RU2449988C1 - 2,2,2-trifluoro-1-trifluoromethyilethyl ester of cyclohexylcarbomine acid as effective agent for selective irreversible carboxylesterase inhibition - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared invention refers to chemical-pharmaceutical industry, and concerns 2,2,2-Trifluoro-1-trifluoromethylethyl ester of cyclohexylcarbamine acid

which is prepared starting with cyclohexylisocyanate and 1-hydrohexafluorisopropanole at 20°C in benzene in the presence of catalytic amounts of triethylamine.

EFFECT: compound (I) is an effective and selective irreversible carboxylesterase inhibitor with low acute toxicity in warm-blooded, and may be used in preclinical studies on rodents for decreasing carboxylesterase hydrolysis rate of the drugs containing ester and/or amide groups, and also as insect carboxylesterase inhibitors in pharmaceutical composition and pesticide formulations for increasing their efficacy.

2 cl

Description

The invention relates to the use of chemical compounds in the field of medicine and can be used in preclinical studies on rodents of drugs containing ester and / or amide groups to reduce the rate of their carboxyl esterase hydrolysis, thereby modulating their pharmacological and / or toxic effects. In addition, the invention can be used in pharmaceutical mixtures and formulations of pesticides to increase their effectiveness by inhibiting carboxyl esterase and thereby reduce the amount of pesticide needed to inhibit the target, acetylcholinesterase, in the case of organophosphorus and carbamate pesticides.

Carboxyl esterases (CE) are a determinant in the pharmacokinetics of most therapeutic agents containing an ester or amide moiety, and many new drugs are being designed with metabolic activation or inactivation due to carboxyl esterase hydrolysis [Prokai L, Prokai-Tatrai K. Metabolism-based drug design and drug targeting. Pharm Sci Technolo Today. 1999; 2 (11): 457-462]. In this regard, CE inhibitors that affect the rate of hydrolysis of such drugs and thus determine, for example, the rate of conversion of a prodrug into an active drug or increase the half-life of an active drug that hydrolyzes CE, have important therapeutic value [Potter PM, Wadkins RM. Carboxylesterases-detoxifying enzymes and targets for drug therapy. Curr Med Chem. 2006, 13 (9): 1045-1054].

In addition, taking into account that preclinical studies of drugs are carried out primarily on rodents, which, unlike humans, have a high level of plasma CE, the problem arises of extrapolating the results from animals to humans. The use of effective irreversible carboxyl esterase inhibitors, which have low acute toxicity for warm-blooded animals, allows the inhibition of plasma CE and thus creates an adequate, effective model in rodents for the study of pharmacologically important drugs that hydrolyze under the action of CE.

Inhibition of insect carboxyl esterases potentiates the action of insecticides hydrolyzing under the action of CEs, such as pyrethroids, as well as anticholinesterase insecticides by reducing their binding to CEs [Shipov A.E., Genkina G.K., Makhaeva G.F., Malygin V.V. ., Volkova R.I., Roslavtseva S.A., Eremina O.Yu., Bakanova E.I., Mastryukova T.A., Kabachnik M.I. Investigation of the mechanism of action of pesticides of the 2-aryloxy-2-thio-1,3,2-oxazaphosphorinan series. Bioorgan. chemistry. 1999, 25 (1): 14-19]. The use of carboxyl esterase inhibitors is one way to overcome insect resistance to insecticides [Yan S, Cui F, Qiao C. Structure, function and applications of carboxylesterases from insects for insecticide resistance. Protein Pept Lett. 2009; 16 (10): 1181-1188]. In addition, inhibition of TBE itself can lead to the death of insects.

A sufficient number of years has been occupied with the solution of the problem of creating selective and low-toxic carboxyl esterase inhibitors in the world, however, due to the large structural similarity of serine esterases, there are only a limited number of reports of selective CE inhibitors. This is a series of analogues of trifluoromethyl ketones (highly hygroscopic compounds that are easily hydrolyzed, see Wheelock CE, Severson TF, Hammock BD. Synthesis of new carboxylesterase inhibitors and evaluation of potency and water solubility. Chem Res Toxicol. 2001, 14 (12): 1563- 1572), recently proposed analogues of benzyl (diphenylethane-1,2-dione) [Wadkins RM, Hyatt JL, Wei X, Yoon KJ, Wierdl M, Edwards CC, Morton CL, Obenauer JC, Damodaran K, Beroza P, Danks MK, Potter PM. Identification and characterization of novel benzil (diphenylethane-1,2-dione) analogues as inhibitors of mammalian carboxylesterases. J. Med. Chem. 2005, 48 (8): 2906-2915] and substituted arylsulfonamides and arylureas [Potter PM, Hyatt JL, Morton CL, Beroza PP, Damoradan KV. Amide, aryl sulfonamide, aryl urea, and a, b-diketone derived carboxylesterase inhibitors, and their methods of use. US Appl. 2008146548, IPC A61K 31/44].

However, these compounds are reversible CE inhibitors.

Irreversible TBE inhibitors are known, the effect of which increases over time and is longer. Representative of such inhibitors are organophosphorus compounds (POIs) - esters and thioesters of pentavalent phosphorus acids of the general formula R ₁ R ₂ P (O) X, where one of the X groups is usually a good leaving group (M. Eto, Organophosphorus Pesticides: Organic and Biological Chemistry, CRC Press, Cleveland, 1974).

The closest analogues to the proposed technical solution are compounds of the general formula (II)

(Ii)

where R = Alkyl, X = CF ₃ , Aryl, C (O) OCH ₃

The inhibitory activity of these compounds with respect to the serine hydrolases of acetylcholinesterase (AChE), butyrylcholinesterase and carboxylesterase (CE) and their synthesis have been studied in detail [Makhaeva G.F., Fetisov V.I., V.B.Sokolov, Yankovskaya V.L., Goreva T.V., Malygin V.V., Beznosko B.K., Galenko T.G., Kolomiyets A.F., Martynov I.V. - Interaction of dialkyl (α-carbomethoxy-β, β, β-trifluoroethyl) phosphates with mammalian esterases. - Bioorganic Chemistry, 1987, Volume 13, no. 1, p. 33-37; Makhaeva G.F., Serebryakova O.G., Boltneva N.P., Galenko T.G., Aksinenko A.Yu., Sokolov V.B., Martynov I.V. Esterase profile and structure-inhibitory selectivity analysis of homologous phosphorylated 1-hydroperfluoroisopropanols. DAN, 2008, Volume 423, Issue 6, p. 826-831; Makhaeva G.F., Aksinenko A.Y., Sokolov V.B., Serebryakova O.G., Richardson R.J. Synthesis of organophosphates with fluorine-containing leaving groups as serine esterase inhibitors with potential for Alzheimer disease therapeutics. Bioorg. Med. Chem. Let. 2009, V.19, P.5528-5530; Aksinenko A.Yu., Sokolov V.B., Goreva T.V., Makhaeva G.F. Synthesis of O-phosphorylated 1-substituted 2,2,2-trifluoroethanol - serine hydrolase inhibitors. Izvestia AN, ser. Chem., 2010, No. 1, pp. 103-106].

However, the practical use of organophosphorus inhibitors of carboxyl esterases causes some caution in connection with their possible delayed neurotoxic effect.

The aim of the invention is to expand the arsenal of tools that can be used as new effective and selective irreversible inhibitors of carboxyl esterase having low acute toxicity to warm-blooded animals.

The problem is solved by the synthesis and use of a low-toxicity for warm-blooded new compound - 2,2,2-trifluoro-1-trifluoromethylethyl ester of cyclohexylcarbamic acid of formula (I) as an effective tool for selective irreversible inhibition of carboxyl esterase.

(I)

The compound of formula (I) - 2,2,2-trifluoro-1-trifluoromethylethyl ether of cyclohexylcarbamic acid is synthesized starting from cyclohexyl isocyanate and 1-hydrohexafluoroisopropanol. Reagents reaction is carried out at ²⁰ ° C in benzene in the presence of catalytic amounts of triethylamine, whereby a desired compound (I) (Scheme 1).

Scheme 1

The synthesis of the compounds of formula (I)

Cyclohexylcarbamic acid 2,2,2-Trifluoro-1-trifluoromethylethyl ester (I)

To a solution of 25.0 mmol of cyclohexyl isocyanate in 10 ml of benzene, 25.5 mmol of 1-hydrohexafluoroisopropanol and 0.05 g of triethylamine are added. The reaction mass is stirred for 2 hours at 20 ° C. The benzene is evaporated and the residue is recrystallized from hexane. Yield 91%. T. pl. 94-96 ° C. ¹ H NMR spectrum (200 MHz, CDCl ₃ , δ, ppm): 1.03-1.45 m (5 N, s-C ₆ H ₁₁ ); 1.54-1.83 m (3 N, s-C ₆ H ₁₁ ); 1.90-2.05 m (2 N, s-C ₆ H ₁₁ ); 3.53 m (1 N, s-C ₆ H ₁₁ ); 4.99 d (1 N, NH, J _HH = 5.1 Hz); 5.68 (m, 1 H, CHO). ¹⁹ F NMR spectrum (188 MHz, CDCl ₃ , δ, ppm): 4.03 (d, J _FH = 5.2 Hz).

Biological Testing Procedure

For kinetic studies, commercial preparations (Sigma, United States) of human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8), and porcine liver carboxyl esterase (EC, EC 3.1.1.1) were used.

Determination of enzyme activity

The activity of AChE and BChE was determined by the Ellman method (λ 412 nm) using acetylthiocholine (1mM) and butyrylthiocholine (1mM) as a substrate, respectively; determination conditions: 0.1 M phosphate buffer pH 7.5, 25 ^° C. CE activity was determined spectrophotometrically by the release of 4-nitrophenol (λ 405 nm), the substrate - 4-nitrophenyl acetate (1mM); determination conditions: 0.1 M phosphate buffer pH 8.0, 25 ^° C. The measurements were carried out on a BioRad Benchmark Plus microplate spectrophotometer.

Determination of IC Values _fifty  for inhibition of AChE, BChE and CE

The initial assessment of the inhibitory activity of the compound was carried out by determining the values of the IC ₅₀ - the concentration of inhibitor, which is required to reduce the activity of the enzyme by 50%. To determine the IC ₅₀ inhibition of AChE, BChE, and CE by compound (I), a sample of the corresponding enzyme was incubated with the test compound (DMSO concentration 2 vol%) for 8 minutes under the conditions of [I] ₀ >> [E] ₀ (I is an inhibitor, E is an enzyme), then the residual activity of the enzyme was determined. The concentration range of the test compound was 1 · 10 ^-12 - 4 · 10 ^-4 M. Each experiment was carried out in triplicate. Measurements were performed on a BioRad Benchmark Plus microplate spectrophotometer. The calculation of the IC ₅₀ was performed using the Origin 6.1 program.

Determination of bimolecular constants of inhibition of CE

To study the kinetics of CE inhibition, an enzyme sample was incubated with the test compound (DMSO concentration 2 vol%) under the condition [I] ₀ >> [E] ₀ , then the residual enzyme activity was determined. During the study, 3-4 inhibitor concentrations were used, and for each concentration 4-5 different incubation time intervals. Measurements were performed on a BioRad Benchmark Plus microplate spectrophotometer. The tangent of the slope angles ( k ' ) of each primary dependence of log (% inhibition) on time was determined by linear regression. Then, the obtained k ′ values were plotted against the inhibitor concentration [ I ] and the slope ( k " ) of the secondary dependence was determined by linear regression. The bimolecular inhibition constant ( k _i ) as a measure of the inhibitory activity of the compound was calculated from the equation k _i = 2.303 k ' / [ I ] = 2.303 k " . Data analysis and all calculations were performed using the Origin 6.1 program.

Determination of acute toxicity of a compound

The experiments were carried out on outbred white mice weighing 25-30 g. The test compound was dissolved in DMSO and was administered once intraperitoneally (iv) in increasing doses in a volume of about 0.1 ml. A minimum of 6 animals were used for each dose. Control animals received only DMSO. The observation period is 24 hours. The LD ₅₀ value was calculated by the probit analysis method (Finney's method) using the BioStat 2006 Professional software.

Biological Test Results of Compound (I) Illustrating the Present Invention

The inhibitory activity of compound (I) against three commercial preparations of serine esterases was determined: human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE), horse serum butyrylcholinesterase (EC 3.1.1.8, BChE) and pig liver carboxyl esterase (EC 3.1.1.1, EC) . An initial assessment of inhibitory activity was carried out by determining the values of IC ₅₀ - the concentration of inhibitor, which is required to reduce the activity of the enzyme by 50%. The study showed that the proposed compound (I) does not inhibit AChE, weakly inhibits BChE: IC ₅₀ = (1.70 ± 0.07) · 10 ^-4 M and exhibits high inhibitory activity against CE: IC ₅₀ = (5.23 ± 0.96) · 10 ^{- 7} M.

The inhibitory selectivity of compound (I) with respect to CE in comparison with BChE is characterized by the ratio of IC ₅₀ values and is equal to 325.

A study of the kinetics of CE inhibition by carbamate (I) showed the irreversible nature of the process with the degree of enzyme inhibition dependent on the incubation time and the linear dependence of the pseudo first order rate constant on the concentration of the inhibitor. The bimolecular CE inhibition constant k _i = (1.42 ± 0.20) · 10 ⁵ M ^-1 min ^-1 , i.e. compound (I) is an effective irreversible CE inhibitor.

The determination of acute toxicity in experiments on outbred white mice with iv administration showed that the proposed compound (I) has low acute toxicity for warm-blooded animals: LD ₅₀ > 2000 mg / kg

Thus, compound (I) is an effective and selective irreversible inhibitor of carboxyl esterase with low acute toxicity for warm-blooded animals and can be used in preclinical studies in rodents to reduce the rate of carboxyl esterase hydrolysis of drugs containing ester and / or amide groups, as well as inhibitors insect carboxyl esterases in pharmaceutical mixtures and formulations of pesticides to increase their effectiveness.

Claims (2)

1.2,2,2-Trifluoro-1-trifluoromethylethyl ether of cyclohexylcarbamic acid (I)

possessing irreversible selective inhibitory activity against carboxyl esterase. 2. The use of compound (I) according to claim 1 as an agent for selective irreversible inhibition of carboxyl esterase.