase, will be selective for this enzyme and relatively inactive against type II topoisomerases in eukaryotes.

Fluoroquinolone antibiotics inhibit bacterial DNA gyrase: they bind to GyrA and stabilize the cleaved complex, thus inhibiting the function of gyrase as a whole, leading to cell death.

Inhibitors that bind to GyrB are less well known. Examples include coumarins, novobiocin and cumericin A, cyclothialidine, cynodin and clerocidin. Coumarins have been found to bind very strongly to GyrB [Maxwell, Trends in Microbiology, 1997, 5, 102].

The synthesis of new compounds combining fragments that inhibit both DNA gyrase subunits in one molecule may be of interest from the point of view of expanding the spectrum of their action on the pathogen.

The objective of the invention is the search for a new compound with high antimycobacterial activity, including in relation to multiresistant strains of mycobacteria, expanding the arsenal of known anti-tuberculosis drugs.

The technical result of this invention is the creation of a new chemical compound - 1-ethyl-6-fluoro-4-oxo-7- (8-ethoxy-2-oxo-2 H -chromen-3-yl) -1,4-dihydroquinolin-3 -carboxylic acid of the formula ( 4 )

Compound 4 , its physicochemical characteristics, and also the method for its preparation are not described in the literature.

The compound of formula ( 4 ) can be obtained in three stages from the available ethyl ester of 6,7-difluoro-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ( 1 ).

Compound 1 is heated with ethyl cyanoacetic acid in dimethyl sulfoxide (DMSO) in the presence of pre-calcined potassium carbonate. As a result, 7- (1-cyano-2-ethoxy-2-oxoethyl) -1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester ( 2 ) is obtained by acid hydrolysis of which get the connection 3 - 7-cyanomethyl-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Compound 3 is reacted with 3-ethoxysalicylic aldehyde in N, N-dimethylformamide at room temperature, then the resulting product is filtered off and in the mixture of sulfuric and acetic acids under heating receive the target compound 4 .

Characteristics of the compounds are given in examples of specific performance.

The structures of the compounds are confirmed by elemental analysis, ¹ H, ¹⁹ F and ¹³ C NMR spectra and mass spectra. The melting points of the compounds are determined on a Stuart SMP3 instrument (Barloworld Scientific). Elemental analysis was performed on a PE 2400 Series II automatic CHNS-O analyzer (Perkin Elmer).

¹ H NMR spectra were recorded on Bruker DRX-400 (400 MHz) or Bruker Avance 500 (500 MHz) instruments in DMSO-d ₆ with SiMe ₄ as an internal standard at 25 ° С. Chemical shifts (δ) are given in parts per million (ppm), and the spin-spin coupling constants ( J ) are in hertz.

¹⁹ F NMR spectra were recorded on Bruker Avance 500 instruments (500 MHz) in DMSO-d ₆ . Chemical shifts (δ) are given in parts per million (ppm), and the spin-spin coupling constants ( J ) are in hertz.

¹³ C NMR spectra were recorded on a Bruker Avance 500 instrument (125 MHz) in DMSO-d ₆ with SiMe ₄ as an internal standard at 25 ° C. Chemical shifts (6) are given in parts per million (ppm).

Mass spectra were recorded on a Shimadzu GCMS-QP 2010 Ultra spectrometer. Shooting conditions: energy of ionizing electrons 70 eV, direct input of the sample into the source.

Examples of specific performance.

Example 1. Synthesis of 1-ethyl-6-fluoro-4-oxo-7- (8-ethoxy-2-oxo-2 H -chromen-3-yl) -1,4-dihydroquinoline-3-carboxylic acid (4) .

Step 1. Synthesis of 7- (1-cyano-2-ethoxy-2-oxoethyl) -1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ( 2 ).

To a solution containing 10.00 g (35.55 mmol) of 6,7-difluoro-1-ethyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ethyl ester (1) in 65 ml of dimethyl sulfoxide, 4 are added 80 g (42.66 mmol) of ethyl cyanoacetic acid and 11.79 g of pre-calcined potassium carbonate are added. The resulting reaction mixture was heated under reflux with a calcium chloride tube at 80 ° C for 4 hours. The reaction mixture was poured into ice water (~ 250 ml) and an HCl solution (18%) was added to the resulting solution with stirring to pH 2-3. The resulting product was extracted with methylene chloride (~ 600 ml). The aqueous layer was separated, and the organic layer was washed 2 times with 200 ml of distilled water and 1 time with a saturated solution of sodium chloride (200 ml), then dried over anhydrous sodium sulfate. Methylene chloride was removed on a rotary vacuum evaporator, diethyl ether (~ 75 ml) was added to the residue, the resulting precipitate of compound 2 was filtered off and dried in air, a colorless substance was obtained from ethyl alcohol with mp. > 250 ° C (decomposition without melting). Yield 11.6 g (87.1%).

In the next step, compound 2 is used without further purification.

Found,%: C 60.92; H 5.12; N, 7.43. Calculated for C ₁₉ H ₁₉ FN ₂ O ₅ (M 374.377),%: C 60.96; H, 5.12; N7.48.

¹ H NMR spectrum (500.1 MHz, DMSO-d ₆ ): 1.22 (t, 3H, J = 7.1 Hz, H-4 '); 1.30 (t, 3H, J = 7.1 Hz, H-3``); 1.41 (t, 3H, J = 7.1 Hz, NCH <sub>2</sub> C H <sub>3</sub> ); 4.24 (q, 2H, J = 7.1 Hz, H-2``); 4.27 (q, 2H, J = 7.1 Hz, H-3 '); 4.41 (d q, 1H, J = 14.1, 7.1 Hz, NCH ^B ); 4.45 (d q, 1H, J = 14.1, 7.1 Hz, NCH ^A ); 6.13 (s, 1H, H-1 '); 8.00 (d, 1H, J = 10.2 Hz, H-5); 8.09 (d, 1H, J = 5.9 Hz, H-8); 8.77 (s, 1H, H-2).

¹⁹ F NMR spectrum (470.5 MHz, DMSO-d ₆ ): 42.97 (dd, J = 10.2, 5.9 Hz, F-6).

¹³ C NMR Spectrum (125.7 MHz, DMSO-d ₆ ): 13.75 (C-4 '); 14.25 and 14.28 (2 * CH ₃ ); 37.94 (C-1 '); 48.46 (NCH ₂ ); 59.88 (C-2``); 63.21 (C-3 '): 109.90 (C-3); 111.93 (d, <sup>2</sup> J <sub>CF</sub> = 22.3 Hz, C-5); 115.19 (CN); 121.41 (d, <sup>3</sup> J <sub>CF</sub> = 2.7 Hz, C-8); 124.21 (d, <sup>2</sup> J <sub>CF</sub> = 17.7 Hz, C-7); 130.53 (d, <sup>3</sup> J <sub>CF</sub> = 6.6 Hz, C-4a); 135.28 (dkv, <sup>4</sup> J <sub>CF</sub> = 1.5 Hz, C-8a); 149.50 (C-2); 156.88 (d, <sup>1</sup> J <sub>CF</sub> = 248.5 Hz, C-6); 163.88 (C-2 '); 164.36 (C-1``); 171.47 (d, ⁴ J _CF = 2.1 Hz, C-4).

Mass spectrum, m / z ( I _rel. ,> 5%): 375 [M + 1] ⁺ (1.35); 374 [M] ⁺ (5.91); 329 (6.76); 303 (18.07); 302 (100); 230 (7.07); 229 (7.09); 228 (6.67); 145 (5.12).

Stage 2. Synthesis of 7-cyanomethyl-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid ( 3 ).

To 10.00 g (26.71 mmol) of ethyl 7- (1-cyano-2-ethoxy-2-oxoethyl) -1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbon acids (2) are added 75 ml of a mixture of acids (sulfuric acid, conc .: acetic acid: dist. water - 1: 20: 20). The reaction mass is heated at a temperature of 125 ° C for 6 hours. Then the reaction solution is cooled to room temperature, the precipitate formed is filtered off, washed on the filter with distilled water and dried in air. The precipitate was recrystallized from ethanol: N, N-dimethylformamide, and a colorless crystalline solid was obtained with a melting point of > 250 ° C (decomposition without melting). Yield: 6.7 g (91.8%).

Found,%: C 60.92; H 5.12; N, 7.43. Calculated for C ₁₄ H ₁₁ FN ₂ O ₃ (M 274.258),%: C 61.31; H 4.04; N 10.22.

¹ H NMR spectrum (500.1 MHz, DMSO-d ₆ ): 1.46 (t, 3H, J = 7.1 Hz, CH ₃ ); 4.34 (s, 2H, CH ₂ CN); 4.60 (q, 2H, J = 7.1 Hz, NCH ₂ ); 8.10 (d, 1H, J = 9.5 Hz, H-5); 8.22 (d, 1H, J = 6.0 H-8); 9.10 (s, 1H, H-2); 14.89 (s, 1H, OH).

¹⁹ F NMR spectrum (470.5 MHz, DMSO-d ₆ ): 44.84 (dd, J = 9.5, 6.0 Hz, F-6).

¹³ C NMR Spectrum (125.7 MHz, DMSO-d ₆ ): 14.47 (CH ₃ ); 17.81 (CH ₂ CN); 49.44 (NCH ₂ ); 107.68 (C-3); 110.87 (d, ² J _CF = 22.8 Hz, C-5); 117.26 (CN); 121.23 (d, ³ J _CF = 3.6 Hz, C-8); 126.67 (d, ² J _CF = 18.8 Hz, C-7); 126.94 (d, ³ J _CF = 7.7 Hz, C-4a); 135.81 (d, ⁴ J _CF = 1.4 Hz, C-8a); 149.42 (C-2); 157.68 (d, ¹ J _CF = 249.9 Hz, C-6); 165.66 (COO); 176.63 (d, ⁴ J _CF = 2.6 Hz, C-4).

Mass spectrum, m / z ( I _rel. ,> 9%): 275 [M + 1] ⁺ (1.26); 274 [M] ⁺ (7.24); 231 (14.36); 230 (100); 215 (53.38); 147 (9.89).

Stage 3. Synthesis of 1-ethyl-6-fluoro-4-oxo-7- (8-ethoxy-2-oxo-2 H -chromen-3-yl) -1,4-dihydroquinoline-3-carboxylic acid ( 4 ) .

To a suspension containing 0.500 g (1.82 mmol) of 7-cyanomethyl-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (3) and 0.303 g (1.82 mmol) 3-ethoxysalicylic aldehyde, add 5 drops of piperidine and stir at room temperature for 1 h. To the reaction mixture add 15 ml of ethanol, the precipitate is filtered off, washed on the filter with a small amount of ethanol and heptane. The filter cake was transferred to a round bottom flask, 20 ml of a 5% sulfuric acid solution was added and heated at 110 ° C for 6 hours. The reaction mixture was cooled to room temperature, the filter cake was washed and the filter was washed with distilled water, then hexane or heptane. Recrystallized from aqueous N, N-dimethylformamide to give 375 mg (48.6%) of the target compound (4). T. pl. 230 ° C (decomposition without melting).

Found,%: C 64.89; H 4.28; N, 3.36. Calculated for C ₂₃ H ₁₈ FNO ₆ (M 423.425),%: C 65.25; H 4.29; N3.31.

¹ H NMR spectrum (500.1 MHz, DMSO-d ₆ ): 1.43 (t, 3H, J = 7.0 Hz, OCH ₂ C H ₃ ); 1.47 (t, 3H, J = 7.1 Hz, NCH ₂ C H ₃ ); 4.23 (q, 2H, J = 7.0 Hz, OC H ₂ CH ₃ ); 4.64 (q, 2H, J = 7.1 Hz, NC H ₂ CH ₃ ); 7.34-7.41 (m, 3H, H-5 ', 6', 7 '); 8.14 (d, 1H, J = 9.7 Hz, H-5); 8.35 (d, 1H, J = 5.9 Hz, H-8); 8.42 (s, 1H, H-4 '); 9.12 (s, 1H, H-2); 14.97 (s, 1H, COOH).

¹⁹ F NMR spectrum (470.5 MHz, DMSO-d ₆ ): 47.91 (dd, J = 9.7, 5.9, F-6).

¹³ C NMR spectrum (125.7 MHz, DMSO-d ₆ ): 14.59 and 14.65 (2 * CH ₃ ); 49.46 (NCH ₂ ); 64.52 (OCH ₂ ); 107.66 (C-3); 110.76 (d, ² J _CF = 24.0 Hz, C-5); 115.97 (C-7 '); 119.29 (C-4'a); 121.46 (C-3 '); 122.24 (d, ³ J _CF = 3.1, C-8); 124.96 (C-6 '); 127.15 (d, ³ J _CF = 7.8, C-4a); 129.80 (d, ² J _CF = 18.1, C-7); 135.58 (d, ⁴ J _CF = 1.2, C-8a); 142.88 (C-8'a); 145.01 (d, ⁴ J _CF = 1.5, C-4 '); 145.67 (C-8 '); 149.50 (C-2); 157.20 (d, ¹ J _CF = 250.5, C-6); 158.43 (C-2 '); 165.77 (COO); 176.66 (d, ⁴ J _CF = 2.7, C-4).

Mass spectrum, m / z ( I _rel. ,> 11%): 424 [M + 1] ⁺ (2.00); 423 [M] ⁺ (6.86); 380 (25.57); 379 (100); 336 (17.50); 176 (11.20).

Biological activity

Tuberculostatic activity was studied in the laboratory of experimental and diagnostic studies of the Federal State Budgetary Institution “Ural Research Institute of Phthisiopulmonology” of the Ministry of Health of Russia, Ekaterinburg.

The study of the tuberculostatic activity of the compounds is carried out by the method of vertical diffusion using a laboratory strain H37Rv in a solid nutrient medium "New".

The nutrient medium is poured into 5 ml tubes, coiled in an inclined position so that 1/2 of the bottom of the tube remains free. Coagulated medium is inoculated with 0.1 ml of suspension of Mycobacterium tuberculosis (MBT) strain H37Rv, diluted according to the turbidity standard of 10 units. GKI, and in an inclined position are placed in a thermostat for 24 hours for growing MBT. After a day, the tubes are placed in an upright position and 0.3 ml of the substance of the solution of compounds in the studied concentrations are instilled along the free edge: 12.5; 6.25; 3.1; 1.5; 0.75 μg / ml. Then the tubes are placed in a thermostat at a temperature of 37 ° C and incubated for 10 days. MBT growth assessment is carried out according to a standard method, where the appearance of MBT growth retardation zones (more than 10 mm) indicates the presence of tuberculostatic properties in the studied concentration of compounds. The size of the MBT growth retardation zone (in mm) is proportional to the degree of tuberculostatic activity of the compounds. A growth retardation of 100 mm or more is regarded as a complete MBT growth retardation. Compound 4 showed high tuberculostatic activity against laboratory strain H37Rv (table 1).

The study of tuberculostatic activity against resistant strains is carried out by the method of vertical diffusion on a solid nutrient medium "New" using laboratory and clinical strains of Mycobacterium tuberculosis with multidrug-resistant (MDR), isolated from patients being treated at the clinic of the Federal State Budgetary Institution UNIIF.

The nutrient medium is poured into 5 ml tubes, coiled in an inclined position so that 1/2 of the bottom of the tube remains free. Coagulated medium is inoculated with 0.1 ml of a suspension of mycobacteria of various strains diluted according to the turbidity standard of 10 units. GKI, and in an inclined position are placed in a thermostat for 24 hours for growing MBT. After a day, the tubes are placed in an upright position and 0.3 ml of a solution of the compounds in the studied concentrations are instilled along the free edge: 12.5; 6.2; 3.1; 1.5; 0.7 mcg / ml. Then the tubes are placed in a thermostat at a temperature of 37 ° C and incubated for 10 days. MBT growth assessment is carried out according to a standard method, where the appearance of MBT growth retardation zones (more than 10 mm) indicates the presence of tuberculostatic properties in the studied concentration of compounds. The size of the MBT growth retardation zone (in mm) is proportional to the degree of tuberculostatic activity of the compounds. A growth retardation of 100 mm or more is regarded as a complete MBT growth retardation. The data are presented in table 1.

From the data of table 1 it follows that compound 4 exhibits high tuberculostatic activity (MIC 0.7-1.5 μg / ml) both in relation to the laboratory strain H37Rv, and in relation to a number of drug-resistant strains of the office. It should be emphasized that compound 4 was active against MDR strain, for which the known anti-TB drug, isoniazid, is not active.

For the claimed compound 4 , acute toxicity was studied and a lethal dose of LD50 was determined in animal experiments ( in vivo ). Determine the maximum tolerated dose (MTD) at which the death of animals was not observed, and the dose that causes the death of 50% of the animals (LD50). In accordance with the methodology (G.N. Pershin, 1971. M.), the calculation of LD50 is carried out according to the formula, determining the average lethal dose:

x average = [(a + b) (m-n)]: 200,

where (a + b) is the sum of adjacent doses; (m-n) -% difference in mortality from the last 2 doses.

The study of acute toxicity and the development of a lethal dose of LD50 of compound 4 is carried out on white mice weighing 17-20 g. Individual doses are selected for the drug: minimum 150 mg / kg, average 300 mg / kg or 600 mg / kg and maximum 900 mg / kg. For each dose, 5 white mice are taken, which are injected with drugs into the stomach in a volume of 0.5 ml in a 1% starch paste solution. Only 1% starch paste solution is administered to the control group.

Observation of animals is carried out for 14 days. In all animals that died during the experiment, macroscopic and histological studies of the internal organs (liver, kidneys, lungs, spleen) are performed. In surviving animals after slaughter, histological examinations of internal organs are also performed.

Data on acute toxicity of compound 4 are presented in table 2.

From the data presented in table 2 it follows that when animals were administered compound 4 at a dose of 300 mg / kg, the death of animals was not observed 24 hours after administration. When animals are administered Compound 4 at a dose of 600 mg / kg, the death of the animals occurs 2 hours after ingestion (20%). When animals are administered Compound 4 at a dose of 900 mg / kg, the death of animals occurs 30-60 minutes after ingestion (60%). At the autopsy of dead animals, macroscopic changes in the internal organs were not detected. As a result of the study for compound 4 , it was found that the maximum tolerated dose (MTD) is 300 mg / kg (no animal deaths were observed), LD50 - 600 mg / kg (animal death in 20%).

Claims (3)

1-Ethyl-6-fluoro-4-oxo-7- (8-ethoxy-2-oxo-2H-chromen-3-yl) -1,4-dihydroquinoline-3-carboxylic acid of the formula (4)

possessing anti-tuberculosis activity.