RU2635112C1 - Halides of 1-(4-tert-butylphenyl)-2-{3-[2-(4-fluoro-phenoxy)ethyl]-2-methyl-3h-benzimidazole-1-yl}ethanone with property of rupture cross-linkers of glycated proteins - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: halides of the formula 1

are described, possessing the property of rupture cross-linkers of glycated proteins.

EFFECT: new halides that have beneficial biological properties.

4 cl, 2 dwg, 1 tbl

Description

The invention relates to new derivatives in the benzimidazole series, namely to the previously undescribed halides 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazole-1 -yl} ethanone of formula 1:

where Hal - including chlorine or bromine.

The invention relates to medicine and biologically active substances capable of functioning as cross-linking breakers (RPS) of glycated proteins, and can be used primarily in the complex treatment of diabetes mellitus and its complications. Pathological processes in diabetes mellitus as one of the most important components include an extremely destructive in its consequences non-enzymatic protein glycation reaction, leading to the accumulation of glycoproteins. The latter are responsible for a sharp decrease in the functional properties of red blood cells and very serious vascular complications of diabetes. In patients with diabetes mellitus, glycation, during which protein macromolecules are glued together with glucose, is primarily subjected to long-lived structural proteins, especially in connection with an increased glucose level [1, 2]. Glycation is a rather slow process, at the final stage of which crosslinking of glycoproteins occurs with the formation of inter- or intramacromolecular bonds of the glycated protein.

It should be noted that glycation of proteins, peptides and other macromolecules is associated not only with diabetes, but also with aging, with neurodegenerative amyloid disease (Alzheimer's disease), as well as with the development of cataracts.

The loss of protein functionality due to their glycation is associated with the fact that the formation of cross-links in proteins, as a rule, leads to a significant decrease in their elasticity. As a result, blood cells, in particular red blood cells, blood vessels, skin become stiff and to a large extent lose their functionality. In healthy people, as they age, this process occurs rather slowly, while in patients with diabetes mellitus, it accelerates sharply due to an increased concentration of glucose. One of the consequences of aging and diabetes is also an increase in the rigidity of the collagen base of the cardiovascular system [3]. Large arteries lose their elasticity and become stiff, which leads to systolic hypertension and, ultimately, heart failure. Non-enzymatic glycosylation of the collagen matrix occurs gradually, both in the aging process and in diabetes. One of the effects of cross-linking collagen is reduced susceptibility to proteolytic and chemical degradation. This leads to increased accumulation of cross-linked collagen, loss of its elasticity and, as a result, to coronary heart disease (CHD) [4]. The results of numerous studies confirm the opinion that a heart attack is the result of glycation of the collagen matrix [4, 5].

The normalizing effect of quaternary azolium salts, including N-phenacyl-4,5-dimethylthiazolium chloride (drug ALT-711, alagebrium), which reduces the level of glycation end products (CNG) in blood vessels, is known to normalize on the glycated channel of the cardiovascular system [ 5]. During clinical trials of the algebra, it was shown that the drug has good tolerance and causes an increase in vascular elasticity in 93% of patients older than 50 years [4].

The closest in structure among the derivatives of 2-methylbenzimidazole are hydrobromides of 1-alkyl-3-phenacyl derivatives of 2-methylbenzimidazole with antimicrobial activity [6], antitumor activity [7].

In the series of phenacyl derivatives of 2-methylbenzimidazole, no compounds exhibiting the properties of crosslinker breakers of glycated proteins are known.

The technical result of the invention is a compound not previously described in the series of phenacyl derivatives of 2-methylbenzimidazole, which exhibits a novel property for this series of crosslinker breakers for glycated proteins.

The technical result is achieved by the compound of formula 1.

Chlorides of 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazol-1-yl} ethanone may include, but not limited to, chloride or bromide.

The synthesis of compounds 1 consists in the alkylation of 2-methylbenzimidazole with 4-fluorophenoxyethyl bromide and the subsequent quaternization of the resulting 1- (4-fluorophenoxy) ethyl-2-methylbenzimidazole 2 2-substituted 1- (4-tert-butylphenyl) ethanones upon boiling in nitromethane:

The following is an example of the synthesis of bromide of the proposed compound.

Stage 1. 1- (4-fluorophenoxy) ethyl-2-methylbenzimidazole

To a solution of 1.32 g (10 mmol) of 2-methylbenzimidazole and 1.3 g (20 mmol) of KOH (calculated as 85% KOH) in 10 ml of DMSO, 2.19 g (10 mmol) of 2- (4-fluorophenoxy) ethyl bromide are added in portions, so that the temperature of the reaction mass does not exceed 45 ° C. The mixture is kept for 1 hour at 45 ° C, then the temperature is raised to 115 ° C and immediately cooled and poured into 20 ml of water. Precipitated slightly pinkish crystals of 1- [2- (4-fluorophenoxy) ethyl] -2-methylbenzimidazole are filtered off, washed with 5 ml of water. Yield 2.37 g (88%). T. pl. 133-135 ° C (from isopropanol).

¹ H NMR spectrum (300 MHz), δ, ppm (CDCl ₃ ): 2.33 (s, 3H, CH ₃ ), 4.25 (t, 2H, J = 4.2, OCH ₂ ), 4.34 (t, 2H, J = 4.3, NCH ₂ ), 6.70-6.77 (m, 2H , Н-2 ' ¹ , Н-6'), 6.89-6.95 (m, 2Н, Н-5, Н-6), 7.10-7.19 (m, 3Н, Н-3 ', Н-5', Н- 4), 7.42 (d, 1H, J = 7.6, H-7).

Found (%): N 10.12. C ₁₆ H ₁₅ FN ₂ O. Calculated (%): N 10.36.

Stage 2. 1- (4-tert-Butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazol-1-yl} ethanone bromide

(The protons of the phenoxyethyl substituent at position 1 of benzimidazole are indicated by ¹ numbers with a dash, and the lines of phenyl at position 3 of benzimidazole by two dashes)

To a solution of 0.54 g (20 mmol) of 2-methyl-1- [2- (4-fluorophenoxy) ethyl] benzimidazole in 15 ml of nitromethane at 35 ° C, 0.53 ml (21 mmol, 0.54 g) of 2-6rom-1- ( 4-tert-butylphenyl) ethanone, boiled for 1 hour, cooled and filtered 0.90 g (87%) of 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2- bromide methyl-3H-benzimidazol-1-yl} ethanone. Colorless crystals with mp 208 ° C (from isopropanol). ¹ H NMR spectrum (600 MHz), δ, ppm (DMSO-d ₆ ): 1-34 (s, 9H, C (CH ₃ ) ₃ ), 2.93 (s, 3H, CH ₃ ), 4.36 (t, 2H, J = 4.5, OCH ₂ ), 5.06 (t , 2H, J = 4.5, NCH ₂ ), 6.42 (s, 2H, CCH ₂ ), 6.88-6.92 (m, 2H, H-3 ', 5'), 7.11 (t, 2H, J = 8.7, H- 2 ', 6'), 7.60-7.70 (m, 4Н, Н-4, Н-5, Н-6, Н-7), 8.00 (d, J = 8.1, 1Н, Н-3 '' or Н- 5``), 8.08 (d, 2H, J = 8.4, H-2 '', 6``), 8.15 (d, 1H, J = 8.1, H-5 '' or H-3``).

Found (%): N 5.04. C ₂₈ H ₃₀ BrFN ₂ O ₂ . Calculated (%): N 5.32.

Bioactivity study

Collagen production

To obtain collagen, Wistar rats (body weight 200 ± 20 g) were anesthetized, after which the tails were excised at 4 ° C and the collagen fibers of the tail tendon were removed, which were washed with physiological saline, the tissues of non-collagen fibers were removed, washed three times with distilled deionized water, cut into pieces and at 4 ° C immersed in a 0.1% solution of acetic acid for one week, during which the immersion liquid was often shaken. The immersion fluid was centrifuged at 8000 g for 30 minutes and the supernatant collagen solution was collected. After dilution, the protein content was measured. A 96-well microplate (Costar) was carefully coated with a 70 μg / well collagen solution at 4 ° C. for 24 hours, then the film-forming solution was removed. The plates were air dried, covered with a film to maintain freshness, and stored at 4 ° C until use.

Obtaining end products of glycation (glycated bovine serum albumin - BSA)

A solution containing 50 mg / ml bovine serum albumin (BSA) (V) (Roch) and 0.5 M glucose in 0.2 M phosphate buffered saline (phosphate buffered saline) (pH 7.4) was incubated at 37 ° C under sterile conditions for 3-4 months for the formation of, thus, glycosylated BSA. At the same time, non-glycosylated BSA with glucose-free BSA was prepared. Then the BSA-AGE solution was dialyzed against a 0.01 M phosphate buffer solution (pH 7.4) to remove unreacted glucose. Used fluorescence scanning (Exi / Em (395/460 nm)).

Study of the activity of 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazol-1-yl} ethanone (1)

A 96-well microplate coated with tail collagen was carefully treated with phosphate buffered saline (pH 7.4) for 1 hour to neutralize acidic collagen. The plate was blocked with Superblock (PIERCE) at 37 ° C for 1 hour and washed with Tween-20 phosphate buffered saline, shaking three times for 1 minute with each wash. 100 μl of a glycated protein solution was added to the wells in rows of a 96-well plate labeled A, B, C and D, and a BSA solution of the same concentration was added to the wells in rows labeled E, F, G and H. Wells were incubated at 37 ° C for 4 hours to ensure crosslinking of the collagen, and washed with phosphate buffered saline with tween-20 four times with shaking for 1 minute with each washing. The test compound was added to duplicated wells with glycated protein and to four duplicated wells with BSA in an amount of 100 μl / well. In the first three wells in each row, instead of the substance, 100 μl / well of a phosphate buffer solution or other solvent (for example, DMSO) was added. Wells were incubated at 37 ° C for 16 hours and washed with a tween-20 phosphate buffered saline, shaking for 1 minute during each wash. Anti-BSA rabbit antibodies (1: 500) of 80 μl / well were added to the wells and the plate was incubated at 37 ° C for 50 minutes. Next, 80 μl / well of horseradish peroxidase labeled goat anti-rabbit IgG (1: 1000) was added to the wells. Wells were incubated at 37 ° C for 50 minutes. After that, a substrate of 3.3 ', 5.5'-tetramethylbenzidine (TMB) was added 100 μl / well. The plate was incubated at room temperature in the dark for 20 minutes. To terminate the reaction, a 2M solution of H ₂ SO _{4 was used} . Within 10 minutes after the reaction, the optical density at 450 nm was recorded on a TECAN M200Pro plate reader.

For each determination, the average optical density (OD) was calculated.

Corrected OD = average OD of the well with glycated protein — average OD of the BSA well.

The percentage of destruction was expressed as a relative decrease in OD:

[(average OD of the hole phosphate buffer solution - average OD of the hole of the test compound) / average OD of the hole phosphate buffer solution] × 100%.

The results are processed in the Microsoft Excel program (Microsoft, USA) with the calculation of basic statistical indicators: arithmetic mean M, standard deviation s, standard error of the arithmetic mean m. Statistical processing using paired Student t-test in the program Statistika 10.0 (StatSoft, USA).

Research results

In FIG. 1 shows the dependence of the tearing activity of 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazol-1-yl} ethanone (1) concentration. The abscissa shows the concentration of compound 1, mmol; along the ordinate axis - inhibition of fluorescence,%; number of experiments - n = 5. R ² is the square of the correlation coefficient for the regression equation (shows the statistical significance of the calculations) = 0, 9402. IC ₅₀ (concentration of half-maximum inhibition) = 0.387 mmol / L. The value is obtained from the regression equation (y = 65.52x + 24.655), which relates the effect of y with the concentration of x, at y = 50. In FIG. Figure 2 shows the dependence of the tearing activity of the ALT-711 comparison drug on its concentration. The abscissa shows the concentration of ALT-711, mmol / l; along the ordinate axis - inhibition of fluorescence,%. (n = 5). IC ₅₀ = 1.89 mmol / L.

Table 1 shows the half-maximum inhibition concentration (IC ₅₀ ) 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazol-1-yl} ethanone bromide (1) in comparison with the reference drug Alabebrium (ALT-711).

As can be seen, for compound 1, the IC ₅₀ value is 0.387 mmol / L, which is 4.9 times higher than the IC ₅₀ for ALT-711.

Thus, benzimidazole derivatives possess the properties of breakers for cross-linking glycated proteins, surpassing the comparison drug Alabrium in 4.9 times.

LITERATURE

1. Ansari N.A., Rashid Z. // Biomedical chemistry. - 2010. - T. 56. - No. 2. - S. 168-178.

2. Lee Song et al. RF Patent 2444517 (2012), Bull. No. 7 (2012).

3. A.A. Spasov, A.I. Rashchenko // Bulletin of Volgograd State Medical University. 2016. - T. 57 - No. 1. - from. 12-15.

4. Vasan S., Foiles P., Founds H. // Archives of Biochemistry and Biophysics. - 2003. - T. 419. - No. 1. - C. 89-96.

5. Kim J. et al. // European journal of pharmacology. - 2015.- T. 748. - C. 108-114. Singh R. et al. // Diabetologia. - 2001. - T. 44. - No. 2. - C. 129-146.

6. Voev V.L. and others, Chem.-farm. Journal, 1990. V. 24, No. 11, p. 40-44.

7. Xiao-Liang Xu, Org. Biomol. Chem., 2015, V. 13, P. 1550; Jin-Mei Liu, RSC Advances, 2015, Issue78.

Claims (5)

1. Halides of 1- (4-tert-butylphenyl) -2- {3- [2- (4-fluorophenoxy) ethyl] -2-methyl-3H-benzimidazol-1-yl} ethanone of formula 1

2. Halides according to claim 1, having the property of breakers cross-linking glycated proteins. 3. The halides of claim 1, wherein Hal is bromo. 4. The halides of claim 1, wherein Hal is chlorine.

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