EA009847B1 - Cystic fibrosis transmembrane conductance regulator protein inhibitors and uses thereof - Google Patents

Abstract

The invention provides compositions, pharmaceutical preparations and methods for inhibition of cystic fibrosis transmembrane conductance regulator protein (CFTR) that are useful for the study and treatment of CFTR-mediated diseases and conditions. The compositions and pharmaceutical preparations of the invention may comprise one or more thiazolidinone compounds, and may additionally comprise one or more pharmaceutically acceptable carriers, excipients and/or adjuvants. The methods of the invention comprise, in certain embodiments, administering to a patient suffering from a CFTR-mediated disease or condition, an efficacious amount of a thiazolidinone compound. In other embodiments the invention provides methods of inhibiting CFTR that comprise contacting cells in a subject with an effective amount of a thiazolidinone compound. In addition, the invention features a non-human animal model of CFTR-mediated disease which model is produced by administration of a thiazolidinone compound to a non-human animal in an amount sufficient to inhibit CFTR.

Description

The transmembrane conduction regulator protein for cystic fibrosis (CPTC) is a chloride (C1 ^- ) channel activated by cyclic adenosine monophosphate (cAMP, cAMP), which is expressed in epithelial cells of the airways, intestines, pancreas, and mammalian testes. CPTC is a chloride channel that is responsible for the secretion of C1 ^- ions mediated by cyclic adenosine monophosphate. Hormones, such as a β-adrenergic agonist, or a toxin, such as cholera enterotoxin, lead to an increase in cAMP, activation of a cAMP-dependent protein kinase and phosphorylation of the C1 ^- channel of CPTC, which leads to the opening of the channel. An increase in the concentration of Ca ^{2+ ions} in the cell can also activate various channels of the apical membrane. Phosphorylation by protein kinase C can also open or close the C1 ^- channels in the apical membrane. IBS predominantly located in epithelia where it provides the formation of conductive paths for the movement of ions C1 ^- across the apical membrane and a key factor in controlling the speed of transepithelial salt and water transport. The functioning of the SRTC chloride channel is associated with a wide range of diseases, including cystic fibrosis (SR), and with some forms of male infertility, polycystic kidney disease and secretory diarrhea.

Hereditary fatal disease cystic fibrosis (SR) is caused by mutations in the IBS. The study of cystic fibrosis (CP) in male patients and in mouse models of CP shows the functional significance of CPTC for fluid transport in the intestines and pancreas, as well as for male fertility (Auger e! A1., 1999, Pb.u8u1. Keu. 79: 8193 -8214; Woid, Ρ.Υ., 1997, Mo1. Thread Kergob. 4: 107110). However, the mechanism by which a defective CRTK causes respiratory tract disease remains unclear, which is the main cause of morbidity and mortality in patients with SR (Рее ^ 8к1 е! А1., 1999, Ryuzyuk Keu. 79: 8215-8255). The main difficulties in understanding the causes of respiratory diseases in SR are the inadequacy of murine models of SR, in which respiratory diseases develop little or do not occur, in the absence of SR models in large animals and in the limited availability of human respiratory tract infections that were not damaged by a chronic infection or inflammation. High affinity CPTK-selective inhibitors were not available to study the mechanisms of respiratory tract disease in SR or to create the phenotype of SR in large animal models.

High affinity CPTK inhibitors also find clinical use in the treatment of secretory diarrhea and polycystic kidney disease, as well as inhibition of fertility in men. Compounds such as diphenylamine-2-carboxylate (ERS) and 5-nitro-2- (3-phenylpropylamino) benzoate (ΝΡΡΒ) inhibit CPTK at high concentrations, however, their inhibitory effect is not specific (CaBa! ., 1992, At. 1. РЬ.у8ю1. 262: С803-С827; МсПоидий е! А1., 1994, Game number 13: 623-634; 8ο1ιιι11ζ е! А1., 1999, Ryuzyuk Keu. 79: 8109-8144 ) Glibenclamide, which is the best CPTK inhibitor available for electrophysiological and other studies based on the study of cells, is used in concentrations> 100 μM (8errag6 e! A1., 1992, 1. Oey. Pb.u8y1. 100: 573-591; Noidge e! A1., 1994, RDidegs Agsy. 426: 284-287). However, at such concentrations, glibenclamide inhibits the action of other C1 ^- ion transporters, as well as K ⁺ channels (Eb ^ arbz e! A1., 1993, Vg. 1. Ryagtaso. 110: 1280-1281; Kabe e! A1., 1995 , RDidegs Agsy. 429: 659-662; ζataζak ^ e! A1., 1997, C1gs. Kez. 81: 101-109). Effective inhibitors of other ion transport proteins are known that have small molecular sizes, however, small sized molecules that have specific inhibitory activity against CPTC and are suitable for the treatment of secretory diseases are not known.

Thus, there is a need for CPTK inhibitor compounds and methods for using such compounds to develop animal models suitable for studying and treating CP, as well as for treating and controlling secretory disorders. The aim of the present invention is the satisfaction of these and other needs, as well as overcoming deficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions and methods for inhibiting a transmembrane conductivity regulator protein for cystic fibrosis (CPTC), which are useful in the study and treatment of CPTC-mediated diseases or conditions. The pharmaceutical compositions of the present invention may contain one or more thiazolidinone compounds or their derivatives and may additionally contain one or more pharmaceutically acceptable carriers, excipients and / or adjuvants. In some embodiments, the methods of the present invention comprise administering to a patient suffering from CPTK-mediated diseases or conditions an effective amount of a thiazolidinone compound or derivative thereof. Other embodiments of the invention relate to methods of inhibiting CPTK, which comprise contacting the subject's cells with an effective amount of a thiazolidinone compound or derivative thereof. In addition, the present invention describes a model of CPTK-mediated disease in a non-human animal, wherein the model is obtained by administering a thiazolidinone compound or derivative thereof to a non-human animal in an amount sufficient to inhibit CPTK.

- 1 009847

These and other objects and advantages of the present invention will become apparent from the following detailed description of the invention.

Brief Description of the Figures

The present invention will become more apparent after reference to the following figures, which are given for illustrative purposes only.

In FIG. 1A schematically illustrates screening techniques that are used to determine CETE inhibitors. CETE is maximally stimulated by many agonists in stably transfected epithelial cells that jointly express human CETE and yellow fluorescent protein (ΥΕΡ), the fluorescence of which is sensitive to CE / G ions. After adding the test compound, the influx of I ^- ions ^is induced by the addition of G-containing solutions.

FIG. 1B is a graphical representation of typical fluorescence data in individual wells, which are obtained using the selection procedure shown in FIG. 1A, and it shows data for controls (without activator, without test compound), for inactive compounds, and for active compounds that inhibit CETE.

In FIG. 1C shows the chemical structure of the CETE inhibitors identified by the screening technique of FIG. 1A.

In FIG. 1Ό shows the chemical structures of ring 2 of a thiazolidinone derivative having the highest inhibitory activity with respect to CETE. The full structure of the thiazolidinone derivative is shown in FIG. 1C. The relative efficiency is 0.2 (set _1pE 020), 0.3 _(1p set -029), 1.0 _(1p set -172), 0.2 _(1p set -185), 0.1 _(1p set -214 ) and 0.1 _(1p set -236).

FIG. 2A is a graphical representation of the dependence of the relative magnitude of fluorescence on time using the sampling technique of FIG. 1A, for the CETE 3 inhibitor [[(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (designated as CETESche-172) at several concentrations.

FIG. 2B is a graphical representation of the amount of inhibition over time, which shows the CETE-mediated rates of ion transfer I ^- at various time intervals after adding 2 μM CETE1bb-172. The inset graphically shows the inverse of the inhibition over time, which shows the transfer rates of I ^- ions at different time intervals after washing out 1 μM CETE1pe-172; the given value is the mean ± standard error of the mean obtained from a series of three experiments.

In FIG. 2C graphically shows the inhibition of CETE after stimulation by various agonists, including benzoflavone and benzimidazolone IS _CE compounds (IS _ce- 029 (2- (4-pyridinium bisulfate) benzo [y] -4H-chromen-4-one) and IS _ce -853 (Sayeia s1 a1. 2001, ί. ΒίοΙ. C11ss. 276: 19723-19728)), genistein, CPT-cAMP, 8-methoxypsoralen (8-MPO), 8-cyclopentyl-1,3-dipropylxanthine (CPX) (all 50 μM each) (± standard error of the mean value obtained from a series of three experiments). The shaded boxes indicate agonist and unfilled rectangles show agonist together with 5 uM set _1p -172.

In FIG. 3A graphically shows the inhibition of short circuit current under the action set _1p -172 EET in permeabilized cells expressing human set. CHAINS are stimulated with 100 μM CPT-cAMP.

In FIG. 3B is a summary charts inhibitory values depending on the dose set to -172 _1p (circles) and glibenclamide (squares) (8E, standard error of the mean values obtained from a series of three experiments).

FIG. 3C graphically illustrates the inhibition of short-circuit current under the action of CETESche-172 in the primary culture of (non-permeabilized) human bronchial epithelial cells. The inhibitor is added to the apical wash solution (left data group) or first to the basolateral and then to the apical solution (right data group).

In FIG. 3Ό graphically presents the magnitude of the potential on the entire cell membrane of EET cells expressing CETE; shows the currents on the membrane obtained at +80 mV (open circles) and minus 100 mV (filled circles). Set stimulatory effect of 5 uM forskolin followed by addition of 2 uM set _1p -172.

In FIG. 3E graphically shows that alternative stimulation is interrupted (ac) to apply a potential gradient.

In FIG. 3E graphically depicts the dependence of current on voltage under basal conditions (control, open circles), after stimulation with forskolin (open circles) and after adding 0.2 μM CETESche-172, providing ~ 50% inhibition (open triangles).

In FIG. 4A graphically shows stimulated by the action of MFI (100 μM) Ca ²⁺ -dependent secretion of C1 ^- ions, which is determined by measuring the short-circuit current in the epithelial cells of the respiratory tract in the absence of CETE _1пЕ -172 and in the presence of 5 μM CETE _1пЕ -172.

In FIG. Figure 4B graphically shows the current of C1 ^- ions activated in the volume (hypotonic 250 mOsm / kg H2O), measured in experiments to determine the potential value over the entire cell membrane

- 2 009847 cells of ECT. Currents are recorded in the absence of CETK _1P b-172 and in the presence of 5 μM CETK _1P b-172.

In FIG. 4C graphically shows the accumulation of ³ H-vincristine in 9 вο- / ϋΧ cells with increasing expression of MEK-1. Intracellular content of vincristine was determined in the absence and in the presence of verapamil (100 uM) or -172 mesh _1p (5 uM) (8Ε. N = 3).

In FIG. Figure 4Ό presents a graph showing the registered typical membrane potential of β-cells of the pancreas (ΙΝ8-1) irrigated extracellularly with CETK _{| mN} -172 solutions. diazoxide (100 μM) and glibenclamide (10 μM).

In FIG. 4E graphically depicts mean changes in membrane potential (DMV). caused by procedures. indicated in FIG. 4Ό (8Ε. N = 4).

In FIG. Figure 5A shows a photograph of isolated mouse ileum loops 6 h after injection of 1 μg of cholera enterotoxin into the intestinal cavity without an intraperitoneal injection of CETK _1P1 172 (upper image) or with an intraperitoneal injection (medium image) CETK _1p41 -172 (150 μg / kg). For comparison, a control sample with physiological saline (without cholera enterotoxin. Bottom picture) is shown.

FIG. 5B graphically shows the weight of the ileum loop after 6 hours as the mean ± standard error of the mean (n = 6-8 mice) for 14-16 studied loops. For the inactive analogue, the 4-carboxyphenyl group in CETK _1P b-172 is replaced by a 3-methoxy-4-methoxyvinylphenyl group (8Ε. 6-8 mice in the same group. * P <0.001. ΑΝΟνΑ).

FIG. 5C graphically explains the total weight of the small intestine 6 hours after oral administration through a probe before removal of intracavitary fluid compared with the state after removal of intracavitary fluid (8 (. 4 mice in one group. P <0.001).

FIG. 5Ό graphically explains the inhibition of the short-circuit current under the action of SETK _1P 17 -172 after the addition of amiloride and stimulation with forskolin (20 μM) in the mucous membrane of the isolated colon. Grid _1P b-172 is applied. as indicated (n = 4) on the surface of the serous membrane. and then the mucous membrane.

In FIG. Figure 6 schematically shows the synthesis of ¹⁴ C-labeled СЕТК _1п41 -172. Label ¹⁴ C is introduced into the thiazolidinone nucleus. using starting compound ¹⁴ C-labeled bromoacetic acid.

In FIG. 7 are photographs. which shows the results of a pharmacokinetic study of S. TTK. ,,, 1-172 in rats after a single intravenous infusion of a bolus of 50 μCi ¹⁴ C-labeled S. TTK. ,,, 1-172. Data are presented as mean values ± standard error of the mean (n = 3-6 rats) for serum radioactivity. The leveling curve corresponds to a 2-chamber model with a half-life of 0.14 hours, a half-removal period of 10.3 hours, and a maximum serum concentration of 3.2 μg / ml. area under the curve 3.8 μg-h / ml. a distribution volume of 1.2 L and a purification coefficient of 99 ml / h.

In FIG. 8 is a series of photographs. which shows the distribution of ¹⁴ C-labeled CETK1b172 after bolus infusion. The results in data group A were obtained for mice. who received a single bolus infusion of 2 μCi ¹⁴ C-labeled СЕТК1Пъ-172. mortified at specified time periods. and for organs. allocated to measure ¹⁴ C-radioactivity. the data are presented as the total ¹⁴ C-radioactivity of the organ at the indicated time periods (excluding skeletal muscles. The results for which are given per 1 g of tissue) after infusion (mean ± standard error of the mean. 4 mice for each point). The results in data group B were obtained for rats. which were injected with a bolus of 50 μCi ¹⁴ C-labeled CETK _1P b-172. and for the number of SETK _1P b-172 throughout the organ. measured 60 min after infusion (3 rats).

In FIG. 9 is a series of photographs. which show the results of a study of the metabolism of CETK _1p11 -172 by thin-layer chromatography of liquids and homogenate of the liver of mice. which were infused with ¹⁴ C-labeled CETK | mN-172. as shown in data group A in FIG. 8. The standard values of ¹⁴ C-SETK1bb-172 are 1. 3 and 6 nCi (data group on the left) and 10. 30 and 60 nCi (data group on the right). The films are autoradiographed for 48 hours (data group on the left) and 12 hours (data group on the right).

In FIG. 10 is a series of photographs. which presents the results of studying the model of the closed intestinal loop of the mouse. Results of data group A: 200 μl of buffer solution is injected into the intestinal loops and the loop weight is measured at the indicated time intervals (mean ± standard error of the mean. 4 mice for each time point). Insert (bottom):% absorption after 30 min without SETK _{| mN-} 172 and together with SETK _11L- 172 (20 μg intraperitoneally. N = 4). Insert (top): chemical structure СЕТК _1П ъ-172. Results of data group B: cholera-enterotoxin-induced fluid secretion over time in a mouse closed loop model. The dashed line shows the control loops (with saline injection). Data for injection loops (1 μg cholera enterotoxin per 1 loop) are presented as mean ± standard error of the mean (4-6 mice).

In FIG. 11 shows a series of graphs. which shows inhibition by CETSCs, 172 secretion of intraintestinal fluid after administration of a cholera enterotoxin mouse. Group given

- 3 009847 A: dose-dependent response to inhibit fluid accumulation in a mouse loop model. Mice are given a single dose of CPTK. _1b -172 by intraperitoneal injection and measure the weight of the loop (mean ± standard error of the mean, 4-6 mice per dose) after 6 hours. The dashed line shows the average weight in control loops of the same mice into which physiological saline was injected. Data Group B: resistance of inhibition under the action of CPTK.pd-172. Mice were injected with 20 μg of CPTC. _{1p |} -172 (intraperitoneally) at the indicated time intervals before or after administration of cholera enterotoxin (4-6 mice at each time point). Data group C: the radioactivity value of ¹⁴ С-СРТК.1п | -172 over time after intravenous injection (caudal vein, left ordinate) and oral administration (СРТК.1п-172 in ТРС8, right ordinate). Data are presented as the number of counts per minute per 1 μCi per injection (4 mice). Data group Ό: accumulation of ¹⁴ C-CPTK.1p | -172 in the gastrointestinal organs 6 hours after intravenous and oral administration of ¹⁴ C-CPT 1p _| -172 (4 mice). Data Group E: Inhibition of cholera-enterotoxin-induced fluid secretion by oral administration of CPT 1p _| -172 (200 μg in TPC8) in a mouse model of an open loop. Data are presented as the total weight of the small intestine 6 hours after oral administration through a probe before removal of intracavitary fluid relative to the state after removal of intracavitary fluid (mean ± standard error of the mean, 4 mice in one group, * p <0.001). Data Group D: CPTK Permeability. _{| m |} -172 through Saso-2 monolayers (mean ± standard error of the mean, 18 inserts) with Papr = 16x10 ^-6 cm / s.

In FIG. 12 is a series of graphs showing inhibition by CPT 1p _| 172 cholera-induced enterotoxin (data group A) and 8Ta toxin (data group B) fluid secretion in a rat closed loop model. Data are presented as mean values ± standard error of the mean (4 rats per group), * p <0.01.

In FIG. 13 shows a series of graphs showing inhibition by CPT 1p _| 172 forskolin and 8Ta toxin-induced short circuit currents in the mouse ileum (data group A) and the human colon (data group B). Data for 8Ta toxin is shown in the inset. The data are the results of a study of 5 mice and 2 sets of human tissues. СРТЕ 1п _| -172 is added on both sides of the fabric. Amyloride (10 μM) is present in apical solutions.

In FIG. Figure 14 shows a series of graphs that show the study of short circuit current during inhibition by the action of SRTC. _{1p |} -172 secretions of C1 ions ^- in T84 colon epithelium cells. Data Group A: Data are shown as recorded curves from experiments with 512 inserts per state. SRTC. _Lb- 172 is added on both sides of the cell layers. SRTC agonists. They include forskolin (left) and 8 W-sSMR (middle) and SRTE _AC1 -16 (right). Data Group B: (Left) Inhibition by CPTK _{1p |} -172 Forskolin-stimulated short-circuit current after basolateral permeabilization with amphotericin B (250 μg / ml). Typical experimental data are given in 6 boxes. (In the middle) the average dose-response for inhibition by CPT 1p _| -172 stimulated forskolin (circles) and stimulated 8-W-cMSP (triangles) short circuit current for permeabilized T84 cells in comparison with non-permeabilized T84 cells (mean ± standard error of the mean, 6-12 inserts). (Right) Inhibition by CPTP 1p _| -172 forskolin stimulated short-circuit current in the presence of high concentrations of K ⁺ (68 mM) to basolateral solution with a low ion content C1 ^- apical solution. Typical data from 4 experiments are presented.

Before the invention is described, it should be emphasized that the present invention is not limited to the specific embodiments presented, which, of course, may have variations. It should also be understood that the terminology used is used only to describe specific embodiments of the invention and should not be construed as limiting the present invention since the scope of the claims of the present invention is limited only by the appended claims.

Unless otherwise specified, all technical and scientific terms that are used in the present description have the same meaning as commonly understood by specialists in the field of technology to which the present invention relates. The following describes methods and materials preferred for carrying out the present invention, although any methods and any materials similar or equivalent to those described herein may be used in the implementation or verification of the present invention. All of these publications are incorporated into this description by reference to disclose or describe the methods and / or materials in connection with which these publications are cited.

It should be noted that the sole forms given in the description and claims include many meanings, unless the context otherwise indicates. So, for example, a reference to the term inhibitor includes many such inhibitors, and a reference to the term cell includes a link

- 4 009847 per one or more cells and their equivalents, known to specialists in this field of technology, etc.

The publications discussed in this description are published prior to the filing date of this application, and they are incorporated into this description by reference. Nothing in the present description should be construed as a recognition that the present invention has a priority later than the specified publication. Moreover, the publication dates shown may differ from the actual publication dates and may require independent confirmation.

The definitions used in the present description are given only for the purpose of a clearer presentation, and should not be construed as limiting the present invention. All technical and scientific terms that are used in the present description have the same meaning as commonly understood by specialists in the field of technology to which the present invention relates.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that thiazolidinone compounds or their derivatives are NET affinity inhibitors. The structure of these compounds and their derivatives of the present invention, as well as pharmaceutical compositions and methods of their use are described in more detail below.

Definitions

A condition or symptom mediated by the transmembrane conductivity regulator protein for cystic fibrosis or NETWORK-mediated condition or symptom means any condition, disorder or disease, or symptom of a similar condition, disorder or disease that results from the activity of the transmembrane conductivity regulator protein for cystic fibrosis (NET) in particular, NETWORK activity in ion transport. These conditions, disorders or diseases or their symptoms can be treated by inhibiting the activity of the NETWORK, in particular by inhibiting the activity of the NETWORK in ion transport. NETWORK activity is manifested, for example, in intestinal secretion in response to the action of various agonists, including cholera enterotoxin (see 8pubet e! A1. 1982 Vi11. \ U'ogk1 Nea Ogdap. 60: 605-613; Failed e! A1. 1994 EMBO 1. 13: 1065-1072; Kltbetd e! A1. 1971 1. Sat. 1p / eU 50: 1218-1230).

The term NETWORK inhibitor as used herein means a compound that reduces the efficiency of ion transport under the influence of NETWORK, in particular, the transport of chloride ions under the influence of NETWORK. Preferred NETWORK inhibitors of the present invention are specific NETWORK inhibitors, i.e., compounds that inhibit NETWORK activity without significantly or adversely affecting the activity of other ion carriers, in particular other chloride carriers, potassium carriers, and the like. The NETWORK inhibitors are preferably high affinity NETWORK inhibitors, in particular inhibitors whose affinity for the NETWORK is at least about 1 μmol, usually about 1 to 5 μmol.

The term treatment as used herein encompasses the treatment of a disease, condition, disorder or symptom thereof in a subject where the disease, condition, disorder or symptom is mediated by the activity of the NETWORK, and includes: (1) prevention of the disease, condition or disorder, i.e., exposure, leading to the fact that a person who is exposed to a disease, condition or disorder, or who is predisposed to the effects of a disease, condition or disorder, but does not yet feel or does not detect its symptoms, does not develop clinical symptoms of the disease, (2) suppression of the disease, condition or disorder, i.e., stopping or slowing down the development of the disease, condition or disorder or its clinical symptoms; or (3) amelioration of a disease, condition or disorder, i.e. exposure that results in regression of the disease, condition or disorder or its clinical symptoms.

A therapeutically effective amount or an effective amount means an amount of a compound of the present invention which, when administered to a mammal or other person in need thereof, is, as indicated above, sufficient to treat diseases, conditions, disorders or symptoms mediated by NETWORK activity. The amount of the compound of the present invention, which is a therapeutically effective amount, will vary depending on the compound, the disease and its severity, as well as on age, weight, and the like. subject, the treatment of which is carried out, however, it can be determined by conventional methods by a person skilled in the art, taking into account his knowledge and information given in the present description.

The terms “subject and patient” mean a representative or representatives of any mammalian species or non-mammalian species that may require pharmaceutical methods, compositions, and methods of treatment described herein. Subjects and patients, therefore, include, without limitation, representatives of the primate family (including humans), canine, feline, ungulates (in particular horses, cows, pigs (eg, domestic pig)), birds and other subjects. Of particular interest are people and other animals other than humans that are of commercial importance (in particular livestock and domestic animals).

Mammal means a representative or representatives of any mammalian species, and includes, for example, members of the canine, feline, horses, cows, sheep, rodents, etc., and primates, in particular humans. Non-human animal models, in particular mammals

- 5 009847 melting animals, for example, families of primates, murine, hare-like, etc., can be used for experimental studies.

The term single dosage form as used herein refers to physically discrete units applicable as single dosages for humans and animal subjects, each unit containing a certain amount of the compound of the present invention, which is calculated so that it is sufficient to obtain the desired effect in combination with a pharmaceutically acceptable diluent, carrier or excipient. The requirements for the new single dosage forms of the present invention depend on the particular compound used and on the effect to be achieved, as well as on the pharmacodynamics associated with the behavior of each compound with respect to the recipient.

It should be understood that the term physiological conditions encompasses conditions that are compatible with living cells, in particular they are predominantly an aqueous medium with temperature, pH, salt content, etc. that are compatible with living cells.

Pharmaceutically acceptable excipient means an excipient suitable for the preparation of a pharmaceutical composition, which is generally safe, non-toxic and is neither biologically nor in any way undesirable, and includes an excipient that is acceptable for use in veterinary medicine, as well as suitable for pharmaceutical use for human. A pharmaceutically acceptable excipient as used herein and as claims includes one or more such excipients.

As used herein, the term pharmaceutically acceptable derivative of a compound of the present invention includes salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemicetals, acids, bases, solvates, hydrates or prodrugs thereof. These derivatives can be easily prepared by those skilled in the art using known methods for preparing said derivatives. The resulting compounds can be administered to animals or humans without causing toxic effects, and are either pharmaceutically active or prodrugs.

A pharmaceutically acceptable salt of a compound of the present invention means a salt that is pharmaceutically acceptable and which possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or salts formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfo hydrochloric acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] oct-2-en-1-carboxylic acid, glucoheptonic acid, 4, 4'-methylene-bis- (3-hydroxy-2-en-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid muconic acid and the like; or (2) salts obtained when the acidic proton present in the parent compound is either substituted by a metal ion, in particular an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or is coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, метил-methylglucamine, etc.

A pharmaceutically acceptable ester of a compound of the present invention means an ester that is pharmaceutically acceptable, has the desired pharmacological activity of the parent compound and includes, without limitation, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroalkyl, cycloalkyl and heterocyclyl esters acid groups, including, without limitation, acid groups of carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids Lot, sulfinic acids and boronic acids.

A pharmaceutically acceptable enol ether of the compound of the present invention means an enol ether which is pharmaceutically acceptable, has the desired pharmacological activity of the parent compound, and includes, without limitation, derivatives of the formula C = C (OK), where K is hydrogen, alkyl, alkenyl, an alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl group.

A pharmaceutically acceptable enol ester of a compound of the present invention means an enol ester that is pharmaceutically acceptable, has the desired pharmacological activity of the parent compound, and includes, without limitation, derivatives of the formula C = C (OS (O) K), where K is hydrogen, alkyl, alkenyl, alkynyl, aryl, ge

- 6 009847 teroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl group.

A pharmaceutically acceptable solvate or hydrate of a compound of the present invention means a solvate or hydrate complex that is pharmaceutically acceptable, has the desired pharmacological activity of the parent compound, and includes, without limitation, complexes of the compound of the present invention with 1 or more solvent molecules or water molecules, or 1 to about 100, or from 1 to about 10, or from 1 to about 2, 3, or 4 solvent molecules or water molecules.

A prodrug means any compound that releases the ίη νίνο active maternal compound of formula (I) when said prodrug is administered to a mammalian subject. Prodrugs of the compounds of formula (I) contain functional groups which, under standard physiological conditions, are hydrolyzed to the corresponding carboxyl groups, hydroxyl groups, amino groups and sulfhydryl groups. Examples of such functional groups include, without limitation, esters (in particular acetate, formate and benzoate derivatives) and carbamates (in particular Ν, Ν-dimethylaminocarbonyl) formed by hydroxyl groups in the compounds of formula (I), and the like . Additional examples include dipeptide or tripeptide esters formed by hydroxyl or carboxyl groups in the compounds of formula (I), and the like. The preparation of such functional groups is well known in the art. For example, a compound of formula (I) that contains a hydroxyl group (in particular when X ₁ , X ₂ , X ₃ , Υ ₁ , Υ ₂ or Υ ₃ denotes a hydroxyl group) can be affected by a carboxylic acid or dipeptide having a free terminal carboxyl group, under the conditions of the esterification reaction, well known in the art, and to obtain the desired ester functional group. Similarly, a compound of formula (I) containing a free carboxyl group can be affected by an alcohol or tripeptide containing a hydroxyl group, such as a serine residue (in particular - ^ H) -C (H) (CH ₂ OH) -C (O) -), under the conditions of the esterification reaction, well known in the art, and to obtain the desired ester functional group. In addition, a compound of formula (I) containing an ester group of a carboxylic acid can be treated with another carboxylic acid ester under standard conditions for the transesterification reaction to obtain a compound of formula (I) containing the desired ester functional group. All of these functional groups are included in the scope of the present invention.

The term organic group or organic radical as used herein refers to any carbon-containing group, including hydrocarbon groups, which are classified as aliphatic groups, cyclic groups, aromatic groups, their functional derivatives and / or various combinations thereof. The term aliphatic group means a saturated or unsaturated linear or branched hydrocarbon group and encompasses, for example, alkyl, alkenyl and alkynyl groups. The term alkyl group means a substituted or unsubstituted, linear or branched hydrocarbon group or chain (in particular, from ₁ to ₈ ) and includes, for example, methyl, ethyl, isopropyl, tert-butyl, heptyl, n-octyl, dodecyl, octadecyl an amyl, 2-ethylhexyl group and the like. Suitable substituents include a carboxyl group, a protected carboxyl group, an amino group, a protected amino group, a halogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a lower alkylthio group, a nitro group, a cyano group, a monosubstituted amino group, a protected mono-substituted disubstituted amino substituted ₇ -substituted , C | -C-acyl group, C1-C ₇ acyloxy group and the like. The term substituted alkyl means a specified alkyl group containing, from 1 to 3 hydroxyl groups, protected hydroxyl groups, amino groups, protected amino groups, cyano groups, halogen atoms, trifluoromethyl groups, monosubstituted amino groups, disubstituted amino groups, lower alkoxy groups, lower alkoxy groups, lower alkoxy groups , carboxyl groups, protected carboxyl groups, or a salt of a carboxyl group, amino group and / or hydroxyl group. As used with substituents for heteroaryl rings, the terms substituted (cycloalkyl) alkyl and substituted cycloalkyl as indicated below are substituted with the same groups as those listed for the substituted alkyl group. The term alkenyl group means an unsaturated linear or branched hydrocarbon group with one or more carbon-carbon double bonds, such as a vinyl group. The term alkynyl group means an unsaturated linear or branched hydrocarbon group with one or more triple carbon-carbon bonds. The term cyclic group means a closed ring hydrocarbon group, which is classified as an alicyclic group, an aromatic group or a heterocyclic group. The term alicyclic group means a cyclic hydrocarbon group whose properties resemble those of an aliphatic group. The term aromatic group or aryl group means a mono or polycyclic aromatic hydrocarbon group and may include 1 or more heteroatoms, and it is discussed in more detail below. The term heterocyclic group means a closed cyclic hydrocarbon in which 1 or more atoms in the ring represent an element other than carbon (in particular, nitrogen, oxygen, sulfur, etc.), and it is discussed in more detail below.

- 7 009847

The organic group may be functionalized or in some other way may contain additional functional groups connected to the organic group, such as a carboxyl group, an amino group, a hydroxyl group and the like, which may or may not be protected. For example, it is assumed that the phrase alkyl group includes not only pure open-chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert-butyl, etc., but also alkyl substituents that contain additional substituents known from the technical field, such as a hydroxyl group, an alkoxyl group, a mercapto group, an alkylthio group, an alkylsulfonyl group, a halogen, a cyano group, a nitro group, an amino group, a carboxyl group, etc. Thus, the alkyl group includes esters, ethers, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms halogen group or halogen are used in the text of the present description as equivalent and refer to fluorine, chlorine, bromine or iodine. Preferred halogens are chlorine and fluorine.

The term haloalkyl refers to an alkyl group as defined above substituted with 1 or more halogen atoms. Halogen atoms may coincide or differ from each other. The term dihaloalkyl refers to an alkyl group as defined above, substituted with 2 halogen atoms, which may be the same or different from each other. The term trihaloalkyl refers to an alkyl group as defined above, substituted with 3 halogen atoms, which may be the same or different from each other. The term perhaloalkyl refers to a haloalkyl group as defined above, wherein each hydrogen atom in the alkyl group is replaced by a halogen atom. The term perfluoroalkyl refers to a haloalkyl group as defined above, wherein each hydrogen atom in the alkyl group is replaced by a fluorine atom.

The term cycloalkyl refers to a mono-, bi- or tricyclic saturated ring that is fully saturated or partially unsaturated. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis or trans decalin, bicyclo [2.2.1] hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl 1,4-cyclooctadienyl and the like.

The term (cycloalkyl) alkyl means an alkyl group, as defined above, substituted by one of the above cycloalkyl groups. Examples of such groups include a (cyclohexyl) methyl, 3- (cyclopropyl) -n-propyl, 5- (cyclopentyl) hexyl, b- (adamantyl) hexyl group, etc.

The term substituted phenyl refers to a phenyl group substituted by one or more moieties, in some cases, one, two or three moieties selected from the groups consisting of halogen, hydroxyl group, protected hydroxyl group, cyano group, nitro group, mercapto group, alkylthio group, trifluoromethyl group, C1 -S7alkilnuyu group, C | -C alkoxy group, a _C1-C7 acyl group, a _C1-C7 acyloxy, carboxyl, oxycarbonyl, protected carboxy, carboxymethyl, protected carboxymethyl, hydro ksimetilnuyu, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino, carboxamide, protected carboxamide, N- (C ₁ -C _b alkyl) carboxamide, protected N- (C ₁ -C _b alkyl) carboxamide, M, S-di (C ₁ -C _b alkyl) carboxamide, trifluoromethyl group, S - ((C ₁ -C _b alkyl) sulfonyl) amino group, S- (phenylsulfonyl) amino group or phenyl group, substituted or unsubstituted in such a way as, for example, to obtain biphenyl or phthyl group.

Examples of the term substituted phenyl group include a mono- or di (halogen) phenyl group such as a 2-, 3- or 4-chlorophenyl, 2, b-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2-, 3 - or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-, 3- or 4-fluorophenyl group and the like; a mono- or di (hydroxy) phenyl group, such as a 2-, 3- or 4-hydroxyphenyl, 2,4-dihydroxyphenyl group, their protected hydroxy derivatives, etc .; a nitrophenyl group such as a 2-, 3- or 4-nitrophenyl group; a cyanophenyl group, for example 2-,

3- or 4-cyanophenyl group; mono- or di (alkyl) phenyl group, such as 2-, 3- or 4-methylphenyl, 2,4-dimethylphenyl, 2-, 3- or 4- (isopropyl) phenyl, 2-, 3- or 4-ethylphenyl , 2-, 3 or 4- (n-propyl) phenyl group and the like; mono- or di (alkoxy) phenyl group, for example 2, b-dimethoxyphenyl, 2-, 3- or 4- (isopropoxy) phenyl, 2-, 3- or 4- (tert-butoxy) phenyl, 3-ethoxy-

4-methoxyphenyl group and the like; A 2-, 3- or 4-trifluoromethylphenyl group; a mono- or dicarboxyphenyl or (protected carboxy) phenyl group, such as a 2-, 3- or 4-carboxyphenyl or 2,4-di (protected carboxy) phenyl group; a mono- or di (hydroxymethyl) phenyl or (protected hydroxymethyl) phenyl group, such as a 2-, 3- or 4- (protected hydroxymethyl) phenyl or 3,4-di (hydroxymethyl) phenyl group; a mono- or di (aminomethyl) phenyl or (protected aminomethyl) phenyl group, such as a 2-, 3- or 4- (aminomethyl) phenyl or 2,4- (protected aminomethyl) phenyl group; or a mono- or di (Y- (methylsulfonylamino)) phenyl group, such as a 2-, 3- or 4- (Y- (methylsulfonylamino)) phenyl group. In addition, the term substituted phenyl includes disubstituted phenyl groups, where the substituents are different from each other, for example

- 8 009847 measures a 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl group etc.

The term (substituted phenyl) alkyl means one of the above-defined substituted phenyl groups which is attached to one of the above-defined alkyl groups. Examples include groups such as 2-phenyl-1-chloroethyl, 2- (4'-methoxyphenyl) ethyl, 4- (2 ', 6'-dihydroxyphenyl) n-hexyl, 2- (5'-cyano-3' -methoxyphenyl) n-pentyl, 3- (2 ', 6'-dimethylphenyl) propyl, 4-chloro-3-aminobenzyl, 3 - (4'-methoxyphenyl) -3-carboxyhexyl, 5- (4'-aminomethylphenyl) -3 - (aminomethyl) pentyl, 5-phenyl-3-oxopent-1-yl, (4-hydroxy-naphth-2-yl) methyl group, etc.

As indicated above, the term aromatic or aryl refers to 5- and 6-membered carbocyclic rings. In addition, as indicated above, the term heteroaryl means optionally substituted 5- or 6-membered rings that contain from 1 to 4 heteroatoms, such as oxygen, sulfur and / or nitrogen atoms, in particular nitrogen atoms, both independently and in combination with sulfur and oxygen atoms in the ring. Said 5- or 6-membered rings may be completely unsaturated.

Moreover, the above optionally substituted 5- or 6-membered rings may optionally be fused to an aromatic 5- or 6-membered ring system. For example, rings may optionally be fused to a 5- or 6-membered ring system, such as a pyridine or triazole system, and preferably to a benzene ring.

The following ring systems are examples of heterocyclic (both substituted and unsubstituted) radicals designated by the term heteroaryl: thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, pyridriol, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazyl, pyrazolid, pyridazinyl, oxazinyl, triazinyl, thiadiazinyl, tetrazole, 1,5- [b] pyridazinyl and purinyl groups, as well as condensed with benzene nym ring derivatives, for example benzoxazolyl, benztiazolilnaya, benzimidazolyl and indolyl group.

Substituents for the above optionally substituted heteroaryl rings are from 1 to 3 halogen atoms, trihalomethyl groups, amino groups, protected amino groups, salts of amino groups, monosubstituted amino groups, disubstituted amino groups, carboxyl groups, protected carboxyl groups, carboxylate salts, hydroxyl groups, protected hydroxyl salts of hydroxyl groups, lower alkoxy groups, mercapto groups, lower alkylthio groups, alkyl groups, substituted alkyl groups, cycloalkyl groups, substitution ennyh cycloalkyl groups, (cycloalkyl) alkyl groups, substituted (cycloalkyl) alkyl groups, phenyl groups, substituted phenyl groups, phenylalkyl groups and (substituted phenyl) alkyl groups. Substituents for the heteroaryl group are as previously defined, and in the case of a trihalomethyl group, they may be a trifluoromethyl, trichloromethyl, tribromomethyl or triiodomethyl group. With respect to use in conjunction with the above substituents for heteroaryl rings lower alkyl means _C1-C4 alkoxy group, similarly, a lower alkylthio group means a C ₁ to C ₄ alkylthio group.

The term (monosubstituted) amino refers to an amino group with one substituent selected from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, _C1-C4 acyl, C ₂ -C ₇ alkenyl, C ₂ to C ₇ substituted alkenyl, C ₂ -C ₇ alkynyl, C ₇ -C _{1 6} alkylaryl, C ₇ -C _{1 6} substituted alkylaryl and heteroaryl groups. The specified (monosubstituted) amino group may additionally have a protecting amino group, which is defined by the term protected (monosubstituted) amino group. The term (disubstituted) amino group refers to amino groups with two substituents selected from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1-C7 acyl, C2-C7 alkenyl, C2-C7 alkynyl, C ₇ -C ₆ alkylaryl, C ₇ -C 1 ₆ substituted alkylaryl and heteroaryl group. The two substituents may be the same or different.

The term heteroaryl (alkyl) means the above-defined alkyl group substituted at any position with a heteroaryl group as defined above.

Optional or optionally means that the event, circumstance, feature or element described below can, but does not necessarily, occur, and this definition includes cases in which the event or circumstance occurs and cases in which they do not occur. For example, a heterocyclic group, optionally mono- or disubstituted by an alkyl group, means that the alkyl group may, but is not necessarily, present, and the description includes situations where the heterocyclic group is mono- or disubstituted by an alkyl group, and situations where the heterocyclic group is not substituted by the indicated alkyl group .

The term electron withdrawing group refers to the ability of a functional group in a molecule to attract electrons to itself more than a hydrogen atom could do if hydrogen occupied the same position in the molecule. Examples of electron withdrawing groups include, but are not limited to, halogen atoms, —C (O) I (where I is alkyl); carboxyl groups and ester groups; groups -ΝΚ ₃ ⁺ (where I is alkyl or hydrogen); azo groups; nitro groups; groups and —OY and —BY (where I is alkyl or hydrogen); and organic groups (as defined in

- 9 009847 in the present description) containing similar electron-withdrawing groups, such as haloalkyl groups (including perhaloalkyl groups), etc.

A compound that has the same molecular formula, but differs in the type or sequence of bonds of their atoms or in the mutual arrangement of their atoms in space, is called isomers. Isomers that differ in the mutual arrangement of their atoms in space are called stereoisomers. Stereoisomers that are not mirror images of one another are called diastereomers, and those stereoisomers that are mirror images of one another that are not compatible with each other are called enantiomers. If the compound has an asymmetric center, for example a center that is associated with 4 different groups, then a pair of enantiomers is possible. The enantiomer can be characterized by the absolute configuration of its asymmetric center and described using the rules of K- and 8-sequences of Kahn and Prelog, or it can be characterized by the way the molecule rotates the plane of polarization of light, and then it is designated as dextrorotatory or levorotatory (i.e. e. as (+) - isomer or (-) - isomer). A chiral compound can exist either as an individual enantiomer or a mixture thereof. A mixture containing equal proportions of enantiomers is called a racemic mixture.

The compounds of the present invention may have one or more asymmetric centers; therefore, such compounds can be obtained as individual (K) or (8) stereoisomers or as a mixture thereof. Unless otherwise indicated, it is understood that the designation or name of a particular compound in the present description and claims includes both individual enantiomers and mixtures thereof — racemic or other mixtures. Methods for establishing stereochemistry and methods for separating stereoisomers are well known in the art (see, in particular, the discussion in chapter 4 of the monograph Abuaseb Ogdashs SNetMgu. 4 ' ¹ ' eB Shoi. 1. MarsE, 1oNi Ubeu aib 8oiz, No. 1992).

Overview

The present invention relates to compositions based on thiazolidinones, compositions based on derivatives of thiazolidinones and their use for a high-affinity inhibition protein transmembrane conductivity regulator in cystic fibrosis (CPTC) and in the study or treatment of CPTC-mediated diseases and conditions. The detection of the desired thiazolidinone compounds and their derivatives is based on the selection of numerous potential candidate compounds by analysis, the purpose of which is to identify CPTC inhibitors that directly interact with CPTC. Without being bound by any particular theory or practice, since the studies that led to the identification of the target compounds included numerous CPTC activators acting on different activating pathways, the authors believe that the inhibitor compounds of the present invention are likely to exert its inhibitory effect due to the impact directly on the pathway of CPTC transport of C1 ions ^- or on the adjacent areas. The selection of 50,000 different compounds revealed several 2-thioxo-4-thiazolidinone compounds and their derivatives as effective CPTC inhibitors. These compounds and their derivatives are not chemically and structurally similar to previously known CPTK activators or previously known CPTK inhibitors such as EDS, No.PB or glibenclamide. The most effective CPTC inhibitors identified by selection have a K ₁ value of ~ 300 nM for inhibition of C1 current ^- human respiratory tract cells. Inhibition is fast, reversible and CPTK-specific.

The compositions and methods of the present invention are described in more detail below. Thiazolidinone Compounds and Their Derivatives

Thiazolidinone compounds and their derivatives, which are used in the compositions and methods of the present invention, include 3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinones of the formula (1b)

where X ₁ represents trifluoromethyl; X ₂ and X _{3 are} independently selected from hydrogen and a halogen atom; Υ1, Υ2 and Υ3 are independently selected from hydrogen, a C1-C8 alkyl group, a C1-C7 alkoxy group, a carbonate group, a carbamate group, a carboxyl group, a halogen atom, a nitro group, an azo group, a hydroxyl group, and a mercapto group. In one embodiment of the invention, X ₁ is in a position selected from 2, 3, or 4; Υ2 occupies a position selected from 2, 3 or 4; and Υ1 and Υ3 can be hydrogen.

More specifically, 3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinones may have the formula (1c)

- 10 009847 where the values of Υ ₁ -Υ ₃ are given above. In one embodiment, the trifluoromethyl group is in a position selected from 2, 3, or 4; Υ ₂ occupies a position selected from 2, 3 or 4; and Υ ₁ and Υ ₃ in this embodiment of the invention may be hydrogen.

In some embodiments, the thiazolidinone compound of the present invention may be

those. 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone;

those. 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-hydroxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone;

those. 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone;

those. 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone;

those. 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; and

those. 3 - [(3-trifluoromethyl) phenyl] -5 - [(3-bromo-4-hydroxy-5-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone.

Alternatively, the trifluoromethyl group in any of the above compounds may occupy position 2 or position 4 in the benzene ring.

Pharmaceuticals

The invention also relates to pharmaceutical preparations of the above thiazolidinone compounds of the present compound. The compounds of the present invention can be included in various formulations for administration in various ways for therapeutic purposes. In particular, the compounds of the present invention may be formulated into pharmaceutical compositions in combination with appropriate pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants, and may be formulated in a solid, liquid or gaseous form, such as tablets , capsules, powders, granules, ointments, solutions, suppositories, injections, dosage forms for inhalation and aerosols. The compositions preferably do not contain detectable amounts of DMSO (dimethyl sulfoxide), which is not an acceptable carrier, diluent, excipient and / or adjuvant for non-local, parenteral or enteral administration. The compositions can be developed for administration to needy subjects or patients through various routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, percutaneous, intratracheal, and the like. introduction.

One embodiment of the present invention is topical application (in particular transdermal administration). Formulations for topical application may be in the form of a transdermal patch, ointment, paste, lotion, cream, gel, etc. Formulations for topical application may include one or more wetting agents, thickeners, diluents, emulsifiers, dispersing agents, or a binder

- 11,009847 generals. In cases where the compounds are prepared for transdermal delivery, the compounds can be prepared using agents that facilitate the penetration of drugs. Drug penetration enhancing agents, which may be drug penetration chemical agents and physical penetration enhancing agents, facilitate the delivery of compounds through the skin, and may equally be referred to as penetration enhancers. Physical penetration aids include, for example, electrophoretic methods such as iontophoresis, the use of ultrasound (or phonophoresis), and the like. Penetration-enhancing chemicals are agents that are administered either before, together, or immediately after the administration of drug compounds and which enhance the permeability of the skin, in particular the outer layer of the epidermis, in order to increase the penetration of the drug through the skin.

Compounds that are used to increase skin permeability include sulfoxides, such as dimethyl sulfoxide (DMSO) and decyl methyl sulfoxide (Xiu M8O); ethers such as diethylene glycol monoethyl ether, decaoxyethylene oleyl ether and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Plohateg (231, 182, 184), T \ heep (20, 40, 60, 80) and lecithin; 1-substituted azacycloheptan-2-ones, in particular 1-n-dodecylcyclazacycloheptan-2-one; alcohols such as ethanol, propanol, octanol, benzyl alcohol and the like; petroleum jelly, such as petroleum jelly (petrolatum), mineral oil (liquid petrolatum), etc .; fatty acids such as C ₈ -C ₂₂ and other fatty acids (in particular isostearic acid, octanoic acid, oleic acid, lauric acid, valerianic acid); C ₈ -C ₂₂ fatty alcohols (in particular, oleyl alcohol, lauryl alcohol); lower alkyl esters of C ₈ -C ₂₂ fatty acids and other fatty acids (in particular ethyl oleate, isopropyl myristate, butyl stearate, methyl laurate, isopropyl myristate, isopropyl palmitate, methyl propionate, ethyl oleate); monoglycerides of C8-C22 fatty acids (in particular glyceryl monolaurate); tetrahydrofurfuryl alcohol ether and polyethylene glycol; 2- (2-ethoxyethoxy) ethanol; diethylene glycol monomethyl ether; polyethylene oxide alkylaryl ethers; polyethylene oxide monomethyl esters; polyethylene oxide dimethyl esters; C ₆ -C ₈ diacid lower alkyl esters (in particular diisopropyl adipate); ethyl acetate; acetoacetate ether; polyols and their esters, such as propylene glycol, ethylene glycol, glycerin, butanediol, polyethylene glycol and polyethylene glycol monolaurate; amides and other nitrogen-containing compounds such as urea, dimethylacetamide (EMA), dimethylformamide (DMF), 2pyrrolidone, Ν-alkylpyrrolidone, in particular 1-methyl-2-pyrrolidone; ethanolamine, diethanolamine and triethanolamine; terpenes; alkanones and organic acids, in particular salicylic acid and salicylates, citric acid and succinic acid. Additional chemical and physical agents that facilitate the penetration of drugs are described, for example, in documents A.R. Kuyosheik (ΕΌ) 1987 SKE Rgekk; Regsi1apeoik Repe! Ha1yup Epyaysegk, ayk. διηίΐΐι e! a1. (SCS Rgekk, 1995); Jeppegiac e! A1., 1. RNagt. Ryagtaso 1. 2002; 54 (4): 499-508; Kagapye e! A1., RNagt. Kek. 2002; 19 (5): 655-60; Wye1 e! A1., 1. RNagt. 8s1. 2002 1i1u; 91 (7): 1639-51; Wow! A1., 1. Aegis of Tagde! 2001; 9 (5): 379-93; 8 Knock e! A1., 1p !. 1. RNagt. 2001; 228 (1-2): 99-107; 8ιιζιι1 <ί е! A1., Vu1. RNagt. Vi11. 2001; 24 (6): 698-700; A1e e! A1., 1. Sop1go1 Ke1eake 2001; 71 (3): 319-27; Oo1yk! Yesh! A1., 2001; 57 (2): 301-5; Kshchauashep e! A1., Eig. 1. RNagt. 8s1. 2000; 10 (2): 97-102; and Tep) ar1a e! A1., 1p !. 1. RNagt. 1999; 192 (2): 147-58.

If the compositions are prepared using a chemical agent that facilitates the penetration of any compound, the penetration facilitating agent is chosen so that it is compatible with this compound and is present in an amount sufficient to facilitate the penetration of the compound through the skin of the subject, in particular with the purpose of delivery of the specified compounds in the circulatory system. In one embodiment of the present invention, a composition is prepared from said compound using a penetration facilitating agent other than DMSO.

In one embodiment of the present invention, said compound is delivered in the form of a therapeutic patch, in particular a patch for delivery through the mucous membrane or through the skin, and may include a penetration enhancing agent. The patch typically contains a protective layer that is not permeable to the compound and other components of the composition, a matrix that contacts one side of the protective layer, wherein the matrix provides a sustained release (this release may be controlled) of the compound, and an adhesive layer, which is located on the same side of the protective layer as the matrix. The matrix can be selected so that it matches the route of administration, and it can be, for example, a polymer matrix or a matrix of hydrogel.

In pharmaceutical dosage forms, the target compounds of the present invention can be administered in the form of their pharmaceutically acceptable derivatives, such as salts, or they can be used either alone or in an appropriate combination thereof, as well as in combination with other pharmaceutically active compounds. The following methods and excipients are given, rather, as an example and do not limit the present invention.

For the preparation of oral preparations, the compounds of the present invention can be used alone or in combination with appropriate additives used for the manufacture of

- 12 009847 tablets. powders. granules and capsules. for example, along with regular supplements. such as lactose. mannitol. corn starch or potato starch; with binders. such as crystalline cellulose. cellulose derivatives. gum. corn starch or gelatins; with baking powder. such as corn starch. potato starch and sodium carboxymethyl cellulose derivative; with facilitating tabletting substances. such as talc or magnesium stearate; and. if necessary. with thinners. buffering agents. humidifiers. preservatives and fragrance. Of particular interest are the preparations of the thiazolidinone compounds of the present invention together with a buffering agent. in order to protect the compound from low pH in the stomach. Preferably, an enteric coating is applied thereto. to prevent the precipitation of the compound during its transit through the stomach.

Dissolution preparations for injection can be prepared from the target compounds of the present invention. suspending or emulsifying the compounds in aqueous or non-aqueous solvents. such as vegetable or similar oils. synthetic glycerides of aliphatic acids. higher aliphatic acid esters or propylene glycol; and. if necessary. along with regular supplements. such as solubilizers. isotonic agents. suspending agents. emulsifiers. stabilizers and preservatives. Solubilizers of particular interest include vitamin E TPO8 (ά-α-tocopheryl polyethylene glycol 1000 succinate). cyclodextrins, etc.

The compounds of the present invention can be used as aerosol formulations for administration by inhalation. Compounds of the present invention can be prepared in admixture with pressurized acceptable carrier gases. such as dichlorodifluoromethane. propane. nitrogen, etc.

Besides. the compounds of the present invention can be prepared in the form of suppositories by mixing with a variety of bases. such as emulsifiable bases or water soluble bases. The compounds of the present invention can be administered rectally with a suppository. A suppository may include a carrier. such as cocoa butter. hydrocarbon waxes and polyethylene glycols. which melt at body temperature. but. harden at room temperature.

Single dosage forms for oral or rectal administration may be prepared. such as syrups. elixirs and suspensions. in which each dosage unit. for example a teaspoon. tablespoon. tablet or suppository. contains a certain amount of composition. including one or more inhibitors. Similarly. single dosage forms for injection or intravenous administration may include the inhibitor (s) in the composition as a solution in sterile water. normal saline or other pharmaceutically acceptable carrier.

Depending on the subject and condition. which treatment is carried out. as well as the route of administration. the compounds of the present invention may be administered in dosage form. components. eg. from 0.1 mcg to 10 mg / kg body weight per day. A wide range is indicated. since in the general case, the effectiveness of the therapeutic effect for different mammals varies widely. at the same time, typical doses for humans are 20. 30 and even 40 times less (per unit body weight). than the dose for the rat. Besides. the route of administration can have a large effect on the dose. The inventors have discovered. that the intestinal secretion of fluid caused by cholera enterotoxin in mice is effectively blocked by a single intraperitoneal dose. component of approximately 10-20 mcg. and the effective dosage for rats is approximately 10 times greater. So. eg. oral dosages may be approximately 10 times the dosage for injection. Higher doses may be used for localized delivery routes.

A typical dosage may be a solution. suitable for intravenous administration; a pill. which is taken 2 to 6 times daily. or capsule or tablet. simultaneously emitting medicine. which is taken 1 time per day and which contains a proportionally larger amount of the active ingredient. etc. The effect of prolonged release can be achieved using capsule materials. which dissolve at different pH values. using capsules. which slowly release the drug under the influence of osmotic pressure. or using another known drug for controlled release of the drug.

For use in the methods of the present invention, formulations with other pharmaceutically active agents can be prepared from the compounds of the present invention. including other SBTC-inhibiting agents.

Pharmaceutically acceptable excipients. suitable for use according to the present invention. such as carriers. auxiliary medicines. fillers or diluents. are publicly available. Besides. readily available auxiliary agents. such as pH regulators and buffering agents. agents to regulate tone. stabilizers. wetting agents, etc.

For a specialist should be clear. that dose levels may vary depending on the particular compound. the severity of the subject’s symptoms and sensitivity to side effects. Preferred dosages for a given compound can be readily determined by one skilled in the art using various techniques.

- 13 009847

Single dose kits of the compounds of the present invention are provided, which are usually oral or injection doses. Such kits, in addition to containers containing single doses, are supplied with embedded information that describes the use and corresponding benefits of drugs for the treatment of a pathological condition of interest. Preferred compounds and unit doses are those described herein.

Conditions that can be treated with the inhibitors of the NETWORK of the present invention

The NETWORK inhibitors disclosed herein are useful in the treatment of a NETWORK-mediated condition, in particular any condition, disorder or disease or symptoms of the condition, disorder or disease that results from an NETWORK activity, such as an NETWORK activity in ion transport. These conditions, disorders or diseases or their symptoms can be treated by inhibiting the activity of the NETWORK, in particular, ion transport under the influence of the NETWORK.

In one embodiment of the invention, the NET inhibitors of the present invention are useful in treating conditions associated with increased excess secretion in the intestine, especially increased excess secretion in the intestine in acute form. The NETWORK takes an active part in intestinal secretion as a response to the action of various agonists, including cholera enterotoxin (see, for example, 8pubeg e! A1., 1982 Vi11. \ U1b Nea1b Ogdap. 60: 605-613; Failed e! A1. , 1994 EMBO 1. 13: 1065-1072; Eltbegd e! A1., 1971 1. Sat. 1pu§ !. 50: 1218-1230). Thus, the NETWORK inhibitors of the present invention can be administered in an amount effective to inhibit ion transport by the NETWORK and, therefore, to reduce the secretion of fluids in the intestine.

Thus, NET inhibitors can be used in the treatment of inflammatory bowel diseases and diarrhea, especially secretory diarrhea. Secretory diarrhea is one of the main causes of child mortality in developing countries and kills about 5 million lives annually (6aLe1 e! A1., 1994 8c1epse 266: 107-109). A number of studies, including with the use of mice suffering from CE, indicate that NET is the usual final metabolic pathway for the secretion of chloridion a (and, consequently, fluid secretion) in the intestine as a response to the action of various agonists (8cube e! A1., 1982 Vi11. \ Wag1b Nea1b Ogdap. 60: 605-613; Sbao e! a1., 1994 EMBO 1. 13: 1065-1072; and Krtbegd e! a1., 1971 1. Sat. 1pu§ !. 50 : 1218-1230). As used herein, a murine model of intestinal fluid secretion shows that inhibition of NET by systemically administering a non-toxic dose of the inhibitor effectively blocks cholera enterotoxin-induced fluid secretion in the intestine (see examples).

Diarrhea that can be treated using the NET inhibitors of the present invention can be the result of exposure to various pathogens or agents, including, without limitation, cholera enterotoxin (U1bgyu Spo1ega), E. coli (in particular its enterotoxigenic strain (ETEC)), 8b1de11a; to increased intestinal secretion, about Mediated NETWORK.

Other diarrhea include diarrhea associated with LH8 (AIDS) (in particular AIDS-related diarrhea) and inflammatory diseases of the gastrointestinal tract, such as ulcerative colitis, inflammatory bowel disease (1VE). Crohn's disease, etc. It has been reported that intestinal inflammation modulates the expression of the three major mediators of salt transport in the intestine and can contribute to ulcerative colitis both by enhancing the transepithelial secretion of C1 ^- ions and by inhibiting the absorption of NaCl in the epithelium (see, for example, Lob1 e! A1., 2002 , At. 1. RBU 1.

The NETWORK inhibitors of the present invention can be used in the treatment of conditions such as polycystic kidney disease, and are further used as drugs against male infertility by inhibiting the NETWORK activity in the testes.

The NETWORK inhibitors of the present invention can be further selected in large animal models (in particular, the rabbit model, which is described in 8p1a e! A1., 1981, 1nGes !. 1typ. 32: 739-747). In addition, to further investigate the properties of the NET inhibitors of the present invention, analysis of isolated stool can be performed using a live cholera embryo.

Non-human animal models and human tissue models with NET deficiency

The NETWORK inhibitors of the present invention can also be used to develop non-human disease models where the disease is associated with increased NETWORK function (in particular, increased ion transport). There is strong evidence that defective secretion of fluid and macromolecules by glands located under the mucous membrane leads to impaired cleansing of the ciliary epithelium and the removal of bacteria in subjects with NET deficiency, in particular those affected by cystic fibrosis (CE, CE); nevertheless, functional studies of the glands in the human respiratory tract are limited to severe respiratory diseases that occurred during lung transplantation (Kuagatap e! A1, 2001, Proc. No. 111. Asab. 8sk I8A 98: 8119-8123). Emergency in

- 14 009847 bending CETE allows you to determine the role of CETE in the secretion of water, salt and macromolecules by the glands located under the mucous membrane. High affinity CETE inhibitors allow pharmacologically to develop non-human animal models that mimic CETE deficiency in humans, for example, mimic the human CE phenotype. In particular, models of SETE deficiency in large animals (in particular, CE) are of particular use for identifying the pathophysiology of initiating the development of respiratory diseases in CE and for evaluating the effectiveness of CE therapy, in particular, in selecting candidates for agents for the treatment of SETE deficiency or its symptoms .

Inhibition of NET transport of ions can occur in diseases of the respiratory tract and pancreas, as well as in infertility in men. For example, inhibition of CETE channels in the lungs and airways affects liquids on the surface of the airways, which leads to the accumulation of mucus, which, in turn, clogs the airways and collects in large quantities on the walls of the lungs, thereby creating the primary environment for infection , which, in turn, can lead to the development of chronic lung diseases. The same phenomenon is observed in the pancreas, where the accumulated mucus disrupts the exocrine function of the pancreas and prevents the most important food-processing enzymes from entering the intestines.

These non-human animal models can be created by administering an amount of a CETE inhibitor effective to reduce the activity of CETE in ion transport. Of particular interest is the use of the CETE inhibitors of the present invention to induce the phenotype of cystic fibrosis (CE) in non-human animal models. The administration of an amount of a CETE inhibitor effective to inhibit CETE receptors, for example, in the lungs, effectively mimics the lack of CETE observed in CE. Routes for administering a CETE inhibitor are discussed in detail above. Depending on the non-human animal model used, the compounds of the present invention can be administered in dosages of, for example, 50 to 500 μg / kg body weight 1 to 3 times a day by intraperitoneal, subcutaneous or other way to create non-human animal models . Oral dosages can be 10 times the intraperitoneal and subcutaneous doses.

Non-human animal models of CETE related diseases can be used as models of any suitable condition associated with reduced CETE activity. Similar conditions include those that are associated with CETE mutations, and these mutations are the result of abnormalities in the transport of ions and water in the epithelium. These abnormalities can, in turn, be associated with a dysfunction of clearing the ciliary epithelium in the airways, as well as in the epithelium of other mucous membranes and duct epithelium. Conditions that can be pharmacologically modeled by inducing CETE deficiency phenotypes in non-human animal models include, but are not limited to, cystic fibrosis (including atypical CE), idiopathic chronic pancreatitis, damage to the vas deferens, mild lung disease, asthma, and t .P. For a review of disorders associated with impaired function of CETE, see, for example, in Eope e1 a1., Ecre. Her§. 2: 232-332 (2001). Non-human animal models generated by the CETE inhibitor can also serve as models of microbial infection (e.g., bacterial, viral or fungal infection, in particular respiratory infection) in a subject with a CETE deficiency. In one embodiment of particular interest, the CETE inhibitors of the present invention are used to pharmacologically induce the phenotype of cystic fibrosis (CE).

Animals that are suitable for use in creating animal models of the present invention include any animals, in particular mammals, for example non-human primates (e.g., monkey, chimpanzee, gorilla, etc.), rodents (e.g., rats, mice, gerbils, hamsters, African ferrets, etc.), rabbit-like, pig (e.g. pigs, pigs), equine, canine, feline, etc. Large animals are of particular interest.

CETE inhibitors can also come in contact with isolated human tissues to create an ex ν моделиνο model of the disease. These tissues are contacted with an amount of CETE inhibitor effective to reduce the activity of CETE in the tissue, which can be observed for at least 15 minutes or up to 2 hours or more. Human tissues of interest include, but are not limited to, lungs (including the trachea and respiratory tract), liver, pancreas, testicles, etc. Physiological, biochemical, genomic, and other studies can be performed with inhibitor-treated tissues to identify new target therapeutic molecules that are important in the pathophysiology of the disease. For example, tissues taken from people without CE can be exposed to an inhibitor in an amount sufficient to induce the CE phenotype, and similar studies can be conducted to identify new targeted therapeutic molecules that are important in the pathophysiology of CE.

The synthesis of compounds of the present invention

Compounds of the present invention can be obtained in accordance with methods known to those skilled in the art, or by methods similar to those disclosed in US Pat. No. 5,327,770 or US Pat. No. 6,380,186 (all of which are incorporated into this description by

- 15 009847 references), or in accordance with methods similar to those described in the present description.

It should be understood that in the following description, combinations of substituents and / or variables in the formulas below are permissible only when such combinations result in the formation of stable compounds.

One skilled in the art will appreciate that in the methods described below it may be necessary to protect the functional groups of the intermediates with suitable protecting groups. Such functional groups include a hydroxyl group, an amino group, a mercapto group and a carboxy group. Suitable protecting groups for the hydroxyl group are a trialkylsilyl or diarylalkylsilyl (e.g. tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl group), tetrahydropyranyl, benzyl group and the like. Suitable protecting groups for an amino group, an amidino group and a guanidino group include a tert-butoxycarbonyl, benzyloxycarbonyl group and the like. Suitable protecting groups for the mercapto group include the groups —C (O) —I (where I represents an alkyl, aryl or aralkyl group), a p-methoxybenzyl, trityl group, etc. Suitable protecting groups for a carboxylic acid are alkyl, aryl and aralkyl groups ethers.

Protecting groups may be introduced or removed in accordance with standard procedures that are well known to those skilled in the art and are also described herein.

The use of protective groups is described in detail in the monograph of Tieobog ^. Steys, RS1sg S.M. \ Yi15. Prgoec1 | ye Sgirk ίη Otdashs 8up1ye518 (1999), 3 ^GB Eb., \ UPeu-1p1eg5C1epse. A protecting group may also be a polymer resin, such as Wang resin or 2-chlorotrityl chloride resin.

For a person skilled in the art it should be clear that, despite the possible lack of pharmaceutical activity as such for such protected derivatives of the compounds of formula (I) described above (in particular, in the sections Overview and Thiazolidinone compounds and their derivatives), the above derivatives can be administered to a mammal, and then they are metabolized to form compounds of the present invention that are pharmacologically active. Therefore, such derivatives will be called prodrugs. All prodrugs of the compounds of formula (I) are within the scope of the present invention.

The following reaction schemes illustrate methods for preparing the compounds of the present invention. It should be understood that a person of ordinary skill in the art is able to obtain the compounds of the present invention using similar methods or using methods known to those skilled in the art. In the general case, the starting components can be obtained from sources such as A1bpsy, or synthesized in accordance with literature known from the technical field (see, in particular, 8th upb Matei, Magsyk Abuapseb Offsets: Yaasyopk, Messyashtk, apb 8yis1ige, 5 ^4th ebayyup (\ UPeu 1n1eg8s1epse, Nei Wogk)). Moreover, various substituted groups (e.g., X ₁ , X ₂ , X ₃ , Υ ₁ , Υ ₂ and Υ ₃ , etc.) in the compounds of the present invention can be attached to the starting components, intermediates and / or final prodrugs in accordance with methods known to those skilled in the art.

In the following reaction schemes, I represents an alkyl or aralkyl group, and represents a halogen atom such as C1, Br or I.

The following reaction scheme 1 is devoted to the preparation of compounds of formula (I) that have already been discussed previously (in particular, in the sections Overview and Thiazolidinone Compounds and Their Derivatives), where the values of X ₁ , X ₂ , X ₃ , Υ ₁ , Υ ₂ and Υ ₃ are indicated above (in particular, in the sections Overview and Thiazolidinone compounds and their derivatives).

Reaction Scheme 1

In general, compounds of formula (I) are prepared by first acting on a compound of formula (a) 1 eq. base, such as NaOH, at room temperature. Then, a compound of formula (s) dissolved in a suitable solvent, such as THF, is added to the reaction mixture. The resulting react

- 16 009847 the reaction mixture is stirred for 1-24 hours. Next, an acid such as HCl is added to the reaction mixture. The resulting reaction mixture was stirred for 1-24 hours. A compound of formula (c) was isolated from the reaction medium using standard isolation and purification techniques. Then, the compound of formula (c) is treated with the compound of formula (b) under standard Knoevenagel condensation conditions and the desired product of formula (I) is obtained.

Otherwise, the compounds of formula (I) can be obtained in accordance with the following reaction scheme 2, where the values of Υ ₁ , Υ ₂ and Υ ₃ are indicated above (in particular, in the sections Overview and Thiazolidinone compounds and their derivatives), and denotes halogen.

Reaction Scheme 2

In general, compounds of formula (I) are prepared by first acting on a compound of formula (e) by a compound of formula (ί) under standard Knevenagel condensation conditions, such as refluxing in the presence of a catalytic amount of piperidine in glacial acetic acid, alcohol or another suitable solvent. The compound of formula (e) is then isolated from the reaction mixture using standard isolation and purification techniques. Next, the compound of formula (e) is treated with the compound of formula (s) under standard Ulmann condensation conditions, in particular in the presence of Cu, or Cu ₂ O, or CuO at elevated temperatures, and the desired product of formula (I) is obtained.

Otherwise, the compounds of formula (I) can be obtained in accordance with the following reaction scheme 3, where the values of Υ ₁ , Υ ₂ and Υ ₃ are indicated above (in particular, in the sections Overview and Thiazolidinone compounds and their derivatives), and denotes halogen.

In this reaction scheme, the first step is Ulmann condensation between a compound of formula (e) and a compound of formula (s) to form a compound of formula (c), which is introduced into a Knevenagel condensation reaction with a compound of formula (b) to obtain the desired product of formula ( I).

The starting compound of formula (e) can be purchased from various suppliers of chemicals or synthesized in accordance with methods known to those skilled in the art or by methods similar to those disclosed in P.C. ΒΐΌ \ νπ c1 a1., 1. At. Siet. Zoe., 78, 384-388 (1956); K.E. Ziyye, Give Zup1ye818, SU 4, 6; K.8. Magk1eu apb E.E. Ke1b, 1. At. Siet. Zoe., 52, 2137-2141 (all of which are incorporated herein by reference in their entirety).

Using methods similar to the above, the synthesis of 3 - [(3-trifluoromethyl) phenyl] -5 - [(4carboxyphenyl) methylene] -2-thioxo-4-tazolidinone (denoted in the present description as CETK _1PY- 172) (see FIG. . 1C) and its analogues with a different arrangement of trifluoromethyl and carboxyl substituents.

- 17 009847 bodies (see, for example, Fig. 1Ό), carried out by Knoevenagel condensation of 2-thioxo-3- [atrifluoromethyl-4-phenyl] -4-thiazolidinone (a = 2, 3 or 4) with L-carboxybenzaldehyde (B = 2, 3 or 4) in the presence of piperidine. The precipitate formed is filtered off, washed with ethanol, dried and recrystallized 2-3 times from ethanol to obtain bright yellow crystals (yield 70-85%). Structures are confirmed by ¹ H-NMR. Purity is> 99% according to thin layer chromatography and HPLC.

Examples

The following examples are provided in order to fully disclose to specialists and describe how the present invention can be obtained and used, and should not be construed as limiting the scope of what, according to the inventors, is included in the claims of the present invention, as well as not they should be considered as the only experiments that were carried out by the authors of the present invention. The authors tried to get the exact values of the given numerical values (for example, quantities, temperature values, etc.), however, some experimental errors and deviations should be taken into account. Unless otherwise indicated, fractional parts are given in mass parts, molecular weights are weight average molecular weights, temperatures are given in degrees Celsius, and pressure is atmospheric or near atmospheric.

The synthesis of the compounds of the present invention is described as examples, however, the examples given do not limit the present invention.

Synthesis Example

Synthesis of 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-tazolidinone.

A. To a stirred solution of 3-trifluoromethylaniline (1.6 g, 10 mmol) and triethylamine (1 g, 10 mmol) in ethyl acetate (10 ml) was added dropwise carbon disulfide (0.8 g, 10 mmol) over 30 minutes. . The weakly exothermic reaction, which begins after approximately half of the reagent is added, is easily controlled using an ice bath from time to time. It is left to mix overnight, a thick yellow suspension is filtered off and the precipitate is washed with 50 ml of diethyl ether and dried in air to obtain 3 g (89%) of a pale yellow dithiocarbamate with a melting point of 92-95 ° C (with decomposition).

B. Sodium dichloroacetate (obtained from chloroacetic acid (0.064 g, 0.46 mmol) and 0.6 ml of solution No. HCO ₃ , pH 8-9) is stirred and cooled to 5-10 ° C, and then for 10 minutes dithiocarbamate (0.3 g, 0.9 mmol) was added. Stirring is continued and the flask is allowed to warm to room temperature. After stirring for 2 hours, the solution was cooled to 10 ° C, acidified with concentrated hydrochloric acid, and the reaction mixture was heated to 90-95 ° C for 30 minutes. The precipitate formed is filtered off, washed with water and recrystallized from ethanol to obtain 0.103 g of 2-thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone in the form of brilliant crystals with a yield of 83%, so pl. 177-178 ° C, ¹ H-NMR (300 MHz, ATP1 ₃ ): δ 4.18 (s, 2H, CH ₂ ), 7.40 (d, 1H, phenyl, 1 = 8.0 Hz), 7 48 (s, 1H, phenyl), 7.64 (t, 1H, phenyl, 1 = 8.0 Hz), 7.72 (d, 1H, phenyl, 1 = 7.6 Hz) ppm.

C. A mixture of the above 2-thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone (0.1 g, 0.36 mmol) and 4-carboxybenzaldehyde (0.054 g, 0.36 mmol) in absolute alcohol (1 ml ) and piperidine (1 drop) are stirred at the boil under reflux for 30 minutes. The yellow precipitate was filtered off, washed with ethanol, dried and recrystallized from ethanol to give 0.108 g (73%) of the title compound as a yellow crystalline solid, mp. 180-182 ° C, 'H-NMR spectrum (300 MHz, SES1 ₃ ): δ 7.78 (d, 2H, carboxyphenyl, 1 = 8.2 Hz), 7.80-8.00 (m, 5H, trifluoromethylphenyl and CH), 8.07 (d, 2H, carboxyphenyl, 4 = 8.31 Hz), 13.20 (s, 1H, COOH, Ό ₂ Θ exchange water) ppm.

Ό. Similarly as described above, the following compounds are prepared: 3 - [(3-trifluoromethyl) phenyl] -5 - [(3-carboxy-4-hydroxyphenyl) methylene] -2-thioxo-4-tazolidinone; 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,4,5-trihydroxyphenyl) methylene] -2-thioxo-4-tazolidinone; 3 - [(3-trifluoromethyl) phenyl] -5 - [(2,3,4-trihydroxyphenyl) methylene] -2-thioxo-4-tazolidinone; 3 - [(3-trifluoromethyl-4-fluoro) phenyl] -5 - [(3-carboxy-4-hydroxyphenyl) methylene] -2-thioxo-4-tazolidinone; 3 - [(4-fluoro-3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-tazolidinone.

The following materials and methods are used in the examples below.

Cell lines, mice and connections

Thyroid cells of rats G18syeg (ECT), coexpressing the wild type of human SETK and halogen indicator UEP-H148O. prepared as previously indicated (Sayeya e (a1., 2001, ί. ΒίοΙ. Seyesh. 276: 19723-19728). Cells are placed on 96-well black-walled microplates (from Sogspd Bochag) with a density of 20,000 cells per well per Kuhn-modified E12 medium supplemented with 5% serum of cow fetus, 2 mM L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin.The tests are carried out 48 hours after being placed on the plate, and during this time the cells only begin to merge ( ~ 40,000 cells per well).

Primary selection is carried out using a diverse collection of 50,000 drug-like compounds from SieshVpbde (San Diego, California), obtained as a 10 mM stock solution in DMSO and diluted in 96-well plates to a concentration of 100 mM. Structural Analysis

- 18 009847 activity is carried out on analogues purchased from SNetVgIde and ΟΊιοιηϋίν (San Diego, California).

Wild mice and mice with cystic fibrosis (homozygous mutant ΔΓ 508) are grown in the center of SG Lsta Soga at the University of California, San Francisco (IS8P). Animal research procedures were approved by the IS8P Animal Research Committee.

T84 and Caco-2 cells are obtained from the IS8P cell culture center. T84 cells are grown in a 1: 1 mixture of medium ΌΜΕΜ and Nutc P12 medium supplemented with 5% cow fetal serum, 100 units / ml penicillin and 100 μg / ml streptomycin, and 8par \\ ^g e11 inserts (from Sogshid Co51ag ) in a humid atmosphere (5% O ₂ /95% CO ₂ ) at a temperature of 37 ° C. Cells are used 10-14 days after placement on the tablet. Saso-2 cells were cultured in medium ΌΜΕΜ containing 10% serum of the fetal cow, 1% non-essential amino acids, 100 units / ml penicillin and 100 μg / ml streptomycin, and cultured at rates of 8pairs \\ 'e11. Cells are used 21-24 days after placement on the tablet. Wild mice with a genetic background of CE1 are grown as previously described. Male rats \ UMag (200-250 g) are bought from .Taskcoi LarvaFpec. Animal research procedures were approved by the IS8P Animal Research Committee. Fragments of the human colon are obtained directly during the excision operation and transported in ice-cold physiological saline for use within an hour after excision.

Forskolin, 8-bromo-cMSP, amiloride, cholera enterotoxin and 8Ta toxin are purchased from 81dta SNetka1 Co. (St. Louis, Missouri). IBS _AC1 -16 were obtained from SNET Vpbde (San Diego, CA).

Selection Techniques

Analyzes are carried out using a custom-designed sampling system (Vesktap), which includes a 3-meter robot arm, a CO ₂ incubator, a tablet washing machine, a manual liquid processing unit, a barcode reader, a lid remover and two fluorescent tablets Reader R1io81ag (Naval Forces company Laesyio1od1e8, Offenburg, Germany), each of which is equipped with two syringe pumps and an Hf500 / 20X filter (500 ± 10 nm) upon excitation and an emission filter (Hf535 / 30M (535 ± 15 nm) (from SNgot company ). Robotizirova The system was integrated using the 8LM1 Wugyui 3.3 (Veschtai) software, which was modified for two tablet readers. subtracting the value for the baseline from the protocols stored in the database.

The analysis is started by placing 40-60 96-well plates containing RKT cells in an incubator (37 ° C, 90% humidity, 5% CO2), and 96-well tablets with test compounds and removable plastic pipette nozzles are placed on the rotary mechanism . To initiate the analysis, each well of a 96-well plate is washed 3 times with phosphate-buffered saline (PB8) (300 μl per wash), leaving 50 μl of PB8 in the well. 10 μl of CPTC-activating cocktail (5 μM forskolin, 100 μM 1BMX, 25 μM apigenin in PB8) is added, and after 5 minutes the test compound (0.5 μl in 1 mM DMSO) is added to each well to obtain a final concentration of 10 μM. After 10 minutes, 96-well plates were transferred to a plate reader to study fluorescence. Each cell is individually examined for CPTC-induced ion transport I ^- by continuously recording fluorescence (200 ms per point) for 2 s (baseline), and then for 12 s after a quick (<0.5 s) addition of 160 μl of isosmolar PB8, in which 137 mM C1 ^{- is} replaced by I ^- .

Analysis of intracellular concentration [cAMP] and toxicity

Assays for [cAMP] and phosphatase are carried out as previously indicated (CaIeKa e1 a1., 2001, 1. Vu1. SN. 276: 19723-19728). Cellular toxicity is studied using the dihydrogenamine method 24 hours after incubation of the cells together with 0-1000 μM inhibitor. Animal toxicity is investigated by studying the chemical composition of serum and hematology (IS8P Clinical Laboratory) in mice after 5 days of daily intraperitoneal injection of the inhibitor in an amount of 0100 μg / kg.

Activity IOC-1

The activity of MEK-1 is evaluated by measuring the accumulation of ³ H-vincristine in the immortalized cell line of the human trachea, 9HTE- / Eh, in which the endogenous expression of MEK-1 is regulated in a controlled manner by selection in increasing concentrations of doxorubicin (Kak1a e1 a1., 1994, 1. Vue 1. SN. 269: 1432-1436). Cells are seeded in 24-well microplates (200,000 cells per well). After 48 hours, the cells are washed with a solution containing (in mM): 130 BaCl, 2 KCl, 1 KH ₂ PO ₄ , CaCl ₂ , 2 MDC1 ₂ , 10 Ba-Nerek (pH 7.3) and 10 glucose, and incubated in for 1 h at 37 ° C along with 200 μl of the same solution containing ³ N-vincristine (0.7 μM; 1 μCi / ml). Then the cells are washed three times with ice-cold solution and subjected to lysis by the action of 0.25 M NaOH solution. Vincristine content is determined by counting the number of scintillations.

- 19 009847

Study of short-circuit currents using ECT cells expressing NETWORKS

Insert 5> pairs \\ 'e11. containing ECT cells expressing NETWORKS or human bronchial epithelial cells are placed in the Ussing chamber system. In the case of ECT cells, 5 ml of a 75 mM solution of No. 101 and 75 mm of sodium gluconate (apical) and 150 mm of No. 101 (basolateral) (pH 7.3) are placed in hemicamers and the basolateral membrane is permeabilized with 250 μM / ml amphotericin B (Sayeia e! a1., 2001, ί. Βίο1. Seet. 276: 19723-19728). In the case of bronchial epithelial cells and T84 cells, both hemicamers contain Krebs bicarbonate solution. Through hemicamers, air (ECT cells) or 5% CO ₂ in the air (bronchial cells and T84 cells) is constantly bubbled and maintained at a temperature of 37 ° C. Short-circuit current is constantly recorded using the IUS-1000-volt clamp (from the company νοτ1ά Ргесткюп 11к1гшпеп1к. Sarasota, Florida), using Ad / AdC1 electrodes and 1M KC1 agar bridges.

Analysis of the activity of the C1 ^- channel by the method of point potential

Membrane current is measured in a full cell configuration. To register the C1 ^- channel, the extracellular (cuvette) solution contains (in mM): 150 No. C1, 1 CaCl ₂ , 1 MdCl ₂ , 10 glucose, 10 mannitol, 10 TE§ (pH 7.4), and intracellular (pipette) the solution contains: 120 SCC1, 1 MdC1 ₂ , 10 TEA-C1, 0.5 ECTA, 1 MD-ATP, 10 Nerek (pH 7.3). CETK is activated with forskolin (5 μM) in an extracellular solution. The change in membrane conductivity over time is controlled as a response to alternating voltage pulses from -100 to +80 mV. At a certain time, the execution of the procedure is interrupted to determine the dependence of current on voltage (voltage pulses from -100 to +100 mV in increments of 20 mV). Volume-sensitive C1 ^- channels are activated with a hypotonic solution (extracellular MK '! Reduce to 120 # C1; 250 mOsm / kg). Calcium-sensitive C1 ^- channels are activated in human bronchial epithelial cells by adding 100 μM MFI to the extracellular solution.

Analysis of the activity of ATP-sensitive K ⁺ channels by the method of point potential

Membrane potential recorded in the β-pancreatic cells lines [N§-1 in which the extracellular solution (cuvette) contains (in mM): 130 №S1 2 KC1, 1 KH ₂ PO _4, 2 SaS1 _2, 2 MdS1 ₂ 10 Νι Nerek (pH 7.3) and 10 glucose. The pipette contains (in mM): 140 KCl, 1 CaCl ₂ , 2 MdC1 ₂ , 10 ECTA, 0.5 MdATP, 10 K-Nerek (pH 7.3). Upon reaching the full-cell configuration, the amplifier is switched to the current fixing mode.

Intestinal fluid secretion and short circuit current

In the first of 3 analyzes, the accumulation of fluid in the loops of the ileum is determined (O1 e! A1., 2002, Pr. !. 50: 881-889). Mice (age 8-10 weeks, body weight 25-35 g) with a genetic background of SI1 (or homozygous DE508 mice) are deprived of food for 24 hours, and then anesthetized with the introduction of ketamine (40 mg / kg) and xylazine (8 mg / kg) ) During the surgery, the body temperature is maintained in the range of 36-38 ° C using a heated pillow. To expose the small intestine, a small abdominal incision is made and closed loops of the ileum (length 20-30 mm) are separated proximal to the cecum by suturing. In the loop, 100 μl of PBV alone or PBV containing cholera enterotoxin (1 μg) are injected. In some experiments, an inhibitor (150 μg / kg) is administered by intraperitoneal injection. The abdominal incision is closed with a suture and allowed the mice to recover after anesthesia. After 6 hours, the mice are anesthetized, intestinal loops are temporarily brought to the surface of the body and the length and mass of the loops are measured after removal of the mesenteric and connective tissues.

In a closed adult mouse model of secretory diarrhea, mice are orally injected with a cholera enterotoxin (10 μg) into a 0.1 ml solution of 7% bicarbonate buffer solution (or buffer solution alone) using an orogastric injection needle (Cuyagcop e! A1). 1986, MGS! Iptip. 54: 522-528; CaLe1 e! A1., 1999, At. Й. Ryukyu.1.276: C58-C63). Four experimental groups are formed: a control (one buffer solution only), treated with cholera enterotoxin, treated with cholera enterotoxin + inhibitor (150 μg / kg intraperitoneally 2 minutes before trans-probe injection) and with only one inhibitor. After 6 hours, the mice are sacrificed and the small intestine (from the pylorus to the cecum) is brought to the surface of the body and freed from the mesenteric and connective tissues. The intestine is weighed, then longitudinally opened to remove fluid from the cavity (blotting) and weighed again. Fluid accumulation is calculated as the ratio of gut mass before and after fluid removal from the cavity. To measure the short circuit current of the colon was isolated strips of rat muscle layers separated direct dissection and placed them in Ussing chamber (area 0.7 ^cm2) and placed in a cell with oxygenated Ringers bicarbonate solution containing 10 .mu.M indomethacin. The short circuit current is measured after suppressing the Νι 'current with amiloride (10 μM), then stimulation with forskolin (20 μM) is carried out, and then an inhibitor is added.

Synthesis of ¹⁴ P-labeled _1p -172 mesh (FIG. 6)

The intermediate 2-thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone is synthesized by adding dropwise carbon disulfide (0.8 g, 10 mM) to a stirred solution of 3-trifluoromethylaniline (1.6 g, 10 mM) and triethylamine ( 1 g, 10 mmol) in ethyl acetate (10 ml) for 30 minutes. An ice bath is used to prevent excessive heat during the reaction. Leave to mix on

- 20 009847 night, a thick yellow suspension is filtered off and the precipitate is washed with 50 ml of ether and dried in air, obtaining 3 g (89%) of solid yellow dithiocarbamate (mp. 92-95 ° C). Br- ¹⁴ C-sodium acetate (obtained from Br- ¹⁴ C-acetic acid (Atetkyat), 55 mCi / mmol, 64 mg, 0.46 mm in 0.6 ml of water, the pH was adjusted to 8-9 with Ν; · ΙΗ0Ό3) are stirred and cooled to 5-10 ° C, and then dithiocarbamate (0.3 g, 0.9 mmol) is added over 10 minutes. Stirring is continued and the flask is allowed to warm to room temperature. After stirring for 2 hours, the solution was cooled to 10 ° C, acidified with concentrated HCl and the reaction mixture was heated to 90-95 ° C for 30 minutes. The precipitate formed is filtered off, washed with water and recrystallized from ethanol to obtain 0.103 g of the desired product in the form of brilliant crystals (yield 83%), mp. 177-178 ° C; specific activity ( ¹⁴ C) 55 mCi / mmol; Ή-NMR spectrum (300 MHz, СПС13): δ 4.18 (s, 2Н, СН ₂ ), 7.40 (d, 1Н, phenyl, 1 = 8.0 Hz), 7.48 (s, 1Н, phenyl), 7.64 (t, 1H, phenyl, 1 = 8.0 Hz), 7.72 (d, 1H, phenyl, 1 = 7.6 Hz) ppm.

For the synthesis of 2-thioxo-3- (3-trifluoromethylphenyl) -5- [4-carboxyphenylmethylene] -4-thiazolidinone ( ¹⁴ С-5) ( ¹⁴ С-СГТК1пЬ-172) a mixture of 2-thioxo-3- (3-trifluoromethylphenyl ) -4-thiazolidinone ( ¹⁴ C-5) (100 mg, 0.36 mmol) and 4-carboxybenzaldehyde (54 mg, 0.36 mmol) in absolute alcohol (1 ml) and piperidine (1 drop) are refluxed within 30 minutes The yellow precipitate was filtered off, washed with ethanol, dried and recrystallized from ethanol to obtain 108 mg (73% yield) of yellow crystals, mp. 180-182 ° C, specific activity ( ¹⁴ C) 54 mCi / mmol; Ή-NMR spectrum (300 MHz, ΌΜδΟ-Ή): δ 7.78 (d, 2H, carboxyphenyl, 1 = 8.2 Hz), 7.80-8.00 (m, 5H, trifluoromethylphenyl and CH), 8 07 (d, 2H, carboxyphenyl, 1 = 8.31 Hz), 13.20 (s, 1H, COOH, Ό 2Ό exchange water) ppm. Purification to> 99.9% is carried out by repeated recrystallizations.

Pharmacokinetic studies

Bolus of ¹⁴ _C-1p SGTK -172 (50 uCi) in RV8 containing 3% DMSO (treated with ΝαΟΗ, to adjust pH to 7.4) was administered intravenously to rats for 1 min (male-OaMeu 8rgadie rats, 360420 g) using a permanent cervical catheter. At certain intervals, blood samples are taken. ¹⁴ C-radioactivity is determined in plasma (isolated from whole blood by centrifugation at 14000 d for 10 min) by scintillation counting (ZstTsuegae 8E, ECset, California) using a B8-6500 MiSh-Ritroke 8ssh1Sha1yui Soip1et counter (Vesstap). Pharmacokinetic analysis is carried out using the software MnAopGp (RyatidY). Rats after sampling the final blood / tissue samples are killed with a large dose of pentabarbital. All animal studies were approved by the IS8H Animal Research Committee.

The study of tissue distribution and leaching

A bolus of ¹⁴ С-СГТЯ1Пъ-172 (2 μCi) is injected into mice (male SER mice of 30-35 g) intravenously for 1 min through the tail vein. Mice are killed in 5, 30, 120 and 240 minutes. The organs are removed, weighed and homogenized in distilled water (10-50 vol.%). Radioactivity is determined by counting homogenizate scintillations (25-50 μl) and expressed as a total of ¹⁴ C-radioactivity per organ (or per gram of tissue for skeletal muscle). At the same time, blood, urine and bile samples are taken (from the gallbladder or duodenum) and ¹⁴ C-radioactivity is measured and converted to 1 ml of liquid. A washout study is carried out by collecting urine and stool samples 24 hours after the administration of ¹⁴ C-CHHB-172. A study is also being made of the tissue distribution for rats prepared for pharmacokinetic studies.

Inhibitor Metabolism Analysis

Aliquots of the body fluids (blood plasma, urine, bile) and liver homogenate are spotted onto silica gel plates and developed by thin layer chromatography using an ethyl acetate: hexane: methanol solvent system (1: 1: 0.1), which gives Ρί — 0.5 value for the original inhibitor. Autoradiography is performed using NuregSht (Atetkyat), using the amplification system Tgapsksep LE (Kobak). Standards ¹⁴ C-CHTBb _- 172 are added to all tablets.

Short circuit current measurements (example 7)

To conduct studies with cells, 8par ^ e11 inserts containing monolayers of T84 cells are placed in the Ussing chamber system (No. 1SBE, Naptagb Arrata1i8, Holliston, Minnesota). The hemocameras are filled with Krebs carbonate solution containing (in mm): No. 1C1 120, NaHCΟ ₃ 25, KH ₂ PO ₄ 3.3, K ₂ NRA ₄ 0.8, MDC1 ₂ 1.2, CaCl ₁ 1.2, glucose 10 (support at a temperature of 37 ° C), and constantly sparged with a mixture of 5% CO ₂ /95% O ₂ . The buffer solution with a high content of K ⁺ ions has the composition (in mm): No. 1C1 65, KC1 67.5, KH2RO4 1.5, CaC12 1, MDC12 0.5, HEPE8 10, glucose 10. Buffer solution with a small content of ions C1 ^- has the composition (in mm): sodium gluconate 120, KH2RO ₄ 3.3, K ₂ NRA ₄ 0.8, MdCl _{1 2} 1,2, CaCl ₂ 1,2, HEPE 10, glucose 10 (support at a temperature of 37 ° C ), and air is constantly sparging through it. For measurements in the colon of mice, mice are anesthetized with intraperitoneal administration of ketamine (40 mg / kg) and xylazine (8 mg / kg). The ileum was removed, washed with ice-cold Krebs solution, opened along the mesenteric border, and mounted in Ussing microchamber (area 0.7 cm ² Uot1b Rgesyop BTststepK Sarasota, FL). For measurements on the human intestine, fragments of the colon are separated from the muscle layers by direct dissection and placed as described above. Hemicameras are filled with saturated oxygen

- 21 009847 Ricers bicarbonate solution containing 10 μm indomethacin. Short-circuit currents are measured using an OUS-1000-volt clamp (from the company \ Wog1b Rteshzyup 1p51gntsp15). using Ad / AdS1 electrodes and 1M KS1 agar bridges. Agonists / inhibitors contribute to hemicamers. as indicated below.

1p νίνο intestinal fluid secretion in the mouse and rat models (examples 5 and 7) Mice (age 8-10 weeks, body weight 25-53 g) with a genetic background of 0Ό1 are given water for 24 hours, but are not fed. Mice are anesthetized as described above, and the temperature during surgery is maintained at 36-38 ° C. using a heated pad. To expose the small intestine, a small abdominal incision is made and closed loops of the ileum (length 20-30 mm) are separated proximal to the cecum by suturing. 100 μl of PB8 or PB8 alone containing cholera enterotoxin (1 μg) is injected into the loops. In some experiments, CPTK.sch-172 (0-200 μg) is administered by intraperitoneal injection at certain times before or after cholera enterotoxin injection. The abdominal incision is closed with a suture and allowed the mice to recover after anesthesia. After 6 hours, the mice are anesthetized, intestinal loops are temporarily brought to the surface of the body and the length and mass of the loops are measured after removal of the mesenteric and connective tissues.

To measure cholera-enterotxin-induced fluid secretion in a closed rat model of male mice \ U | Chag (body weight 200-250 g), sodium pentobarbital (45 mg / kg) is anesthetized. Loops (40-60 mm) are isolated and injected with PB8 or PB8 alone containing cholera enterotoxin (10 μg) or 8Ta toxin (0.1 μg). In some experiments, SETK. _1p1 -172 (200 μg) is administered by intraperitoneal injection after injection of cholera enterotoxin or 8Ta toxin. The length and weight of the loops are measured after 3 hours (8Ta) or after 6 hours (cholera enterotoxin).

In the study of the orally administered CETK _1p1 -172, a mouse model with an open loop is used, in which mice are orally administered with a 7% bicarbonate buffer solution or only cholera enterotoxin (1 μg in 7% bicarbonate buffer solution) or cholera enterotoxin along with the grid. _Lb -172 (200 mcg in vitamin E TRC8, see below) using an orogastric needle for injection nutrition. After 6 hours, the small intestine (from the pylorus to the cecum) is brought to the surface of the body and freed from the mesenteric and connective tissues. The intestine is weighed, then longitudinally opened to remove fluid from the cavity and again weighed to quantify the accumulation of fluid.

Saso-2 permeability study

Saso-2 cells are cultured at porous rates and monolayers with a resistance of 400-600 Ω cm ^-1 are obtained. To study ion transport, the culture medium is replaced with an equal volume of Hank's buffered saline (HB88) containing 15 mM glucose and 25 mM HEPE8 (pH 7.3). After 1 h, CETK _1p1 -172 (25 μM) was added to the upper chamber and the plates were gently shaken at a temperature of 37 ° C. At certain time intervals, 50 μl of the solution is taken from the lower (receiving) chamber to determine the concentration of CETK. _1p11 -172 by UV absorption (385 nm). The apparent permeability (Papp) was calculated according to the equation Papp = e C / WT X (Vm / AFR) wherein 6C / 6T denotes the rate of increase of concentration _1p -172 mesh in the receiving chamber, Vr denotes the volume of the receiving chamber, A is the area of the monolayer, and SB denotes initial concentration _1p -172 mesh in the flow chamber.

Pharmacokinetic studies and the study of oral bioavailability

Mice are rapidly anesthetized with halotran and ¹⁴ C-labeled СЕТК1пЬ-172 (12 μCi), solubilized with vitamin E TPO8 (α-α-tocopheryl polyethylene glycol 1000 succinate, 0.5% w / v, СЕТК.1п11) is orally administered via a probe -172 in a 10% w / v suspension of TPO8 in water). For comparison, ¹⁴ C-SETK.1p11-172 (2 μCi) was intravenously administered to other mice by infusion into the tail vein. Blood samples are taken from the tail vein at certain intervals to measure the radioactivity of ¹⁴ C in plasma. After 6 hours, the mice were killed by a large dose of pentobarbital and organs were removed to determine the radioactivity in the homogenates.

Biological Example 1. Selection of CETK inhibitors.

The primary screening technique used to identify the compounds of the present invention was developed with the aim of identifying C1 ^- conduction inhibitors of CETC through direct interaction of the CETC-inhibitor. CETK is preliminarily stimulated in ECT cells expressing CETK with an activating cocktail containing forskolin, 1BMX and apigenin, as shown schematically in FIG. 1A. Activation of CETC by a multiple mechanism (increasing cAMP, inhibition of phosphodiesterase and direct binding of CETC) allows the identification of inhibitors that are active by directly blocking the transport pathway of CETC C1 ^- rather than by a greater number of proximal steps in the signal transmission pathway. ECT cells coexpress the C1 ^- / 1 ^- sensor based on a yellow fluorescent protein, which allows a quantitative assessment of the effectiveness of inhibition (see, in particular, 1auatatap e1 a1., 2000, 1. Vu1. C1et., 275: 6047-6050; OaPeya e1 a1., 2001, At. 1. Pb.u8yu.1.281: С1734-С1742). After pre-stimulation with CETC and addition of the compound in cells, an inward gradient of I ^- ions ^{is formed} in order to ensure an influx of I ^- and

- 22 009847 receive a decrease in fluorescence. Each analysis consists of recording for 2 s the baseline level of fluorescence, followed by continuous recording of fluorescence for 12 s after a quick addition of a solution containing I ^- ions. Compounds were tested separately at a concentration of 10 μM in a 9-well format using a fully automated high-performance screening device (see Example 2 below).

In FIG. 1B is a graph of characteristic curves showing relative fluorescence ΥΕΡ versus time for the initial selection of 50,000 compounds using the analysis shown in FIG. 1A. As quantified by the tilt angle of decreasing fluorescence after addition of I ^- ions, 49993 compounds do not significantly affect the kinetics of the influx of I ^- ions (<10% decrease in tilt angle). Seven compounds give a slight decrease in the negative tilt angle (10-52%), and almost all of them have a similar central core, including a 2thioxo-4-thiazolidinone heterocycle with substituted phenylmethylene and phenyl fragments (see Fig. 1C). Then, more than 250 analogues having the thiazolidinone structure of the central core are selected to identify the most potent inhibitors of CETK.

As shown in FIG. 1Ό, the most effective thiazolidinone SETK inhibitors identified during the selection are 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4thiazolidinone (referred to as SETK _1pb -172 in this description ) along with its five analogues, which have similar potency for inhibition. Thus, as a grid identifying inhibitors of 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone _(1p -172 mesh); 3 - [(3triftormetil) phenyl] -5 - [(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone _(1p -020 mesh); 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-hydroxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (SETK _1pb -029); 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone (SETK _1pb -185); 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone (SETK _1pb -214) and 3 - [(3-trifluoromethyl) phenyl] -5 - [(3-bromo-4-hydroxy-5-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone (SETK _1pb -23b).

The most effective CETK inhibitors contain one or more electron-withdrawing groups, such as a 3-trifluoromethyl group, in ring 1 and an electron-withdrawing group or polar solvents in ring 2, as described above. Grid _11L- 172 selected for further analysis. The following values of the relative magnitudes of effectiveness at inhibiting agents: 0.2 _(1PB 020 mesh), 0.3 _(1p -029 mesh), 1.0 _(1PB -172 mesh), 0.2 _(1p -185 mesh) 0 1 _(1p -214 mesh) and 0.1 _(1p mesh -23b).

In order to study the effect that the arrangement of trifluoromethyl and carboxyl substituents in the ring has, 8 CETK _1pb- 172 analogues were synthesized, in which the substituents shifted relative to their unique positions in rings 1 (trifluoromethyl group) and 2 (carboxyl group). Compared to CETK _1pb -172 (potency 1.0), the relative potency for inhibition for 3 - [(a-trifluoromethyl) phenyl] -5 - [(b-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone analogues : 0, b9 (a = 2, b = 2), 0.70 (2, 3), 0, bb (2, 4), 0.74 (3, 2), 0.90 (3, 3), 0, b7 (4, 2), 0, b4 (4, 3) and 0.5b (4, 4).

Biological Example 2: Determination of properties -172 mesh _1p.

The level of inhibition of CETK for specific dosages of the thiazolidinone compounds of the present invention is determined using the fluorescence assay shown in FIG. 1A and as described above. In FIG. 2A presents the data for SETK _1pb -172 as a function of dose as relative values of UEP fluorescence as a function of time. Significant inhibition of CETK is observed at concentrations of 0.3-0, b μM of the indicated thiazolidinone compound. From FIG. 2B, it follows that inhibition under the action of SETK _1pb -172 (shown graphically as the relative transport speed versus time after administration or when washing) ends within about 10 minutes (1 _1/2 is 4 minutes) and reverses after washing with a value of 1 _{1 / 2} equal to approximately 5 min (insert). The relative values of the transport speed shown in FIG. 2C show that СЕТКщъ-172 effectively inhibits the activation of СЕТК under the influence of various types of agonists that were not included in the activating cocktail used in the initial selection. Said agonists include genistein, CPT-cAMP, CPX, 8-MPO and such a potent benzoflavone activator CETK, which is IS _ce- 029 (2- (4-pyridinium bisulfate) benzo [b] -4H-chromene-4-one), and a SETK benzimidazolone activator such as IS _ce -853 (see Sabeya s1 a1., 2001, 1. ΒίοΙ. Sb., 27b: 19723-19728).

In order to determine the efficacy in the inhibition and specificity of SETK _1pb- 172, electrophysiological experiments are also carried out. FIG. 3A shows rapid dose-dependent inhibition of short-circuit current in ECT cells expressing CETK by adding CETC _1pb -172 to a solution washing the apical surface of cells. FIG. 3B shows the average dose-dependent inhibition for CETK _1pb -172 (K _b ~ 300 nM, Hill coefficient ~ 1) and glibenclamide (K _b ~ 200 μM), which are tested under identical conditions.

A similar inhibition effect for the indicated thiazolidinones is found in cells naturally expressing the wild type of CETK, including T84 cells and primary cultures of human bronchial epithelium cells, as well as in transformed ECT cells expressing C55YU-CETK and AE508-CETK (after low-temperature correction). In order to conduct research in bronchial cells

- 23 009847 hove No. 1'-channel is blocked by amiloride in such a way that the current value corresponding to the baseline strongly depends on CPTC. After the maximum activation of CPTC using CPT-cAMP, the effect of CPTC _1iE- 172 from the apical side strongly suppresses the short circuit current (Fig. 3C, left). CPTK _111b 172 also strongly suppresses short circuit current when introduced from the basolateral side (Fig. 3C, right).

Currents across the entire membrane are measured in PKT cells expressing CPTK, as shown in FIG. 3Ό. Stimulation by the action of 5 μM forskolin causes a current in the membrane equal to 381 ± 47 pA / pF (u = 4) at +100 mV (total membrane capacity 21 ± 3 pF). The dependence of current on voltage is linear, which could be expected for a clean SRTC stream (Fig. 3P). Extracellular infusion of a solution of 2 μm CPTK _111E- 172 causes a rapid decrease in the current at all membrane potentials, based on which it can be concluded that the inhibition of CPTK is independent of voltage. The absence of the dependence of channel blocking on voltage is confirmed by using low concentrations of CPTK _1iE- 172 (0.2 μM), obtaining ~ 50% inhibition (Fig. 3P).

The specificity of CPTK _1iE- 172 in the inhibition of CPTK is also investigated. Learn two non-IBS C1 ^- channel subunit. CPTK1iE-172 with a concentration of 5 μM does not inhibit Ca ²⁺ -activated secretion of C1 ^- ions, which is caused by the addition of MFI (100 μM) to the apical wash solution in polarized epithelial cells of the human bronchi (Fig. 4A). The maximum values of the ITR-dependent short circuit are 9.9 ± 0.5 μA / cm ² and 10.0 ± 0.2 μA / cm ² , respectively, in the absence and in the presence of CPTK1i-172 (8E, u = 4). CPTC _1II -172 at a concentration of 5 μM does not inhibit C1 ^- activated flows that are induced in the cells by extracellular wetting with hypotonic solution of 250 mOsm / kg (Fig. 4B).

The activity of the CPTC homolog, ATP-binding cassette carrier MEK-1 (multidrug resistance protein-1), is measured in 9HTE- / Ox, where overexpression of MEK1 is observed (Kazo1a e! A1., 1994, 1. Vyu. Seet. 269: 1432 -1436). The accumulation of vincristine, which is inversely proportional to the displacement of the active drug under the action of IEC-1, increases significantly under the influence of a verapamil inhibitor (100 μM) (Fig. 4C). SRTC _1iE- 172 (5 μM) does not affect the accumulation of vincristine and, therefore, does not inhibit MEK-1.

Another SRTC homologue is the sulfonylurea receptor (8IR), which regulates the activity of ATP-sensitive K ⁺ channels (K-ATP channels) (Ad1ag-Vguai aib Vguai 1999 Eibos Keu. 20: 101-135). 8IC1 is expressed by pancreatic β-cells, where it controls the membrane potential and insulin release. Sulfonylureas, such as glibenclamide, cause insulin release (and a hypoglycemic response) by blocking K-ATP channels and depolarizing the membrane. In order to determine whether CPTK1iE-172 also blocks K-ATP channels, the membrane potential of the β8-1 cell line of rat pancreatic β cells is measured (Figs. 4Ό, 4E). Unlike significant membrane depolarization caused by glibenclamide, CPTK1iE-172 (2 and 5 μM) does not cause depolymerization of the membrane potential. CPTK1iE-172 with a concentration of 5 μM causes a slight hyperpolymerization, which is much smaller than the hyperpolymerization caused by the activator of K-ATP channels with diazoxide (100 μM). Additional studies show that CPTK111E-172 with a concentration of 5 μM does not block the water channel (AOR1). urea carrier (IT-B), ion exchanger No. '/ H' (ΝΗΕ3) and ion exchanger C1 ^- / НСО3 ^- (АЕ1).

Further analysis shows that 5 μM CPTC _1iE- 172 does not affect cAMP production by cells or phosphatase activity. In RKT cells, the basal content of cAMP is 225 ± 22 fmol / cell, which increases 30 min after stimulation with 20 μM forskolin to 1290 ± 190 fmol / cell (without inhibitor) and 1140 ± 50 (+ SRTC _{1 and E-} 172) (and = 3). As can be concluded from the analysis using dihydrorodamine, CPTK _111E- 172 with concentrations up to 100 μM is not toxic to _PKT cells after 24 hours. In mice, an intravenous injection of 1000 μg / kg CPTK _1iE- 172 is not daily causes apparent toxicity. Water consumption did not decrease, and the concentration in serum of the electrolyte, glucose level, liver function index, concentration in serum creatinine, amylase and hematocrit did not change. In addition, a single, very large systemic dose of CPTK _1iE- 172 (10 mg / kg) does not cause obvious toxicity.

Biological example 3. The effectiveness of w u1uo.

The efficacy of CPTK _11t- 172 was tested by means of two analyzes on the secretion of fluid in the intestine induced by cholera enterotoxin and in the isolated intestine by the method of measuring short circuit current. In the first analysis, w1u0 form a series of closed loops of the small intestine, and small volumes of saline or saline containing cholera enterotoxin are injected into the cavity of alternating loops. The accumulation of fluid in the cavity is determined after 6 hours. As can be seen from FIG. 5A, marked fluid accumulation and bloating are observed in loops exposed to cholera enterotoxin, while adjacent control (with saline) loops remain empty. A single administration of CPTK _11t -172 (150 μg / kg ip) before infusion of cholera enterotoxin effectively prevents the accumulation of fluid in intestinal loops exposed to cholera enterotoxin.

- 24 009847

Results. obtained in a series of these experiments. graphically summarized in FIG. 5B. SETK. ,,, 1, -172 significantly reduces the secretion of fluid to a level in control loops with saline. while the inactive thiazolidinone analog does not inhibit fluid secretion. As follows from the previously obtained data (CaLps1 s1 a1 .. 1994. 8psups 266: 107-109). intestinal loops exposed to cholera enterotoxin. obtained from homozygous AE508-SETK mice. also remain empty. pointing to that. that CETK is involved in the secretion of fluid in the intestine. In a second assay, fluid secretion in the intestine is induced by the oral administration of cholera enterotoxin (10 μg). and SETK _1P b-172 is administered systemically. After 6 hours, a clear accumulation of fluid is observed. as measured by weighing the entire small intestine. Maintaining SETK _1P b-172 markedly reduces the accumulation of fluid in the intestine. what is observed visually and quantitatively evaluated by the ratio of the mass of the intestine before and after removal of fluid from its cavity (Fig. 5C).

In FIG. 5Ό shows the suppression under the action of SETK _1pp -172 of a short circuit current through an unexposed mucous membrane of the rat colon. After inhibition of the Ν; ί flux by forskolin amyloride, a noticeable increase in short circuit current occurs. Grid _1p41 -172. added to the mucous solution. suppresses short circuit current with greater efficiency. than in that case. when it is added to a serous solution. which may be associated with poor access to the cells of the colon epithelium through residual submucosal tissue. Adding 5 μM CETK. ,,, 1-172 only in the mucous solution reduces the short circuit current by> 80%. These results provide electrophysiological evidence of this. that CETE _1p41 -172 inhibits the CETC C1 ^- channel in the intestine.

Biological example 4. Pharmacokinetic analysis.

Pharmacokinetic analysis in rats is carried out in the form of a series of measurements of ¹⁴ C-radioactivity after a single intravenous infusion of a ¹⁴ C-labeled SETK1p41-172 bolus. The total amount of inhibitor administered (400 mcg. ~ 1 mg / kg) is effective as an anti-diarrhea agent in rats. FIG. 7 shows. that the kinetics of ¹⁴ C-radioactivity in serum is in good agreement with the 2-chamber model. the volume of distribution is 1.2 liters. and the AIS value (area under the curve) is 3.8 μg-h / ml. The half-periods of time are 0.14 hours (redistribution) and 10.3 hours (removal). No trace of ¹⁴ Marked CETK1P1-172 is detected in plasma 72 hours after administration or in liver or kidney homogenates 14 days after administration.

The distribution of ¹⁴ C-labeled CETK1bb-172 in tissues is determined from the radioactivity of organ homogenates and body fluids after a single bolus infusion. In FIG. 8 in data group A, the distributions of ¹⁴ C in the main organs of mice at the indicated time are summarized. the past after the infusion of SETK1P-172. After 5 min ¹⁴ C-radioactivity is detected. mainly. in the liver and kidneys and decreases over time. Minor radioactivity is found in the brain. a heart. skeletal muscles and testicles. After longer periods of time (30-240 min), ¹⁴ C-radioactivity accumulates in the intestine. In FIG. 8 in data group B shows a similar distribution of ¹⁴ C-radioactivity in rats. measured 60 min after intravenous bolus infusion. however, little radioactivity is found in the brain. heart and skeletal muscle. In some experiments, rats were euthanized 10 days after infusion of ¹⁴ C-labeled CETK _1p41 -172 (50 μCi).

In order to establish the mechanism of accumulation of CETC. ,,, 1-172 in the kidney. liver and intestines ^14. Radioactivity is measured in blood serum. urine and bile. The average radioactivity in the urine of mice after the first 2 hours after infusion is 4.2 ± 1.2x10 ⁵ counts per minute per milliliter. The ratio of ¹⁴ C-radioactivity in the urine to ¹⁴ C-radioactivity in the blood is in the range of 5-7: 1 and is comparable to the ratio of the osmolarity of urine to the osmolarity of serum. which is in the range of ~ 5: 1 (1550 mOsm vs. 310 mOsm). and from this we can assume. that washing out СЕТК1п41172 is carried out by the kidneys by glomerular filtration without absorption or secretion in the renal tubules. The mechanism of excretion of СЕТК1п41-172 by leaching by the kidneys is confirmed by an approximately parallel kinetic decrease in ¹⁴ C-radioactivity in blood serum. urine and kidney tissue (data not shown). The possibility of accumulation of СЕТК1п41-172 in bile is studied on the basis of the observed rapid accumulation of ¹⁴ C-radioactivity in the liver and later accumulation in the intestine. 60 minutes after administration to mice, the concentration of ¹⁴ C-radioactivity in the bile is approximately 9 times higher than the concentration of ¹⁴ C-radioactivity in the blood. To determine that. whether CETK _{| mN} -172 is secreted from bile with stool or continues to circulate. urine and bile samples were taken from mice within the first 24 hours after infusion of the inhibitor containing the radioactive label. 93 ± 3% of the radioactivity excreted along with feces is found in urine. and this suggests. that the main mechanism of excretion in the kidneys is hepatoenteric circulation.

In order to determine that. whether ¹⁴ C radioactivity corresponds. measured in organs and fluids. original or chemically modified SETK _1p41 -172. method of thin layer chromatography and autoradiography conduct studies of urine samples. blood serum and bile. as well as liquids above the sediment of liver homogenates. isolated by centrifugation. In FIG. Figure 9 shows one spot with a KE-0.5 value for the initial CETK _1P b-172. which is administered by bolus infusion. Autoradiography of fluid and organ homogenates shows a single spot with an identical value

- 25 009847 by YAG. and this indicates that NETWORK _1P -172 is not chemically modified.

Setjan -172 _1p is a weak acid with a pKa of 5.5 as determined by spectrophotometric pH titration. At a pH value of ~ 1% under physiological conditions, _CPTNW11 -172 is present in the form of a non-ionized acid with a low polarity and high ability to penetrate through membranes. The rapid uptake of CPTI _1P b-172 in the cell models discussed above suggests the possibility of developing bioavailable oral preparations, but it must be borne in mind that, in order to avoid precipitation, protection of the preparations against low pH in the stomach may be required. The results of these pharmacokinetic studies show that CPTR _1p41 172 is slowly excreted by rodents by cleaning the kidneys without undergoing chemical modification, and that CPTR1-172 is concentrated in bile and accumulates in the intestine. CPTI _1p41 -172 is not able to overcome the blood-brain barrier in a significant amount, and a small amount of CPTR-172 was found in other vital organs, including the heart, lung, skeletal muscle, and testicles. Slow excretion from the kidneys, accumulation in the liver, and low ability of CPTR _W11 -172 to penetrate the blood-brain barrier are favorable for use against diarrhea.

Biological example 5. The response depending on the dose and the duration of the inhibitory effect of CPTI _1P b-172

This example is intended to further expand the above observations regarding the ability of a single intraperitoneal injection of CPIT _1p41 -172 to inhibit secretion induced by cholera enterotoxin in a closed intestinal loop model in mice. More specifically, the purpose of this example is to determine the dose-response response and establish the apparent half-life of the inhibitory effect of NETWORK _1p41 -172.

First, in order to establish the properties of the mouse model, the kinetics of absorption and secretion of fluid within the intestine is determined. To study the absorption, the liquid content in the loop is measured at certain intervals after the injection of 200 μl of RVB into individual loop injections. In FIG. 10 group of data A shows a rapid absorption of the liquid, and after ~ 25 min 50% of the liquid remains. Intraperitoneal administration of NETWORK _11L- 172 with a dose that substantially inhibits cholera enterotoxin-induced fluid secretion (20 μg) does not change the rate of fluid absorption (measured after 30 min) compared to controls (Fig. 10, data group A, insert) . To study secretion, cholera enterotoxin injections (1 μg in 0.1 ml of RVB) are injected into the loop of the intestine. In FIG. 10 group of data B shows the slow onset of fluid secretion after 6 hours, which corresponds to previous studies on rodent models (Ogbacia c1 a1., 1. C11P. 1pus51. 1971, 50881-889; O1 eG a1., Proc. Ναΐΐ. Asab. 8c1. I8A 2002, 99: 3042-3046). Rapid absorption of fluid in the intestine under normal conditions indicates that fluid accumulated in the intestinal cavity after active secretion can be rapidly absorbed if secretion is blocked, and indicates that inhibition of NETWORK may be effective to prevent fluid accumulation even after administration of cholera enterotoxin.

Data Group A in FIG. 11 summarizes the results of studying the dose-response setjan _1p -172 mice, a single dose of the inhibitor which are administered by intraperitoneal injection immediately after infusion of the cholera toxin into the closed intestinal loops. The base fluid content (dashed line), which is measured in loops not exposed to cholera enterotoxin, is approximately zero. NETWORK _1p41 -172 inhibits the accumulation of fluid in the intestinal loops treated with cholera enterotoxin by approximately 90%, while 50% inhibition is achieved with the introduction of ~ 5 μg NETWORK _1P b-172 (150 μg / kg). The duration of inhibition is measured during the study of the dose-response response, with the exception that a single dose of 20 μg NETWORK _1p41 -172 is administered at different time periods before or after administration of cholera enterotoxin. The data group B in FIG. 11 shows a significant inhibition of fluid accumulation in the cavity when MES _1p41 -172 is administered within 3 hours before or after administration of cholera enterotoxin. However, significantly less inhibition is observed 6 hours before the introduction of cholera enterotoxin. Taking into account the duration of the action of cholera enterotoxin of 6 hours, which is established using a provocative test, the value of G _{1/2 the} inhibition stability under the action of NETWORK _1P 1 ₁ -172 is ~ 9-10 hours

Biological example 6. Oral bioavailability of NETWORK _1P b-172.

To investigate the efficacy of orally administered anti-diarrhea setjan _1p determine pharmacokinetics setjan -172 -172 mice _1p and _1p, the transfer setjan -172 through Caco-2 monolayers. Since SETH-172 is a relatively non-polar weak acid (pKa 5.5), which can be expected to precipitate in the stomach, oral administration is carried out using two agents that are usually used to solubilize drugs intended for oral administration , - Vitamin E TPO8 and cyclodextrin. Measurements are carried out using ¹⁴ Marked NETWORKS _w11 -172.

Data Group C of FIG. 11 shows the pharmacokinetics of ¹⁴ C-NET _1p11 -172 after oral administration compared with intravenous administration in mice. Intravenous administration leads to initially high concentrations in serum, which decrease over ~ 30 min (overdosing

- 26 009847 distribution in tissues), while serum radioactivity is low immediately after oral administration, reaches a peak value in ~ 60-90 minutes, and then decreases. The data group Ό in FIG. 11 summarizes the distribution of ¹⁴ C-CPTB1bb-172 in organs 6 hours after oral and intravenous administration and shows accumulation in the gastrointestinal tract, as well as in the liver and kidneys. ¹⁴ C-radioactivity is concentrated in bile approximately 10 times more than in serum, with a negligible amount of ¹⁴ C-radioactivity being excreted along with stool (<10% of the total amount of radioactivity released over a period of 24 hours), which suggests that the accumulation of CETK111-172 in the intestine is carried out through hepatoenteric circulation. Comparison of the oral and intravenous administration of CPTV _1pp- 172 (content in tissues / serum after 4-6 hours) shows that the oral bioavailability of CPTV _1Gl- 172 in TP68 preparations is 15-20%.

The data group P in FIG. 11 shows a linear increase in the number of SRTK _1PY- 172 on the other side of the Saso-2 monolayers and allows us to establish that the permeability coefficient for SRTK _1PY- 172 is 16x10 ^-6 cm / s. The obtained value fits into the range obtained for a variety of orally prescribed drugs (in particular, 36 x 10 ^-6 cm / s for pindolol and 48 x 10 ^-6 cm / s for sildenafil) (31epet e! A1., 1. Furniture. , 2001, 44: 1927-1937).

Biological example 7. Inhibition of cGMP and cAMP-mediated fluid secretion.

To determine the effectiveness of CPTK _1yp- 172 in the inhibition of cGMP and cAMP-mediated secretion of fluid, as well as to study the effectiveness of CPTK _1yp- 172 in an alternative animal model, a closed rat loop model was used. The guanylyl cyclase C receptor is expressed by rat intestinal cells and allows you to bind the 8Ta toxin and increase the level of cGMP (Mapp e! A1., Vusyet. Vyuruk. Vek. Sottip., 1997, 239: 463-466). After 3 hours, it was shown that the 8Ta toxin causes fluid secretion in the ileum of rats (Soiep e! A1., At. 1. Ryuk1o1., 1989, 257: 6118-123). CPTV _1π- 172 prevents cholera-enterotoxin-induced fluid secretion in the intestinal loops of rats (Fig. 12, data group A) at a dose (600 μg / kg) that is effective for mice. In order to induce 8Ta toxin-induced secretion of fluid, injections of 8Ta toxin (0.1 μg in 300 μl of PB8) are introduced into the intestinal loops and the loops are weighed after 3 hours. Data group B in FIG. 12 shows ~ 75% inhibition of fluid secretion in the intestine under the action of CPTV _1π- 172.

Short circuit current measurements are carried out for mouse and human epithelial layers in order to assess the inhibition of transepithelial secretion of ions under the action of CPTV _1π- 172. Data Group A in FIG. 13 shows inhibition of short-circuit current in the ileum of the mouse, after stimulation with foscolin or 8Ta toxin (insert), depending on the dose of CPTV _1π- 172. 50% inhibition is found for a concentration of ~ 5 μM CPTV _1π- 172 for both cGMP and cAMP-mediated chloride secretion. The data group B in FIG. 12 shows the efficacy of CPTV _1π- 172 in suppressing short-circuit current in the human colon.

It was unexpectedly found that the apparent efficacy of CPTV _1π- 172 in the inhibition of short-circuit current in the intestine (2-5 μM) was significantly less than that found in electrophysiological studies carried out on several cell lines, including PBT cells expressing CPTV (0, 2-0.5 μM), and Ca1i-3 cells (0.5 μM). Provides some explanation to this difference, including those due to differences in cell type restricted SRTV _1p -172 to intestinal cells for the initial condition of the intestine, the influence of the membrane potential (interior negative cell potential decreases the concentration of _[1p SRTV -172]) and competitive interaction between ATP and SRTV _1p -172.

In order to study this phenomenon, a short circuit current is measured on the colon T84 epithelial cells. In typical experiments of FIG. 14 for data group A, it was shown that, using a high-performance selection method, ~ 3 μM CPTV _1π- 172 causes 50% suppression of the short-circuit current in non-permeabilized monolayers of T84 cells after stimulation with forskolin, a cAMP agonist (left) , using 8-Вгс6МР, which is permeable to the cellular analogue of GMF (in the middle), or using СРТВ _1пі 16, which is a direct activator of СРТВ chloride conductivity. In order to determine whether intact cells are necessary for the relative decrease in the effectiveness of CPTV _1π- 172 in T84 cells, the short circuit current is measured after permeabilization of the cell basolateral membrane with amphotericin B and in the presence of a C1 ^- ion gradient (in order to generate currents that can be measured ) In FIG. 14 group of data B (left) shows significantly greater efficacy of CPTC. ,,, 1-172 in the inhibition of short-circuit current after permeabilization. Dose response data, which are summarized in FIG. 14 for data group B (in the middle) indicate a decrease in the apparent value of CG for inhibition under the influence of CPTC. _{| M} | -172 in the range from ~ 3 to 0.3 μM after permeabilization of cells. To investigate whether the decrease in the efficacy of CPTV _1π- 172 in intact cells is a consequence of the negative internal membrane potential (decrease in the concentration of [CPTV _1π- 172] in the cytoplasm compared with the external environment), the short-circuit current is measured on T84 cells after depolarization using washing basolateral solution with a high content of K ⁺ ions. In FIG. 14th group

- 27 009847 pa of data C shows that the increased efficacy of _{NETI 1n1} -172 (K1 ~ 0.3 μM) is restored in depolarized cells, indicating that the membrane potential is important for the effectiveness of NETI _1n- 172.

Based on the data presented above, it can be expected that the thiazolidinone compounds of the present invention, represented by the NETWORK compound _IIb- 172, have antidiarrheal efficacy in enterotoxin-induced secretory diarrhea caused by enterotoxigenic organisms such as E. coli and Clinella with cholera-associated diarrhea Traveler complex and AIDS-related complex.

Inhibition of NETWORKS can be used as adjunctive therapy for diarrhea caused by entero-invasive bacteria, such as the S1o8tbst bIysbe and Baltope11a species; however, mucosal damage caused by these organisms will not decrease with inhibition of NET. Similarly, it cannot be assumed that inhibition of NETs will lead to the correction of the pathology underlying inflammatory bowel disease, but it can reduce the amount of fluid secretion in the intestine. Recent data suggest that the secretion of fluid due to diarrhea by the action of viruses, such as rotavirus, may include other mechanisms, such as Ca ²⁺ -mediated C1 ^- channel opening, although the role of networks in fluid secretion remains unknown and, therefore, can be tested in suitable animal models using the compounds of the present invention.

In short, the NETWORK _1p1 -172 compound, which is a thiazolidinone blocker for NETWORK, prevents cAMP and cGMP-induced ion / fluid secretion in rodent and human tissues without affecting fluid absorption in the intestine. Its favorable pharmacological profile and profile of its activity contribute to the further expansion of its use against diarrhea.

Although the present invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalent replacements may be made that do not change the nature and scope of the claims of the present invention. In addition, many modifications can be made in order to adapt a particular situation, substance, composition of compounds, method, individual step or steps of the method to the purpose, nature and scope of the claims of the present invention. It is assumed that all such modifications are included in the scope of the following claims.

Claims (39)

1. A method of treating a subject in which a condition is mediated by a protein regulator of transmembrane conductivity in cystic fibrosis (NET), treatable by inhibiting ion transport mediated by NET, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of formula (1b) wherein X ₁ is trifluoromethyl; X ₂ and X _{3 are} independently selected from hydrogen and a halogen atom; Υ _1, Υ Υ ₂ and ₃ are independently selected from hydrogen, _C1-C8 alkyl group, a C ₁ -C ₇ alkoxyl group, a carbonate group, a carbamate group, a carboxyl group, a halogen atom, a nitro group, an azo group, a hydroxyl group and a mercapto group; or a pharmaceutically acceptable salt thereof in the form of an individual stereoisomer or a mixture thereof. 2. The method according to claim 1, where the condition is increased above normal secretion in the intestine. 3. The method according to claim 2, where the condition is secretory diarrhea. 4. The method according to claim 1, where the compound of formula (1b) is 3 - [(3-trifluoromethyl) phenyl] -5 [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone. 5. The method according to claim 1, where X ₁ is located in the 2, 3 or 4 position of the phenyl group to which it is attached. 6. The method according to claim 1, where Υ _{2 is} selected from a hydroxyl, carboxyl group, nitro group, carbonate group and halogen. 7. The method according to claim 5, where the trifluoromethyl group is located in 3 position. 8. The method according to claim 1, where Υ2 is a hydroxyl group. 9. The method of claim 8, where Υ ₁ is a hydroxyl group. 10. The method of claim 8, where Υ1 is a bromine atom. 11. The method of claim 8, where Υ ₃ is a nitro group. 12. The method according to claim 1, where the compound of formula (1b) is selected from - 28 009847 13. A method of inhibiting in a subject’s cell the activity of a transmembrane conductivity regulator protein for cystic fibrosis, which comprises contacting the cell with a compound of the formula (! B) where X ₁ is trifluoromethyl; X ₂ and X _{3 are} independently selected from hydrogen and a halogen atom; Υι, Υ Υ ₂ and ₃ are independently selected from hydrogen, _C1-C8 alkyl group, a _C1-C7 alkoxy group, a carbonate group, a carbamate group, a carboxyl group, a halogen atom, a nitro group, an azo group, a hydroxyl group and a mercapto group; or a pharmaceutically acceptable salt thereof in the form of an individual stereoisomer or a mixture thereof; in an amount sufficient to inhibit CETE ion transport in the cell. 14. The method according to item 13, where the compound of formula (1b) is 3 - [(3-trifluoromethyl) phenyl] -5 [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone. 15. The method according to item 13, where X ₁ is located in the 2, 3 or 4 position of the phenyl group to which it is attached. 16. The method according to clause 15, where X1 is located in 3 position. 17. The method according to item 13, where Υ _{2 is} selected from a hydroxyl, carboxyl group, nitro group, carbonate group and halogen. 18. The method according to item 13, where Υ ₂ is a hydroxyl group. 19. The method according to p, where Υ ₁ is a hydroxyl group. 20. The method according to p, where Υ1 is a bromine atom. 21. The method according to p, where Υ3 is a nitro group. 22. The method according to item 13, where the compound of formula (1b) is selected from - 29 009847 23. The method according to item 13, where the contacting of the cell includes the ingestion of a compound of formula (1b) by the subject. 24. The method according to item 23, where the ingestion also includes the ingestion of a pharmaceutically acceptable carrier along with a compound of formula (1b). 25. A method of inhibiting in a subject's cell the activity of a protein-regulator of transmembrane conductivity in cystic fibrosis during w νίνο analysis, which involves contacting the cell with a compound of formula (1b) where X ₁ is trifluoromethyl, X ₂ and X _{3 are} independently selected from hydrogen and a halogen atom ; Υ1, Υ2 and Υ3 are independently selected from hydrogen, a C1-C8 alkyl group, a C1-C7 alkoxy group, a carbonate group, a carbamate group, a carboxyl group, a halogen atom, a nitro group, an azo group, a hydroxyl group and a mercapto group; or a pharmaceutically acceptable salt thereof in the form of an individual stereoisomer or a mixture thereof; in an amount sufficient to inhibit CETE ion transport in the cell. 26. A method for inducing the phenotype of cystic fibrosis (CE) in an animal other than human, the method comprising administering to a non-human animal a compound of formula (1b) wherein X1 is trifluoromethyl, X2 and X3 are independently selected from hydrogen and a halogen atom; Υι, Υ Υ ₂ and ₃ are independently selected from hydrogen, _C1-C8 alkyl group, a C ₁ -C ₇ alkoxyl group, a carbonate group, a carbamate group, a carboxyl group, a halogen atom, a nitro group, an azo group, a hydroxyl group and a mercapto group; or a pharmaceutically acceptable salt thereof as an individual stereoisomer or a mixture thereof; in an amount sufficient to induce the phenotype of cystic fibrosis (CE) in an animal other than human. 27. A method of treating a subject who has a condition characterized by increased excess transport of ions under the influence of a protein regulator of transmembrane conductivity in cystic fibrosis (CETE), the method comprising administering to the subject an effective amount of thiazolidinone compound 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; in this case, NET is inhibited by ion transport, and the condition is cured. 28. A pharmaceutical composition for treating a condition characterized by increased excess transport of ions under the influence of a protein transmembrane conductivity regulator for cystic fibrosis (CETE), containing a thiazolidinone compound independently selected from 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone; 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-hydroxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3 - [(3-trifluoromethyl) phenyl] -5 - [(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone and 3 - [(3-trifluoromethyl) phenyl] -5- [ (3-bromo-4-hydroxy-5-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone; and at least one pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, and a pharmaceutically acceptable excipient, wherein the NET-mediated ion transport is inhibited. 29. The composition according A.25, where the composition does not contain detectable amounts of dimethyl sulfoxide. 30. A pharmaceutical composition for treating a condition mediated by a protein regulator of transmembrane conductivity in cystic fibrosis (CETE), comprising a compound of formula (1b) o ¹ 3 where X1 is trifluoromethyl, X2 and X3 are independently selected from hydrogen and a halogen atom; Υι, Υ ₁ and Υ _{3 are} independently selected from hydrogen, a C ₄ -C ₈ alkyl group, a C1-C ₇ alkoxy group, a carbonate group, a carbamate group, a carboxyl group, a halogen atom, a nitro group, an azo group, a hydroxyl group and a mercapto group; or its pharmaceutically acceptable salt in the form of indie - 30 009847 visual stereoisomer or mixtures thereof and at least one pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent and a pharmaceutically acceptable excipient. 31. The composition of claim 30, wherein X _£ is located at the 2, 3, or 4 position of the phenyl group to which it is attached. 32. The composition of claim 30, wherein Υ2 is selected from a hydroxyl, carboxyl group, nitro group, carbonate group, and halogen. 33. The composition according to p. 30, where X1 is located in 3 position. 34. The composition of claim 30, wherein Υ2 is a hydroxyl group. 35. The composition according to clause 34, where Υ1 is a hydroxyl group. 36. The composition according to clause 34, where Υ1 is a bromine atom. 37. The composition according to clause 36, where Υ ₃ is a nitro group. 38. The composition of claim 30, wherein the compound is 3 - [(3-trifluoromethyl) phenyl] -5 - [(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone. 39. The composition of claim 30, wherein the composition does not contain detectable amounts of dimethyl sulfoxide.