SK288508B6 - Use of 5-carboxymethyl-3-mercapto-1,2,4-triazino-[5,6-b]indoles and pharmaceutical preparation containing them - Google Patents

Abstract

It is described the use of 5-carboxymethyl-3- mercapto-1,2,4-triazino-[5,6-b]indoles of the general formula (I) and their pharmaceutically acceptable salts, hydrates and solvates for the preparation of a medicament for treatment of human and veterinary diseases in which activities of aldo- keto reductases AKR1B1 and AKR1B10 are key etiological factors for their development and progress.

Description

The invention relates to the use of 5-carboxymethyl-3-mercapto-1,2,4-triazino- [5,6-b] indoles and a pharmaceutical composition thereof for the treatment of human and veterinary diseases.

BACKGROUND OF THE INVENTION

Aldo-keto reductases are NAD (P) H-dependent oxidoreductases, which are best characterized as glucose reducing agents. They are involved in the pathophysiology of diabetic complications. These enzymes also metabolize lipid peroxidation products, thereby contributing to the formation of an inflammatory response in certain circumstances.

Aldose reductase (AR, ALR2, AKR1B1), in addition to being involved in diabetic complications through glucose reduction, effectively reduces aldehydes resulting from lipid peroxidation and their glutathione conjugates (Ramana, BioMol Concepts 2: 103-114, 2011). Lipid peroxidation aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and their glutathione conjugates (e.g. GS-HNE) are effectively reduced by the action of AR on the corresponding alcohols, DHN (1,4-dihydroxynonene) and GS -DHN (glutationyl-1,4-dihydroxynonene), which mediates inflammatory signals. The reduced GS-DHN conjugate is considered to be a novel signaling intermediate in the transduction of cellular signals initiated by reactive oxygen species that may ultimately lead to an inflammatory response (Srivastava et al. Chem Biol Interaction 191: 330-338,

2011). In cell and animal models, AR inhibition effectively eliminated inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens, and auto-immune responses (Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010) .

Aldose reductase inhibitors (ARIs) thus represent a new therapeutic approach to the treatment of a wide range of inflammatory diseases such as atherosclerosis, asthma, uveitis, sepsis, arthritis, periodontitis and other disorders that have the potential to stimulate the immune system and generate large amounts of inflammatory cytokines and chemokines. colon cancer (Ramana, BioMol Concepts 2: 103-114, 2011; Srivastava et al. Chem Biol Interaction 191: 330-338, 2011; Tammali et al. Curr Cancer Drug Targets. 11 (5): 560-571, 2011) Kador et al. J Periodontol. 2011; 82: 926-933, 201; Yadav et al. Curr Mol Med 10, 540-549, 2010).

It is well documented that chronic inflammation is associated with cancer progression (Solinas et al. Cancer Metastasis Rev. 29, 243-248, 2010) and increased expression of aldoketoreductases has been reported in lung, breast, prostate, cervical, testes and gross tumors. gut (Liu et al. Recent Pat Anticancer Drug Discov. 4 (3): 246-53, 2009; Laffin and Petrash, Front Pharmacol. 3: 104, 2012; Terzig et al. Gastroenterology. 138 (6): 2101-2114 , 2010). In addition, overexpression of AKR1B1 in various types of cancer causes resistance to doxorubicin, which is explained by the increased detoxification of doxorubicin by AKR1B1-mediated carbonyl reduction (Lee et al. Anticancer Drugs (2): 129-32 2001).

In addition to AKR1B1, the related enzyme AKR1B10 is involved in several cancers (Penning, Clin Cancer Res. 11 (5): 1687-90, 2005; Fukumoto et al. Clin Cancer Res. 11 (5): 1776-1785, 2005; Yan; et al. Int J Cancer 121 (10): 2301-6 2007; Zhao et al. Eur J Med Chem. 45 (9): 4354-7, 2010; Matsunaga et al. Front Pharmacol. 3: 5.2012; Laffin and Petrash, Front Pharmacol 3: 104, 2012; Liu et al., Biochem J. 442 (2): 27382, 2012). AKR1B10 is a 36-kDa cytosolic reductase that is similar to the AKR1B1 amino acid sequence (71% identity) as well as a tertiary structure with a (α / β) 8 barrel topology. Like AKR1B1, the enzyme AKR1B10 reduces a variety of aromatic and aliphatic aldehydes, dicarbonyl compounds and some ketone drugs using NADPH as a coenzyme.

Overexpression of both AKR1B1 and AKR1B10 has been observed in different types of cancer, and great variability has been reported by cancer type and tissue origin (Laffin and Petrash, Front Pharmacol. 3: 104,

2012). A significant increase in AKR1B10 expression was observed in lung and liver cancer. Overexpression of AKR1B1 is mostly observed in human cancer, although generally at a lower level. Pharmacological inhibition or genetic ablation of AKR1B1 and AKR1B10 prevents the spread of inflammatory signals induced by cytokines and growth factors, as well as the proliferation of cancer cells in cell cultures and nude mouse xenograft models. E.g. The antitumor activity of the anti-inflammatory drug sulindac has recently been interpreted in the context of inhibition of AR Steuber, ChemMedChem. 6 (12): 2155-7 (2011). The silencing of AKR1B10 expression led to inhibition of colorectal carcinoma cells (Yan et al. Int J Cancer. 121 (10): 2301-6 2007).

The contribution of AKR1B10 to the development of resistance to anticancer agents has been suggested (Jin et al. Front Biosci. 11: 2767-73 2006; Matsunaga et al. Front Pharmacol. 3: 5.2012; Laffin and Petrash, Front Pharmacol. 3: 104, 2012).

These findings suggest that inhibitors of AKR1B1 and AKR1B10 are expected to be used as a novel agent in cancer therapy.

AKR1B1 synthase activity has also been described where it catalyzes the transformation of PGH2 to PGF2α in the presence of the NADPH cofactor. PGF2a plays various physiological roles in the body such as uterine contraction, bronchial vascular and arterial smooth muscle, regulation of ocular pressure, renal filtration, stimulation of hair growth, regulation of the ovarian cycle through induction of luteolysis and others (Nagata et al., FEBS Journals 278: 1288- 98, 2011). It has been associated with diseases such as diabetes, osteoporosis, menstrual disorders and has a significant effect on the female reproductive system (Madore et al., J.Biol.Chem. 278 (13): 11205-12, 2003). Elevated levels of PGF2α have been detected during labor as a means of inducing myometrial contractions and cervical dilatation (Breuiller-Fouche'et al., Biol.Reprod. 83: 155-162, 2010).

The enzyme AKR1B1 is also capable of catalyzing the isomerization of PGH2 to PGD2 in the absence of NADPH (Nagata et al., FEBS Journals 278: 1288-98, 2011). PGD2 is a vasodilator and prevents platelet aggregation. It is mainly produced in the brain, where it plays an important role in the central nervous system (sleep regulation, allergic or pain response ...) (Cebola at Peinado, Progress in Lipid Research 51: 301-313, 2012). It is also involved in the regulation of the menstrual cycle and an inflammatory mediator (Catalano et al., Mol.Hum.Reprod. 17 (3): 182-192, 2011).

AKR1B1 inhibitors such as acetic acid derivatives (tolrestat, zenarestat), spirohydantoins (sorbinil) or succinimide compounds (ranirestat) have been mainly investigated for their role in preventing diabetic complications (Hotta, Biomed Pharmacother. 5, 244-250 1995; Costantino et al. Expert Opin Ther Patents 10, 1245-1262, 2000; Miyamoto, Expert Opin Ther Patents 2002, 12, 621-631 2002; Srivastava et al. Endocr Rev. 26, 380-392 2005). In search of better AKR inhibitors, interest has shifted in recent years towards new chemotypes by Alexia et al. Curr Med Chem. 16 (6): 734-52, 2009; Chatzopoulou et al. Expert Opin Ther Pat. 11: 1303-23 2012).

In addition to the treatment of chronic diabetic complications, the use of aldoketoreductase inhibitors AKR1B1 and AKR1B10 in the treatment of the following medical conditions has been reported: sepsis (Srivastava et al. Chem Biol Interaction 191: 330-338, 2011; Ramana, BioMol Concepts 2: 103-114, 2011; Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010, Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010, Reddy et al. Cytokine 48: 170-176 2009), periodontitis (Kador et al. J Periodontol. 2011; 82: 926-933,2011), asthma (Yadav et al. Chemico-Biological Interactions 191: 339-345,2011; Srivastava et al. Chem Biol Interaction 191). Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010;) uveitis (Srivastava et al. Chem Biol Interaction 191: 330-338, 2011; Yadav et al. IOVS 48 (10): 4634-4642, 2007; Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010; Yadav et al. Curr Mol Med 10, 540-549, 2010), atherosclerosis Ramana and Srivastava, I nt J. Biochem Cell Biol. 42: 17-20, 2010), endotoxemia (Pandey et al. Expert Opin Investig Drugs 21 (3): 329-339, 2012) and cancer (Tammali et al. Carcinogenesis.32 (8): 1259-1267, 2011; Fukumoto et al. Clin Cancer Res. 1; 11 (5): 1776-85, 2005; Laffin and Petrash, Front Pharmacol. 3: 104, 2012; Ma et al. Int. J. Cancer: 131, E862-E871, 2012; Kang et al. J Int Med Res 39: 78-85, 2011) and neurological and psychiatric disorders or mood disorders (Alexiou et al., Curr.Med.Chem. 16: 734-752, 2009; Regenold et al. (Mol. Psych. 9: 731-733, 2004).

US 2011092566 relates to methods for treating lung, breast and prostate cancer and suppressing metastasis associated with colon cancer using aldose reductase inhibitors.

US 2013023529, US 8273746 relate to methods and compositions comprising aldose reductase inhibitors for the treatment of inflammation, including uveitis and asthma.

US 20100292287 relates to methods for preventing and treating periodontitis in mammals, including humans and dogs, based on the administration of an effective amount of an aldose reductase inhibitor.

CN 102346193 relates to AKR1B10 as a diagnostic marker of breast cancer and drug target.

Patent RU 2374647 discloses a diagnostic technique for colon cancer based on detecting the mRNA level of the AKR1B10 gene.

US 20111236394 discloses a method of modulating and monitoring the level of PGF2α by modulating the level of AKRI BI, respectively. its PGFS activity, wherein AKR1B1 is presented as a therapeutic target.

WO 02/098421 is concerned with the treatment of psychiatric and neurological lesions and diseases by inhibiting aldose reductase.

U.S. Patent No. 6,696,407 discloses methods of modulating neurotrophic factor-associated activity using aldose reductase inhibitors.

Although a number of aldo-ketoreductase inhibitors AKR1B1 and AKR1B10 have been extensively studied, none of them have demonstrated sufficient efficacy in human clinical trials without undesirable side effects. Thus, there remains a need for novel, potent and safe aldo-ketoreductase inhibitors AKR1B1 and AKR1B10 as potential drugs for diabetic complications, inflammatory diseases and cancer.

5-Carboxymethyl-3-mercapto-1,2,4-triazino- [5,6-b] indole (Ia) was subjected to HTS screening in 372 assays. Biological activity was recorded in 12 cases, none of which represented inhibition of AKR1B1 and AKR1B10 aldo-ketoreductases (http://www.ncbi.nlm.nih.gov/sites/ entrez? Db = pccompound & cmd = Link & Link Name = pccompound pcassav active & uid) = 685,919).

(Ia)

The most disclosed and patented compounds so far described are the following documents: WO2013041859 A1, US2004127499 A1, RU1487415 C, RU1804067 C, RU1487413 C, RU1665679 C, GB 1023720 A and Aswar et al. Asian Pac J Trop Biomed 2, 992-998 (2012). In the structures defined in the above documents, the substituent at the 5-position is not defined as the carboxymethyl group -CH ₂ COOH. In M Stefek et al. J Med Chem 58: 2649 (2015) has been clearly shown that the presence of the -CH ₂ COOH group at the 5-position of the 1,2,4-triazino [5,6-b] indole skeleton unexpectedly and unpredictably affects pharmacological effects. It has been shown in the cited publication that compound 19 in which the carboxymethyl group CH ₂ COOH is absent at the 5-position is ineffective as an aldose reductase inhibitor. Inhibition of the enzyme aldose reductase is critical to the pharmacological effects of the claims. The compounds defined in the present application cannot be derived from the compounds claimed in WO2013041859 A1, US2004127499 A1, RU1487415 C, RU1804067 C, RU1487413 C, RU1665679 C, GB 1023720 A and Aswar et al. Asian Pac J Trop Biomed 2, 992-998 (2012).

The aldose reductase inhibitors described so far most closely related to the present invention include structurally related triazines (DaSettimo et al. J Med Chem. 44 (25): 4359-4369, 2001) as well as structurally different triazines (Mavel et al. Chem Pharm Bull (Tokyo) 40). (6): 1411-1414,1992. Aldose reductase inhibitory activity has been reported in a number of tricyclic carbolines (Minehira et al. Bioorg Med Chem 20 (1): 356-67, 2012). The compounds defined in the present application cannot be derived from the compounds claimed herein.

SUMMARY OF THE INVENTION

The present invention relates to 5-carboxymethyl-3-mercapto-1,2,4-triazino- [5,6-b] indoles of formula (I),

R 2 is -H, or R 1 is and R 2 is -CH 3.

The invention also relates to a pharmaceutical composition comprising as active ingredient one of the compounds of formula (I) and a pharmaceutically acceptable carrier for use in the treatment of human and veterinary diseases in which aldo-keto reductase activities AKR1B1 and AKR1B10 are key etiological factors for their development and progression such as diabetic complications of macro- and microangiopathy, atherosclerosis, retinopathy, cataract, nephropathy, neuropathy and bone loss, inflammatory diseases such as uveitis, sepsis, periodontitis, asthma and colon cancer, abnormal proliferation of vascular smooth muscle cells and restenosis of smooth muscle cells and , lung cancer in smokers, lung, breast, liver, prostate, pancreas, endometrial cancer, cervical cancer and cervical adenocarcinoma, diseases of the female reproductive system, such as disorders of the menstrual cycle of apertility, menstrual problems, time not delivery, mood disorders, psychiatric and neurological diseases.

The invention also relates to a pharmaceutical composition comprising as active ingredient one of the compounds of formula (I) in solid, oral or injectable dosage form for systemic administration or in the form of drops, pastes, gels or ointments for topical administration, for use in the treatment of human and veterinary diseases in which aldo-keto reductase activities AKR1B1 and AKR1B10 are key etiological factors for their development and progression such as diabetic complications of macro- and microangiopathy, atherosclerosis, retinopathy, cataract, nephropathy, neuropathy and bone loss, inflammatory diseases, are uveitis, sepsis, periodontitis, asthma and colon cancer, abnormal proliferation of vascular smooth muscle cells during atherosclerosis and restenosis, lung cancer in smokers, lung, breast, liver, prostate, pancreas, endometrial cancer, cervical cancer and cervical cancer female of the reproductive system such as menstrual cycle and fertility disorders, menstrual problems, childbirth timing, mood disorders, psychiatric and neurological diseases.

The invention also relates to pharmaceutical compositions comprising as active ingredient one of the compounds of formula (I), their use as well as therapeutic methods for administering the compound (I) to a mammal.

The compounds of formula (I) of the present invention are useful as an agent with the ability to inhibit aldo-ketoreductases AKR1B1 and AKR1B10.

Compounds of formula (I) may be prepared by synthetic procedures well known to those skilled in the art, as described in standard papers such as Romanchick and Joullie Heterocycles 9, 1631 (1978); Neunhoeffer, H.; Wiley, P. E. In Chem. Heterocycl. Compd .; Wiley-Interscience: New York, 1978, 33, 749; E1 Ashry, E. S. H .; Rashed, N .; Taha, M. In Advances in Heterocyclic Chemistry; Katritzky, A.R. ed .; Academic Press: New York, 1994, 59.

The general scheme for the preparation of 5H- [1,2,4] triazino [5,6] indolo-3-thiols consists of the following steps:

λ.

h ₃ ca

where R 1 is h ₃ c

ch ₃ ch ₃ ch ₃

R 1 is -H, or R 1 is

and Rz is -CH 3.

1H-indole-2,3-dione (1, substituted isatin) plus thiosemicarbazide to give isatin thiosemicarbazone (2) which is cyclized in an alkaline solution to give the substituted 5H- [1,2,4] triazino [5,6] indole- 3-thiol (3a) (tautomeric form of 2,5-dihydro-3H- [1,2,4] triazino [5,6] indole-3-thione, 3b). Alternatively, the product is alkylated with a suitable halogen derivative to give a derivative with the desired substituent on the sulfur atom (4). Reaction conditions as described in Gupta et al. Eur J Med Chem 45 (6): 2359-2365 (2010); Hamid J. Chem. Res. 183-185 (2004); Shelke and Bhosale Bioorg Med Chem Lett 20 (15): 4661-4664 (2010); Abdel-Sayed Bulgarian Chem. Commun 41 (4) 362 (2009); Mohan and Kumar Indian J. Chem 41B, (11), 2364-2370 (2002); Pal et al. Indian J. Chem. B 1991, 30, 1098-1102 (1991), US 5830894, GB 1023720, are well known to a person skilled in the art.

For oral therapeutic administration, the active compound may be combined with one or more carriers and used in the form of ingestive tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically used compositions is such that an effective dosage level is achieved.

Tablets, lozenges, pills, capsules and the like. they may also contain the following: binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid and the like; lubricants such as magnesium stearate; and sweetening agents such as sucrose, fructose, lactose or aspartate, or essential oils such as menthol, wintergreen oil or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to the listed ingredients, a solvent such as a vegetable oil or polyethylene glycol. Various other substances may be present, such as surface coatings or substances otherwise modifying the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, and the like. The syrup or elixir form may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, dyes and a cherry or orange flavoring as flavoring agents. Naturally, any agent used in the preparation of any unit dosage form should be pharmaceutically acceptable and non-toxic in the amount used. In addition, the active compound may be incorporated into controlled release formulations and compositions.

The compounds or mixtures may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or salt thereof may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations contain preservatives to protect against the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion may contain sterile aqueous solutions or dispersions or sterile powders containing the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions or are encapsulated in liposomes. In all cases, the final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion comprising e.g. water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyedtylene glycols, and the like), vegetable oils, non-toxic esters of glycerol, and suitable mixtures thereof. Proper fluidity can be maintained e.g. liposome formation by maintaining the desired particle size in the case of dispersions or by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, e.g. parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to use isotonic agents, e.g. sugars, buffers or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use of agents which delay absorption, such as e.g. aluminum monosterate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound in the required amount into a suitable solvent with various other ingredients and, if necessary, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum and freeze-drying techniques which leave the active ingredient powder plus the desired additive present in previously sterile filtered solutions.

For topical administration, the present compounds may be administered in pure form, i. j. if they are fluids. However, it is generally desirable to administer them to the skin as mixtures or kits in combination with a dermatologically acceptable carrier, which may be solid or liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, quartz, and the like. Useful liquid carriers include water, alcohols or glycols, or water-alcohol / glycol mixtures in which the present compound can be dissolved or dispersed to an effective level, optionally with the help of non-toxic surfactants. Adjuvants such as perfumes and other antimicrobial agents can be added to optimize properties for a given application. The resulting liquid compounds can be applied from absorbent pads, to impregnate bandages and other dressings, or spray onto the affected area by means of pump or aerosol sprays. Thickeners, such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified celluloses or modified mineral materials can also be used with liquid carriers to form disintegrating pastes, gels, ointments, soaps, and the like. for application directly to the user's skin.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by the following examples which present measurements of inhibition of aldo-keto-reductases by (Ia) - (Ie). The examples do not limit the scope of the claims. It will be apparent to those skilled in the art that many modifications in terms of materials and methods may be practiced without limiting the purpose and content of the invention.

N

SH .O

OH (Ial

OH (Ibl

Ile ")

He ')

Example 1

Inhibition of aldose reductases (ALR2) at the level of isolated enzyme in vitro

Preparation of ALR2. ALR2 from rat lenses was partially purified by the adaptive method of

Hayman and Kinoshita [J Biol Chem. 240: 877-882 (1965)] as follows: the lenses were rapidly dissected from rats after their euthanasia and homogenized in a Teflon plunger glass homogenizer in 5 volumes of cold distilled water. The homogenate was centrifuged at 10,000 g at 0-4 ° C for 20 min. The supernatant was precipitated with saturated ammonium sulfate successively at 40%, 50%, then at 75% saturation.

After the first two precipitations, the supernatant was used. The sediment from the last step containing the ALR2 activity was dispersed in 75% ammonium sulfate and stored in smaller aliquots in a liquid nitrogen container.

Enzyme test. ALR2 activity was measured spectrophotometrically [Stefek et al. Bioorg Med. Chem. 16: 4908-4920 (2008)] by determining NADPH consumption at 340 nm and expressed as a decrease in optical density (O.D.) / s / mg protein. The reaction mixture contained 4.67 mM d, 1-glyceraldehyde as a substrate, 0.11 mM NADPH, 0.067 M phosphate buffer, pH 6.2, and 0.05 ml enzyme preparation in a total volume of 1.5 ml. The reference blank sample contained all of the aforementioned components except the d, l-glyceraldehyde substrate to correct NADPH oxidation associated with substrate reduction. The enzyme reaction was initiated by addition of d, 1-glyceraldehyde and monitored for 4 min. after an initial 1 minute period at 30 ° C. Enzyme activities were adjusted by diluting the enzyme preparations with distilled water so that 0.05 ml of the preparation gave an average reaction rate for the control samples in the range of 0.020 ± 0.005 absorbance units / min. The effect of inhibitors on enzyme activity was determined by adding the inhibitor to the reaction mixture at the desired concentrations. An inhibitor of the same concentration was added to the reference sample. IC 50 values (inhibitor concentration required to achieve 50% inhibition of the enzyme reaction) were determined by the least squares method from the linear portion of the semi-logarithmic inhibition curves. Each curve was generated at least using four inhibitor concentrations causing inhibition ranging from at least 25 to 75%.

Table 1

Inhibitory effect of Ia-Ie on ALR2 from rat lens

substance IC (g M) la 0.106 + 0.004 lb 2.25 + 0.06 ic 4.18 ± 0.80 ld 0.319 + 0.031 Ie 2.06 + 0.17

Results are mean ± SD from at least three measurements.

Example 2

Inhibition of aldehyde reductase (ALR1) at the level of isolated enzyme in vitro Preparation of ALR1. ALR1 was partially purified from rat kidney as described by Constantino et al. [Chem. 42: 1881-1893 (1999)] as follows: the kidneys were rapidly removed from rats after euthanasia and homogenized with a knife homogenizer followed by treatment in a Teflon plunger glass homogenizer in 3 volumes of 10 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2.0 mM EDTA dipotassium salt, and 2.5 mM β-mercaptoethanol. The homogenate was centrifuged at 16,000 g at 0-4 ° C for 30 min. and the supernatant was subjected to fractional precipitation with ammonium sulfate at 40%, 50% and 75% saturation. The sediment obtained from the last precipitation, having ALR1 activity, was redissolved in 10 mM sodium phosphate buffer, pH 7.2, containing 2.0 mM EDTA dipotassium salt and 2.0 mM β-mercaptoethanol to achieve a protein concentration of approximately 20 mg / ml. DEAE DE 52 resin (33 mg / ml) was added to the solution and after gentle stirring for 15 min. was removed by centrifugation. The supernatant containing ALR1 was then stored in smaller aliquots in liquid nitrogen. No significant contamination of ALR2 in ALR1 preparations was noted since no consumption of NADPH was recorded in the presence of glucose up to a concentration of 150 mM.

Enzyme test. ALR1 activity was measured spectrophotometrically [Stefek et al. Bioorg Med. Chem. 16: 4908-4920 (2008)] by determining NADPH consumption at 340 nm and expressed as a decrease in optical density (O.D.) / s / mg protein. The reaction mixture contained 20 mM D-glucuronate as a substrate, 0.12 mM NADPH in 0.1 M phosphate buffer pH 7.2, and 0.05 mL of enzyme preparation in a total volume of 1.5 mL. The reference blank sample contained all of the above components except the D-glucuronate substrate to correct NADPH oxidation associated with substrate reduction. The enzyme reaction was initiated by the addition of D-glucuronate and monitored for 4 min. after an initial period after an initial 1 minute period at 37 ° C. Enzyme activities were adjusted by diluting the enzyme preparations with distilled water so that 0.05 ml of the preparation gave an average reaction rate for the control samples in the range of 0.020 ± 0.005 absorbance units / min. The effect of inhibitors on enzyme activity was determined by adding the inhibitor to the reaction mixture at the desired concentrations. An inhibitor of the same concentration was added to the reference sample. IC 50 values (inhibitor concentration required to achieve 50% inhibition of the enzyme reaction) were determined by the least squares method from the linear portion of the semi-logarithmic inhibition curves. Each curve was generated at least using four inhibitor concentrations causing inhibition ranging from at least 25 to 75%.

Table 2

Inhibitory effect of Ia-Ie on rat kidney ALR1

substance IC 50 / I (%, c = 10 pM) la 40.6 + 2.1 / 22.71 +4.41 lb > 10 / 1.18 + 4.35 lc > 10 / 23.48 + 2.11 Id 19.56 + 5.38 / 27.06 + 7.38 Ie 14.13 + 0.36 / 35.67 + 0.89

Results are mean ± SD from at least three measurements.

Example 3

Inhibition of AKR1B1 and AKR1B10 aldo-ketoreductases at the level of isolated enzymes in vitro

Preparation of cell fractions enriched for recombinant enzyme AKR1B10. COS-7 cells obtained from ATCC were cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS (fetal bovine serum), 2 mM glutamine, 100 U / ml penicillin and 100 µg / ml streptomycin. For transfection, cells were grown to 70% -80% confluency. Just prior to transfection, the culture medium was replaced with DMEM supplemented with 10% FBS. Transient transfection of cells with plasmid pCEFLKZ-AU5 containing AKR1B10 ORF was performed using Lipofectamine 2000 reagent at a ratio of 1 µg DNA to 3 µl Lipofectamine. After incubation for 5 hours, the transfection medium was replaced with complete medium. The cells were incubated for 48 hours. Subsequently, cells were scraped and collected in cold isotonic phosphate buffer and lysed in 50 mM phosphate buffer, pH 6.8 in the presence of 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM β-mercaptoethanol, 320 pg / µl Pefablock , 0.1 mM sodium ortovanadate, and protease inhibitors (leupeptin, aprotinin and trypsin inhibitor, 2 µg / µl each), by forced passage through a 261 / 2G syringe.

Enzyme assay AKR1B1 and AKR1B10. The test mixtures contained sodium phosphate buffer (0.1 M; pH 6.8), defined weight (pg) of proteins, 0.2 mM NADPH cofactor, 10 mM _DL- glyceraldehyde. Compound I was added as a solution in DMSO at a final DMSO concentration of 1%. The activity assay was initiated by adding AKR protein to the cuvette. After 4 min. by incubation at room temperature, enzyme activity was evaluated based on NADPH consumption at 340 nm (ε ₃₄₀ nadph = 6200 M'AM ¹ ) on an Ultrospec 4300 spectrophotometer (GE Biosciences). Blanking was performed for the contribution of inhibitors to absorbance at 340 nm. Enzyme activity was expressed in nmol / min / mg protein. S100 fractions from non-transfected COS-7 cells did not contain AKR activity.

Table 3

Inhibitory effect of 1a on human AKRI B1 and AKR1B10 enzymes

substance IC (pM) AKR1B1 AKR1B10 la 0.48 + 0.29 3.69 + 0.53

Results are mean ± SD from at least three measurements.

Example 4

In vitro inhibition of aldose reductase (ALR2) (isolated eye lens)

Determination of sorbitol in ophthalmic lenses. Animals under light ether anesthesia were sacrificed by bleeding after cervical artery disruption and the eyeballs were separated. The ophthalmic lenses were rapidly dissected and rinsed with saline. Compound I dissolved in DMSO was added to tubes containing freshly prepared lenses (1 lens per tube) in M-199 medium at pH 7.4, bubbled at 37 ° C with pneumoxide (5% CO ₂ , 95% O ₂ ), in the desired concentrations, 30 min. before adding glucose. The final DMSO concentration in all incubations was 1%. Incubations were initiated by the addition of glucose at a final concentration of 50 mM and continued at 37 ° C, with occasional (approximately 30 min. Intervals) bubbling the mixture (over 30 s) with pneumoxide. Incubations were terminated after 3 hours by cooling the mixture in an ice bath, followed by brief washing three times with cold isotonic phosphate buffer (1 mL). Short-term cultures were preferred to avoid substantial changes in lens permeability. The washed lenses were kept deep frozen until the sorbitol assay was performed as previously described [Juskova et al. General Physiology and Biophysics, 28, 325-330 (2009). After subtracting the appropriate blank, the sorbitol content in nmol / g wet weight of the lens was determined by comparison with a calibration line of sorbitol standards.

Table 4

Effect of substance 1a on sorbitol accumulation in isolated rat lenses incubated with glucose '

incubation Sorbitol (nmol / g) n - Glucose 233.99 + 7.80 ^b 15 + Glucose 772.90 + 19.70 17 + Glucose + la (1.0 μΜ) 668.66 + 54.15 4 + Glucose + Ia (10 μΜ) 536.01 + 25.90 ° 7 + Glucose + la (50 μΜ) 478.71 + 30.99 ° 4 + Glucose + la (100 μΜ) 395.06 + 23.83 ° 10

Results are mean ± SEM from independent measurements. Glucose, 50 mM; incubation time, 3 h; Deň: 32 ° C. ^b p <0.001 (+) Glucose (Student's t-test).

° p <0.001 (+) Glucose (Student's t-test).

PATENT CLAIMS

Claims (3)

1. 5-Carboxymethyl-3-mercapto-1; 2,4-triazino- [5,6-b] indoles of general formula (I) in which R 1 is and pharmaceutically acceptable salts, hydrates and solvates thereof, for use in the treatment of human and veterinary diseases in which aldo-keto reductase activities AKR1B1 and AKR1B10 are key etiological factors for their development and progression such as diabetic complications of macro- and microangiopathy, atherosclerosis, retinopathy, cataract, nephropathy, neuropathy and bone loss, inflammatory diseases such as uveitis, sepsis, 20 periodontitis, asthma and colon cancer, abnormal proliferation of vascular smooth muscle cells during atherosclerosis and restenosis, lung cancer in smokers, lung, breast, liver, prostate, pancreas, endometrial cancer, cervical cancer, and cervical adenocarcinoma, maternal cervical disease, such as menstrual cycle disturbances, menstrual problems, timing of labor, mood disorders, psychiatric and neurological diseases. 2. A pharmaceutical composition comprising, as an active ingredient, one of the compounds of formula (I) and a pharmaceutically acceptable carrier for use in the treatment of human and veterinary diseases in which aldo-keto reductase activities AKR1B1 and AKR1B10 are key etiological factors for their development and progression, such as diabetic complications of macro- and microangiopathy, atherosclerosis, retinopathy, cataract, nephropathy, neuropathy and bone loss, inflammatory diseases such as uveitis, sepsis, periodontitis, asthma and colon cancer, abnormal proliferation of vascular smooth muscle cells and restenosis, lung cancer in smokers, lung, breast, liver, prostate, pancreas, endometrial cancer, cervical cancer and cervical adenocarcinoma, diseases of the female reproductive system such as menstrual cycle and fertility disorders, menstrual problems, timing of labor, malfunction Moods, psychiatric 5 and neurological diseases. A pharmaceutical composition comprising an effective amount of a compound of formula (I), in solid, oral or injectable dosage form for systemic administration, or in the form of drops, pastes, gels or ointments for topical administration, for use in the treatment of human and veterinary diseases, whose aldo-keto reductase activities AKRI BI and AKR1B10 are key etiological factors for their development and progression, such as diabetic complications of macro- and microangiopathy, atherosclerosis, retinopathy, cataract, nephropathy, neuropathy and bone loss, inflammatory diseases such as uveitis , sepsis, periodontitis, asthma and colon cancer, abnormal proliferation of vascular smooth muscle cells during atherosclerosis and restenosis, lung cancer in smokers, lung, breast, liver, prostate, pancreas, endometrial cancer, cervical cancer, and adenocarcinoma system as with ú faults 15 menstrual cycle and fertility, menstrual problems, birth timing, mood disorders, psychiatric and neurological diseases.