WO2015057175A1 - Use of 5-carboxymethyl-3-mercapto-1,2,4-triazino-[5,6-b]indoles and their pharmaceutical composition - Google Patents

Use of 5-carboxymethyl-3-mercapto-1,2,4-triazino-[5,6-b]indoles and their pharmaceutical composition Download PDF

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WO2015057175A1
WO2015057175A1 PCT/SK2014/000021 SK2014000021W WO2015057175A1 WO 2015057175 A1 WO2015057175 A1 WO 2015057175A1 SK 2014000021 W SK2014000021 W SK 2014000021W WO 2015057175 A1 WO2015057175 A1 WO 2015057175A1
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cancer
diseases
alkyl
atherosclerosis
cyano
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French (fr)
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Superior De Investigaciones Cientificas Consejo (60%)
Vedecko-Technických Informácií Centrum Sr
Milan ŠTEFEK
Ivana MILÁČKOVÁ
Beatriz DIEZ-DACAL
Dolores PÉREZ-SALA GOZALO
Marta ŠOLTÉSOVÁ PRNOVÁ
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Ústav Experimentálnej Farmakológie A Toxikológie Sav
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to the use of 5-carboxymethyl-3-mercapto-l,2,4-triazino- [5,6-b]indoles and a pharmaceutical composition containing this compounds for treatment and control of human and veterinary diseases.
  • Aldo-keto reductases are NAD(P)H-dependent oxidoreductases, best characterized as glucose reducing agents. They have been implicated in the pathophysiology of diabetic complications. These enzymes have also been reported to metabolize lipid peroxidation products, contributing in some settings to the inflammatory response.
  • Aldose reductase (AR, ALR2, AKR1B1), apart from its involvement in diabetic complications via reducing glucose, was found to efficiently reduce lipid peroxidation- derived aldehydes and their glutathione conjugates (Ramana, BioMol Concepts 2:103-114, 2011).
  • Lipid peroxidation-derived lipid aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) and their glutathione conjugates (e.g. GS-HNE)
  • HNE 4-hydroxy-trans-2-nonenal
  • GS-HNE glutathione conjugates
  • the reduced aldehyde glutathione conjugate GS-DHN is considered a novel signaling intermediate in the transduction of reactive oxygen species-initiated cell signals, leading eventually to an inflammatory response (Srivastava et al. Chem Biol Interaction 191:330-338, 2011). Inhibition of AR was found to prevent significantly the inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens and auto-immune reactions in cellular as well as animal models (Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010).
  • Aldose reductase inhibitors thus present a novel therapeutic approach to treat a wide array of inflammatory diseases such as atherosclerosis, asthma, uveitis, sepsis, arthritis, periodontitis and other injuries that have the potential of stimulating the immune system and generating large amounts of inflammatory cytokines and chemokines including 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. 1 1 (5):560-571 ,2011; Kador et al. J Periodontol. 201 1 ;82:926-933,2011; Yadav et al. Curr Mol Med 10, 540-549, 2010).
  • inflammatory diseases such as atherosclerosis, asthma, uveitis, sepsis, arthritis, periodontitis and other injuries that have the potential of stimulating the immune system and generating large amounts of inflammatory
  • AKR1B1 overexpression of AKR1B1 in various cancers caused resistance to doxorubicin which was explained by increased detoxification of doxorubicin via carbonyl reduction mediated by AKR1B1 (Lee et al. Anticancer Drugs (2):129-32 2001).
  • AKR1B1 the closely related AKR1B10 has been implicated in various types of cancer (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):273-82, 2012).
  • AKRIBIO is a 36-kDa cytosolic reductase that is similar to AKRIBI in both amino acid sequence identity (71%) and tertiary structure with the ( ⁇ / ⁇ )8 barrel topology. Like AKRIBI, AKRIBIO reduces a variety of aromatic and aliphatic aldehydes, dicarbonyl compounds and some drug ketones using NADPH as the coenzyme.
  • AKRIBI AKRIBIO
  • AKRIBI AKRIBIO
  • AKRIBI AKRIBIO
  • Pharmacological inhibition or genetic ablation of AKRIBI and AKRIBIO has been shown to prevent cytokine and growth factor induced inflammatory signals and proliferation of cancer cells in culture as well as in nude mice xenograft models.
  • PGF2a plays a variety of physiological roles in the body, such as the contraction of uterus, bronchial and arterial and vascular smooth muscles, regulation of pressure in the eye, renal filtration, stimulation of hair growth, regulation of ovarian cycle through the induction of luteolysis and others (Nagata et al. FEBS Journals 278:1288 - 98, 2011). It is associated with diseases such as diabetes, osteoporosis, menstrual disorder and has important role in the female reproductive system (Madore et al. J. Biol.
  • AKRIBI enzyme is also able to catalyze the isomerisation of PGH2 to PGD2 in the absence of NADPH (Nagata et al. FEBS Journals 278:1288-98, 2011).
  • PGD2 is vasodilatator and inhibits platelet aggregation. Preferentially produced in the brain, it play an important role in the central nervous system modulation (regulation of sleep, allergic and pain responses%) (Cebola at Peinado, Progress in Lipid Research 51: 301-313, 2012). It is also member of the regulation of the menstrual cycle and mediators of inflammation (Catalano et al. Mol.Hum.Reprod. 17 (3) :182-192, 2011).
  • AKRIBI inhibitors such as the acetic acid derivatives (tolrestat, zenarestat), spiro hydantoins (sorbinil), or the succinimide class of compounds (ranirestat) have been primarily investigated for their role against 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 the search for better AKR inhibitors, the focus has been shifted in recent years towards novel chemotypes (Alexiou et al. Curr Med Chem. 16(6):734-52, 2009; Chatzopoulou et al. Expert Opin Ther Pat. 11:1303-23 2012).
  • Patent US2011092566 relates to methods of treating lung, breast and prostate cancer or suppressing metastasis of colon cancer by using aldose reductase inhibitors.
  • Patents US2013023529, US 8273746 claim methods and compositions involving aldose reductase for treatment of inflammation, including uveitis and asthma.
  • Patent US20100292287 relates to a method for the prevention and treatment of periodontitis in mammals, including humans and dogs, based on administering an effective amount of an aldose reductase inhibitor.
  • Patent CN102346193 deals with AKR1B10 as a breast cancer diagnostic marker and drug target.
  • Patent RU 2374647 describes diagnostic technique for colon cancer based on detection of mRNA level of gene AKR1B10.
  • Patent US 20111236394 is related to a method for modulating and monitoring PGF2a levels and activity in a subject in need thereof by modulating AKRIBI leves or ots PGFS activity. Also, AKRIBI is a therapeutic target.
  • Patent WO 02098421 is related to the use of aldose reductase inhibitors to treat psychiatric and neurological disorders and diseases.
  • Patent US 6696407 relates to methods of modulating neurotrophic factor -associated activity using aldose reductase inhibitors.
  • aldose reductase inhibitors most closely related to the present invention include structurally related triazines (DaSettimo et al. J Med Chem. 44(25):4359- 4369, 2001) and structurally more distinct triazines (Mavel et al. Chem Pharm Bull (Tokyo) 40(6):1411-1414, 1992).
  • a series of tricyclic carbolines revieled aldose reductase inhibitory activity Minehira et al. Bioorg Med Chem 20(l):356-67, 2012).
  • the subject matter of the invention relates to 5-carboxymethyl-3-mercapto-l,2,4- triazino-[5,6-b]indoles of a general formula I in which
  • Ro is hydrogen atom, an alkyl, alkenyl, alkynyl, cykloalkyl, aryl or aralkyl radical, thienyl, or a radical of a general formula -AlkOH, -AlkNXY,
  • Alk is a straight or branched chain alkylene radical containing 2 to 10 carbon atoms and X and Y are H atoms or alkyl radicals or together with the adjacent nitrogen atom form a heterocyclic ring which may contain additional hetero atom;
  • cycloalkyl which is optionally substituted with cyano, nitro, fluoro or alkyl;
  • R 6 and R 7 are each independently selected from hydrogen, methyl, ethyl, phenyl, benzyl or R 6 and R 7 are linked so that, together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring optionally comprising one, two or three additional heteroatoms selected from N, O or S;
  • Q 2 is optionally substituted by one or more substituents selected from halo, trifluoromethyl, trifluoromethoxy, cyano, isocyano, nitro, hydroxy, mercapto, amino, formyl, carboxy, carbamoyl, ureido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylamino, di-alkyl-amino, alkoxycarbonyl, N- alkylcarbamoyl, N,N-di-alkyl-carbamoyl, alkynoyl, alkanoyloxy, alkanoylamino, sulphamoyl, N-alkylsulphamoyl, N,N-di-alkyl-sulphamoyl or a phenyl ring which is optionally further substituted by halo, meth, me
  • Ri is a protected or unprotected carboxyl group; groups R 2 , R3, R4 and R 5 are each independently hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl, cykloalkyl, alkylene, aralkyl, hydroxy, alkoxy, nitro, amino, trifluoromethyl, thiophene or aryl, wherein the aryl ring is optionally substitutedby one or more substituents selected from halo, trifluoromethyl, trifluoromethoxy, cyano, isocyano, nitro, hydroxy, mercapto, amino, formyl, carboxy, carbamoyl, ureido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylamino, di-alkyl- amino, alkoxycarbonyl, N-alkyl
  • AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system (menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases.
  • diabetic complications micro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis
  • inflammatory diseases uveitis, se
  • a further embodiment of the present invention relates to a pharmaceutical composition containing one of the compounds of the general formula I for use in the treatment and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system(menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases.
  • a further embodiment of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a compound of the general formula I, the formulation of which is a solid oral form or injection form for systemic administration or it is in the form of drops, pastes, gels or ointments for topical administration for its use in the treatment, control and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometri
  • a further embodiment of the present invention relates to pharmaceutical compositions comprising an effective amount of a compound of the general formula I, their use as therapeutic methods by means of administering compound I to mammals.
  • the compounds of the general formula I are useful as means with the ability to inhibit aldo-keto reductases AKR1B1 and AKR1B10.
  • the compounds of the general formula I may be prepared by synthetic processes known to the person skilled in the art and described in standard works 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; El Ashry, E. S. H.; Rashed, N.; Taha, M. In Advances in Heterocyclic Chemistry; Katritzky, A.R. Ed.; Academic Press: New York, 1994, 59.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained- release preparations and devices.
  • the compounds or compositions can also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • ARR2 Aldose reductase
  • ALR2 from rat lens was partially purified using a procedure adapted from Hayman and Kinoshita [Hayman S, Kinoshita J H. Isolation and properties of lens aldose reductase. J Biol Chem. 240: 877-882 (1965)] as follows: lenses were quickly removed from rats following euthanasia and homogenized in a glass homogenizer with a teflon pestle 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 at 40 %, 50 % and then at 75 % salt saturation. The supernatant was retained after the first two precipitations. The pellet from the last step, possessing ALR2 activity, was dispersed in 75 % ammonium sulfate and stored in smaller aliquots in liquid nitrogen container.
  • Enzyme assay ALR2 activities were assayed spectrophotometrically [Stefek et al. Bioorg Med Chem. 16:4908-4920 (2008)] by determining NADPH consumption at 340 nm and were expressed as the decrease of the optical density (O.D.)/s/mg protein.
  • the reaction mixture contained 4.67 mM D,L-glyceraldehyde as a substrate, 0.11 mM NADPH, 0.067 M phosphate buffer, pH 6.2 and 0.05 ml of the enzyme preparation in a total volume of 1.5 ml.
  • the reference blank contained all the above reagents except the substrate D,L-glyceraldehyde to correct for oxidation of NADPH not associated with reduction of the substrate.
  • the enzyme reaction was initiated by addition of D,L-glyceraldehyde and was monitored for 4 min after an initial period of 1 min 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 sample of 0.020 ⁇ 0.005 absorbance units/min.
  • the effect of inhibitors on the enzyme activity was determined by including in the reaction mixture each inhibitor at required concentrations. The inhibitor at the same concentration was included in the reference blank.
  • IC50 values (the concentration of the inhibitor required to produce 50 % inhibition of the enzyme reaction) were determined from the least-square analysis of the linear portion of the semilogarithmic inhibition curves. Each curve was generated using at least four concentrations of inhibitor causing an inhibition in the range from at least 25 to 75 %.
  • Result is a mean values ⁇ SD from at least three measurements.
  • the homogenate was centrifuged at 16 000 g at 0-4°C for 30 min and the supernatant was subjected to ammonium sulfate fractional precipitation at 40 %, 50 % and 75 % salt saturation.
  • the pellet obtained from the last step, possessing ALRl activity was redissolved in 10 mM sodium phosphate buffer, pH 7.2, containing 2.0 mM EDTA dipotassium salt and 2.0 mM ⁇ -mercaptoethanol to achieve total protein concentration of approximately 20 mg/ml.
  • DEAE DE 52 resin was added to the solution (33 mg/ml) and after gentle mixing for 15 min removed by centrifugation.
  • the supernatant containing ALRl was then stored in smaller aliquots in liquid nitrogen. No appreciable contamination by ALR2 in ALRl preparations was detected since no activity in terms of NADPH consumption was observed in the presence of glucose substrate up to 150 mM.
  • Enzyme assay ALRl activities were assayed spectrophotometrically [Stefek et al. Bioorg Med Chem. 16:4908-4920 (2008)] by determining NADPH consumption at 340 nm and were expressed as decrease of the 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 the enzyme preparation in a total volume of 1.5 ml.
  • the reference blank contained all the above reagents except the substrate D-glucuronate to correct for oxidation of NADPH not associated with reduction of the substrate.
  • the enzyme reaction was initiated by addition of D-glucuronate and was monitored for 4 min after an initial period of 1 min 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 sample of 0.020 ⁇ 0.005 absorbance units/min.
  • the effect of inhibitors on the enzyme activity was determined by including in the reaction mixture each inhibitor at required concentrations. The inhibitor at the same concentration was included in the reference blank.
  • IC 50 values (the concentration of the inhibitor required to produce 50 % inhibition of the enzyme reaction) were determined from the least-square analysis of the linear portion of the semilogarthmic inhibition curves. Each curve was generated using at least four concentrations of inhibitor causing an inhibition in the range from at least 25 to 75 %.
  • Result is a mean value ⁇ SD from at least three measurements.
  • COS-7 cells obtained from ATCC, were cultured in DMEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. For transfection cells were grown to 70%-80% confluence. Just prior to transfection, cell culture medium was replaced by DMEM supplemented with 10% FBS. Cells were transiently transfected with the plasmid pCEFL-KZ-AU5 containing AKRIBIO ORF, using Lipofectamine 2000 reagent, in a proportion of 1 ⁇ g DNA per 3 ⁇ of Lipofectamine. After 5 h incubation, the transfection medium was replaced by complete medium.
  • Cells were incubated for 48 h. Following, cells were scraped and collected in cold phosphate buffered saline and lysed in 50 mM sodium phosphate buffer, pH 6.8 in the presence of 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM ⁇ -mercaptoethanol, 320 ⁇ g/ ⁇ l Pefablock, 0.1 mM sodium orthovanadate, and protease inhibitors (leupeptin, aprotinin and trypsin inhibitor at 2 ⁇ g/ ⁇ l each one), by forced passes through a 261 ⁇ 2G syringe.
  • protease inhibitors leupeptin, aprotinin and trypsin inhibitor at 2 ⁇ g/ ⁇ l each one
  • Cell lysate was ultracentrifugated for 30 min at 200,000 g at 4°C in a Beckman TLA 100.2 rotor. The soluble fraction S100 (supernatant) was collected for further analysis and pellet was discarded. Protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific, IL, USA) according to the manufacturer's protocol.
  • AKR1B1 and AKRIBIO enzyme assays contained sodium phosphate buffer (0.1 M; pH 6.8), defined weight ⁇ g) of proteins, 0.2 mM NADPH cofactor, 10 mM D,L-glyceraldehyde.
  • the compounds 1 and 2 were added as DMSO solutions at 1% DMSO final mixture concentration.
  • Results are mean values ⁇ SD from at least three measurements.
  • Aldose reductase (ALR2) inhibition at the organ level (isolated eye lens) in vitro Eye lenses sorbitol assay The animals in light ether anesthesia were killed by exsanguinations of the carotid artery and the eye globes were excised. The lenses were quickly dissected and rinsed with saline. The compound 1 dissolved in DMSO was added into the tubes containing freshly dissected eye lenses (1 lens per tube) in M-199 medium at pH 7.4, bubbled at 37 °C with pneumoxid (5% C0 2 , 95% 0 2 ), to the final concentrations as reported, 30 min before adding glucose. The final concentration of DMSO in all incubations was 1%.
  • the incubation was initiated by adding glucose to the final concentration of 50 mM and then continued at 37 °C with occasional (in about 30-min intervals) bubbling the mixture for approximately 30-s periods with pneumoxid.
  • the incubations were terminated after a 3-h period by cooling the mixtures in an ice bath, followed by washing the lenses three times with ice-cold phosphate buffered saline (1 ml).
  • the short term cultivations were preferred to avoid substantial permeability changes of the eye lenses.
  • the washed lenses were kept deep-frozen for sorbitol determination which was performed as described before [Juskova et al. General Physiology and Biophysics, 28, 325-330 (2009). After the appropriate blanks were subtracted from each sample, the amount of sorbitol in nmol per gram of lens wet weight in each sample was determined by comparison with a linear regression of sorbitol standards.
  • Results are mean values ⁇ SEM from n independent incubations.
  • aGlucose 50 mM; time of incubation, 3 hours; 37 °C.

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Abstract

The present invention relates to the use of 5-carboxymethyl-3- mercapto-1,2,4-triazino-[5,6-b]indoles (the general formula (I)) and their pharmaceutically acceptable salts hydrates and solvates thereof for the use in treatment, control and prevention of human and veterinary diseases in which activities of aldo- keto reductases AKR1B1 and AKR1B10 are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic, endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system (menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases. The present invention relates to pharmaceutical compositions comprising effective amount of 5-carboxymethyl-3-mercapto-1,2,4-triazino-[5,6-b]indoles of the general formula (I) and a pharmaceutically acceptable carrier for the use in treatment, control and prevention of human and veterinary diseases. (Formula (I))

Description

Use of 5-carboxymethyl-3-mercapto-l,2,4-triazino-[5,6-b]indoles and their pharmaceutical composition
Technical Field
The present invention relates to the use of 5-carboxymethyl-3-mercapto-l,2,4-triazino- [5,6-b]indoles and a pharmaceutical composition containing this compounds for treatment and control of human and veterinary diseases.
Background of the Invention
Aldo-keto reductases are NAD(P)H-dependent oxidoreductases, best characterized as glucose reducing agents. They have been implicated in the pathophysiology of diabetic complications. These enzymes have also been reported to metabolize lipid peroxidation products, contributing in some settings to the inflammatory response.
Aldose reductase (AR, ALR2, AKR1B1), apart from its involvement in diabetic complications via reducing glucose, was found to efficiently reduce lipid peroxidation- derived aldehydes and their glutathione conjugates (Ramana, BioMol Concepts 2:103-114, 2011). Lipid peroxidation-derived lipid aldehydes, such as 4-hydroxy-trans-2-nonenal (HNE) and their glutathione conjugates (e.g. GS-HNE), were found to be efficiently reduced by AR to the corresponding alcohols, DHN (1,4-dihydroxy-nonene) and GS-DHN (glutathionyl-1,4- dihydroxynonene), which mediate inflammatory signals. The reduced aldehyde glutathione conjugate GS-DHN is considered a novel signaling intermediate in the transduction of reactive oxygen species-initiated cell signals, leading eventually to an inflammatory response (Srivastava et al. Chem Biol Interaction 191:330-338, 2011). Inhibition of AR was found to prevent significantly the inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens and auto-immune reactions in cellular as well as animal models (Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42: 17-20, 2010).
Aldose reductase inhibitors (ARIs) thus present a novel therapeutic approach to treat a wide array of inflammatory diseases such as atherosclerosis, asthma, uveitis, sepsis, arthritis, periodontitis and other injuries that have the potential of stimulating the immune system and generating large amounts of inflammatory cytokines and chemokines including 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. 1 1 (5):560-571 ,2011; Kador et al. J Periodontol. 201 1 ;82:926-933,2011; Yadav et al. Curr Mol Med 10, 540-549, 2010).
It has been well documented that chronic inflammation is associated with the progression of cancer (Solinas et al. Cancer Metastasis Rev. 29, 243-248, 2010) and increased expression of AKRs has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon (Liu et al. Recent Pat Anticancer Drug Discov. 4(3):246-53, 2009; Laffin and Petrash, Front Pharmacol. 3:104, 2012, Terzig et al. GastroenterologyA W^yM W ^ A, 2010). Moreover, overexpression of AKR1B1 in various cancers caused resistance to doxorubicin which was explained by increased detoxification of doxorubicin via carbonyl reduction mediated by AKR1B1 (Lee et al. Anticancer Drugs (2):129-32 2001).
In addition to AKR1B1, the closely related AKR1B10 has been implicated in various types of cancer (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):273-82, 2012). AKRIBIO is a 36-kDa cytosolic reductase that is similar to AKRIBI in both amino acid sequence identity (71%) and tertiary structure with the (α/β)8 barrel topology. Like AKRIBI, AKRIBIO reduces a variety of aromatic and aliphatic aldehydes, dicarbonyl compounds and some drug ketones using NADPH as the coenzyme.
Overexpression of both AKRIBI and AKRIBIO has been observed in various types of cancer and was reported to vary greatly by cancer type and tissue of origin (Laffin and Petrash, Front Pharmacol. 3:104, 2012). AKRIBIO was found to be significantly over- expressed in cancers of the lungs and liver. AKRIBI is more broadly over-expressed in human cancers than AKRIBIO, albeit at a generally lower magnitude. Pharmacological inhibition or genetic ablation of AKRIBI and AKRIBIO has been shown to prevent cytokine and growth factor induced inflammatory signals and proliferation of cancer cells in culture as well as in nude mice xenograft models. E.g. the antitumor activity of the antiinflammatory drug sulindac was recently interpreted in the context of AR inhibition (Steuber, ChemMedChem. 6(12):2155-7, 2011). Silencing of AKRIBIO gene resulted in the inhibition of colorectal cancer cell growth (Yan et al. Int J Cancer. 121(10):2301-6 2007).
The involvement of AKRIBIO in the development of resistance toward 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).
The above mentioned findings suggest that AKRIBI and AKRIBIO inhibitors hold great potential as novel cancer therapeutics.
Also, it has been previously described synthase activity of AKRIBI which catalyzes the transformation of PGH2 to PGF2a in the presence of cofactor NADPH. PGF2a plays a variety of physiological roles in the body, such as the contraction of uterus, bronchial and arterial and vascular smooth muscles, regulation of pressure in the eye, renal filtration, stimulation of hair growth, regulation of ovarian cycle through the induction of luteolysis and others (Nagata et al. FEBS Journals 278:1288 - 98, 2011). It is associated with diseases such as diabetes, osteoporosis, menstrual disorder and has important role in the female reproductive system (Madore et al. J. Biol. 278 (13) :11205-12, 2003). Increased level of PGF2a was detected during parturition as a means of inducing myometrial contractions and cervical dilation (Breuiller-Fouche'et al., Biol.Reprod. 83: 155-162, 2010).
AKRIBI enzyme is also able to catalyze the isomerisation of PGH2 to PGD2 in the absence of NADPH (Nagata et al. FEBS Journals 278:1288-98, 2011). PGD2 is vasodilatator and inhibits platelet aggregation. Preferentially produced in the brain, it play an important role in the central nervous system modulation (regulation of sleep, allergic and pain responses...) (Cebola at Peinado, Progress in Lipid Research 51: 301-313, 2012). It is also member of the regulation of the menstrual cycle and mediators of inflammation (Catalano et al. Mol.Hum.Reprod. 17 (3) :182-192, 2011).
AKRIBI inhibitors, such as the acetic acid derivatives (tolrestat, zenarestat), spiro hydantoins (sorbinil), or the succinimide class of compounds (ranirestat) have been primarily investigated for their role against 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 the search for better AKR inhibitors, the focus has been shifted in recent years towards novel chemotypes (Alexiou 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 inhibitors of aldo-keto reductases AKRIBI and AKRIBIO for the treatment of following health disorders has been reported and extensively reviewed: 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 191:330-338, 2011; 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. 10 VS 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, Int. J. Biochem. Cell Biol. 42: 17-20, 2010), endotoxemia (Pandey et al. Expert Opin Investig Drugs 21(3):329-339, 2012), cancer (Tammali et al. Carcinogenesis.32(8): 1259-1267, 2011; Fukumoto et al. Clin Cancer Res. l;ll(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 the neurological and psychiatric disorders and mood disorders (Alexiou et al. Curr. Med. Chem. 16: 734-752, 2009; Regenold et al. Mol. Psych. 9: 731-733, 2004).
Patent US2011092566 relates to methods of treating lung, breast and prostate cancer or suppressing metastasis of colon cancer by using aldose reductase inhibitors.
Patents US2013023529, US 8273746 claim methods and compositions involving aldose reductase for treatment of inflammation, including uveitis and asthma.
Patent US20100292287 relates to a method for the prevention and treatment of periodontitis in mammals, including humans and dogs, based on administering an effective amount of an aldose reductase inhibitor.
Patent CN102346193 deals with AKR1B10 as a breast cancer diagnostic marker and drug target.
Patent RU 2374647 describes diagnostic technique for colon cancer based on detection of mRNA level of gene AKR1B10.
Patent US 20111236394 is related to a method for modulating and monitoring PGF2a levels and activity in a subject in need thereof by modulating AKRIBI leves or ots PGFS activity. Also, AKRIBI is a therapeutic target.
Patent WO 02098421 is related to the use of aldose reductase inhibitors to treat psychiatric and neurological disorders and diseases.
Patent US 6696407 relates to methods of modulating neurotrophic factor -associated activity using aldose reductase inhibitors.
Although a number of inhibitors of aldo-keto reductases AKRIBI and AKR1B10 have been extensively studied, none of them have demonstrated sufficient efficacy in human clinical trials without undesirable side effects. Consequently there is still a significant need for new, efficacious and safe inhibitors of aldo-keto reductases AKRIBI and AKRIBIO as medications for the treatment of diabetic complications, inflammatory disorders and cancer.
Compound la was subjected to HTS screening in 372 tests. Biological activity was recorded in 12 cases, none of them being aldo-keto reductase AKRIBI or AKRIBIO inhibition (http:/ /www.ncbi.nlm.nih.gov/sites / entrez?db=pccompound& cmd=Link& Link Name=pccompound pcassay active&from uid=685919).
Figure imgf000005_0001
la
Previously described aldose reductase inhibitors most closely related to the present invention include structurally related triazines (DaSettimo et al. J Med Chem. 44(25):4359- 4369, 2001) and structurally more distinct triazines (Mavel et al. Chem Pharm Bull (Tokyo) 40(6):1411-1414, 1992). A series of tricyclic carbolines revieled aldose reductase inhibitory activity (Minehira et al. Bioorg Med Chem 20(l):356-67, 2012).
Disclosure of the Invention
The subject matter of the invention relates to 5-carboxymethyl-3-mercapto-l,2,4- triazino-[5,6-b]indoles of a general formula I in which
Ro is hydrogen atom, an alkyl, alkenyl, alkynyl, cykloalkyl, aryl or aralkyl radical, thienyl, or a radical of a general formula -AlkOH, -AlkNXY,
where Alk is a straight or branched chain alkylene radical containing 2 to 10 carbon atoms and X and Y are H atoms or alkyl radicals or together with the adjacent nitrogen atom form a heterocyclic ring which may contain additional hetero atom;
or a group of a general formula -(CH2)„-Q\ (n = 1-8),
wherein Q1 is selected from
cycloalkyl which is optionally substituted with cyano, nitro, fluoro or alkyl;
or phenyl which is substituted in the ortho or para position (relative to the point of attachment to the -CH2- group) by cyano, nitro, methyl, -C02H and tetrazole and optionally further substituted with cyano, nitro, fluoro, or methyl;
or naphthyl which is optionally substituted with cyano, nitro, fluoro or methyl;
nitro, fluoro or methyl;
or
Figure imgf000006_0001
alkyl, cycloalkyl, aryl, a carbon-linked heterocyclyl, a carbon-linked heteroaryl or -NR6R7 where R6 and R7 are each independently selected from hydrogen, methyl, ethyl, phenyl, benzyl or R6 and R7 are linked so that, together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring optionally comprising one, two or three additional heteroatoms selected from N, O or S;
and wherein Q2 is optionally substituted by one or more substituents selected from halo, trifluoromethyl, trifluoromethoxy, cyano, isocyano, nitro, hydroxy, mercapto, amino, formyl, carboxy, carbamoyl, ureido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylamino, di-alkyl-amino, alkoxycarbonyl, N- alkylcarbamoyl, N,N-di-alkyl-carbamoyl, alkynoyl, alkanoyloxy, alkanoylamino, sulphamoyl, N-alkylsulphamoyl, N,N-di-alkyl-sulphamoyl or a phenyl ring which is optionally further substituted by halo, methoxy, ethoxy, methyl, ethyl, cyano or hydroxy;
Ri is a protected or unprotected carboxyl group; groups R2, R3, R4 and R5 are each independently hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl, cykloalkyl, alkylene, aralkyl, hydroxy, alkoxy, nitro, amino, trifluoromethyl, thiophene or aryl, wherein the aryl ring is optionally substitutedby one or more substituents selected from halo, trifluoromethyl, trifluoromethoxy, cyano, isocyano, nitro, hydroxy, mercapto, amino, formyl, carboxy, carbamoyl, ureido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylamino, di-alkyl- amino, alkoxycarbonyl, N-alkylcarbamoyl, NN-di-alkyl-carbamoyl, alkynoyl, alkanoyloxy, alkanoylamino, sulphamoyl, N-alkylsulphamoyl, N,N-di-alkyl-sulphamoyl or a phenyl ring which is optionally further substituted by halo, methoxy, ethoxy, methyl, ethyl, cyano or hydroxyl, and their pharmaceutically acceptable salts for use in the
Figure imgf000007_0001
( I ) treatment, control and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system (menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases.
A further embodiment of the present invention relates to a pharmaceutical composition containing one of the compounds of the general formula I for use in the treatment and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system(menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases.
A further embodiment of the present invention relates to a pharmaceutical composition comprising an effective amount of a compound of the general formula I, the formulation of which is a solid oral form or injection form for systemic administration or it is in the form of drops, pastes, gels or ointments for topical administration for its use in the treatment, control and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases (uveitis, sepsis, periodontitis, asthma and colorectal cancer), abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system (menstrual disorders and fertility problems), timing of parturition, mood disorders, psychiatric and neurological diseases.
A further embodiment of the present invention relates to pharmaceutical compositions comprising an effective amount of a compound of the general formula I, their use as therapeutic methods by means of administering compound I to mammals.
The compounds of the general formula I are useful as means with the ability to inhibit aldo-keto reductases AKR1B1 and AKR1B10.
The compounds of the general formula I may be prepared by synthetic processes known to the person skilled in the art and described in standard works 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; El 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 preparation of compounds of the general formula I comprises steps as indicated below:
Figure imgf000008_0001
lH-indole-2,3-dione (1, substituted isatin) plus thiosemikarbazide gives isatin thiosemi- karbazone (2), which in the alkaline solution undergoes cyclization to yield substituted 5H- [l,2,4]triazino[5,6]indolo-3-tiol (3a) (tautomeric form 2,5-dihydro-3H-[l,2,4]triazino [5,6]indolo-3-thione, 3b). Alternatively this product is alkylated with an appropriate halo- derivative to provide the sulfur substituent desired in the final product. The 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 those skilled in the art.
For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained- release preparations and devices.
The compounds or compositions can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Best Mode for Carrying out the Invention
The invention is illustrated by reference to the following examples comprising measurements of aldo-keto reductases inhibition by the compounds la - Ie. However, the examples are not intended to limit the scope of the patent claims. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the purpose and interest of this invention.
Figure imgf000010_0001
la
Figure imgf000010_0002
lb
Figure imgf000011_0001
 Example 1
Aldose reductase (ALR2) inhibition at the enzyme level in vitro
Preparation of ALR2. ALR2 from rat lens was partially purified using a procedure adapted from Hayman and Kinoshita [Hayman S, Kinoshita J H. Isolation and properties of lens aldose reductase. J Biol Chem. 240: 877-882 (1965)] as follows: lenses were quickly removed from rats following euthanasia and homogenized in a glass homogenizer with a teflon pestle 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 at 40 %, 50 % and then at 75 % salt saturation. The supernatant was retained after the first two precipitations. The pellet from the last step, possessing ALR2 activity, was dispersed in 75 % ammonium sulfate and stored in smaller aliquots in liquid nitrogen container.
Enzyme assay. ALR2 activities were assayed spectrophotometrically [Stefek et al. Bioorg Med Chem. 16:4908-4920 (2008)] by determining NADPH consumption at 340 nm and were expressed as the decrease of the optical density (O.D.)/s/mg protein. The reaction mixture contained 4.67 mM D,L-glyceraldehyde as a substrate, 0.11 mM NADPH, 0.067 M phosphate buffer, pH 6.2 and 0.05 ml of the enzyme preparation in a total volume of 1.5 ml. The reference blank contained all the above reagents except the substrate D,L-glyceraldehyde to correct for oxidation of NADPH not associated with reduction of the substrate. The enzyme reaction was initiated by addition of D,L-glyceraldehyde and was monitored for 4 min after an initial period of 1 min 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 sample of 0.020 ± 0.005 absorbance units/min. The effect of inhibitors on the enzyme activity was determined by including in the reaction mixture each inhibitor at required concentrations. The inhibitor at the same concentration was included in the reference blank. IC50 values (the concentration of the inhibitor required to produce 50 % inhibition of the enzyme reaction) were determined from the least-square analysis of the linear portion of the semilogarithmic inhibition curves. Each curve was generated using at least four concentrations of inhibitor causing an inhibition in the range from at least 25 to 75 %.
Table 1.
Inhibitory effect of compounds Ia-Ie on rat lens ALR2
Figure imgf000012_0001
Result is a mean values ± SD from at least three measurements.
Example 2 Aldehyde reductase (ALRl inhibition at the enzyme level in vitro
Preparation of ALRL ALRl from rat kidney was partially purified according to the reported procedure of Constantino et al. Chem. 42:1881-1893 (1999), as follows : kidneys were quickly removed from rats following euthanasia and homogenized in a knife homogenizer followed by processing in a glass homogenizer with a teflon pestle 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 ammonium sulfate fractional precipitation at 40 %, 50 % and 75 % salt saturation. The pellet obtained from the last step, possessing ALRl 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 total protein concentration of approximately 20 mg/ml. DEAE DE 52 resin was added to the solution (33 mg/ml) and after gentle mixing for 15 min removed by centrifugation. The supernatant containing ALRl was then stored in smaller aliquots in liquid nitrogen. No appreciable contamination by ALR2 in ALRl preparations was detected since no activity in terms of NADPH consumption was observed in the presence of glucose substrate up to 150 mM.
Enzyme assay. ALRl activities were assayed spectrophotometrically [Stefek et al. Bioorg Med Chem. 16:4908-4920 (2008)] by determining NADPH consumption at 340 nm and were expressed as decrease of the 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 the enzyme preparation in a total volume of 1.5 ml. The reference blank contained all the above reagents except the substrate D-glucuronate to correct for oxidation of NADPH not associated with reduction of the substrate. The enzyme reaction was initiated by addition of D-glucuronate and was monitored for 4 min after an initial period of 1 min 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 sample of 0.020 ± 0.005 absorbance units/min. The effect of inhibitors on the enzyme activity was determined by including in the reaction mixture each inhibitor at required concentrations. The inhibitor at the same concentration was included in the reference blank. IC50 values (the concentration of the inhibitor required to produce 50 % inhibition of the enzyme reaction) were determined from the least-square analysis of the linear portion of the semilogarthmic inhibition curves. Each curve was generated using at least four concentrations of inhibitor causing an inhibition in the range from at least 25 to 75 %.
Table 2.
Inhibitory effect of the compounds la - Ie on rat kidney ALRl
ICso / I (%, c = 10 μΜ)
Compound la 40.6 ± 2.1 / 22.71 ± 4.41 lb > 10 / 1.18 ± 4.35
Ic > 10 / 23.48 ± 2.11
Id 19.56 ± 5.38/ 27.06 ± 7.38 Ie 14.13 ± 0.36 / 35.67 ± 0.89
Result is a mean value ± SD from at least three measurements.
Example 3
Aldo-keto reductase AKR1B1 and AKRIBIO inhibition at the enzyme level in vitro
Preparation of recombinant AKRIBlO-enriched cell fractions. COS-7 cells, obtained from ATCC, were cultured in DMEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. For transfection cells were grown to 70%-80% confluence. Just prior to transfection, cell culture medium was replaced by DMEM supplemented with 10% FBS. Cells were transiently transfected with the plasmid pCEFL-KZ-AU5 containing AKRIBIO ORF, using Lipofectamine 2000 reagent, in a proportion of 1 μg DNA per 3 μΐ of Lipofectamine. After 5 h incubation, the transfection medium was replaced by complete medium. Cells were incubated for 48 h. Following, cells were scraped and collected in cold phosphate buffered saline and lysed in 50 mM sodium phosphate buffer, pH 6.8 in the presence of 0.1 mM EDTA, 0.1 mM EGTA, 0.1 mM β-mercaptoethanol, 320 μg/μl Pefablock, 0.1 mM sodium orthovanadate, and protease inhibitors (leupeptin, aprotinin and trypsin inhibitor at 2 μg/μl each one), by forced passes through a 26½G syringe. Cell lysate was ultracentrifugated for 30 min at 200,000 g at 4°C in a Beckman TLA 100.2 rotor. The soluble fraction S100 (supernatant) was collected for further analysis and pellet was discarded. Protein concentration was determined using BCA Protein Assay Kit (Thermo Fisher Scientific, IL, USA) according to the manufacturer's protocol.
AKR1B1 and AKRIBIO enzyme assays. Assay mixtures contained sodium phosphate buffer (0.1 M; pH 6.8), defined weight ^g) of proteins, 0.2 mM NADPH cofactor, 10 mM D,L-glyceraldehyde. The compounds 1 and 2 were added as DMSO solutions at 1% DMSO final mixture concentration. The activity assay started when the indicated AKR protein was added to the cuvette. After 4 min of incubation at room temperature, enzymatic activity was monitored by measuring NADPH consumption at 340 nm (ε340 nm, NADPH = 6200 M 'cm"1) in an Ultrospec 4300 pro spectrophotometer (GE Biosciences). Blanking for absorbance of the inhibitors at 340 nm was performed. The enzymatic activity was expressed in nmol/min/mg of protein. S100 fractions from non-transfected COS-7 cells were devoid of AKR activity.
Table 3.
Inhibitory effect of compound la on human AKR1B1 and AKRIBIO
Figure imgf000014_0001
Results are mean values ± SD from at least three measurements.
Example 4
Aldose reductase (ALR2) inhibition at the organ level (isolated eye lens) in vitro Eye lenses sorbitol assay. The animals in light ether anesthesia were killed by exsanguinations of the carotid artery and the eye globes were excised. The lenses were quickly dissected and rinsed with saline. The compound 1 dissolved in DMSO was added into the tubes containing freshly dissected eye lenses (1 lens per tube) in M-199 medium at pH 7.4, bubbled at 37 °C with pneumoxid (5% C02, 95% 02), to the final concentrations as reported, 30 min before adding glucose. The final concentration of DMSO in all incubations was 1%. The incubation was initiated by adding glucose to the final concentration of 50 mM and then continued at 37 °C with occasional (in about 30-min intervals) bubbling the mixture for approximately 30-s periods with pneumoxid. The incubations were terminated after a 3-h period by cooling the mixtures in an ice bath, followed by washing the lenses three times with ice-cold phosphate buffered saline (1 ml). The short term cultivations were preferred to avoid substantial permeability changes of the eye lenses. The washed lenses were kept deep-frozen for sorbitol determination which was performed as described before [Juskova et al. General Physiology and Biophysics, 28, 325-330 (2009). After the appropriate blanks were subtracted from each sample, the amount of sorbitol in nmol per gram of lens wet weight in each sample was determined by comparison with a linear regression of sorbitol standards.
Table 4.
Effect of compound la on sorbitol accumulation in isolated rat lenses cultivated with high glucose3.
Figure imgf000015_0001
Results are mean values ± SEM from n independent incubations.
aGlucose, 50 mM; time of incubation, 3 hours; 37 °C.
b p<0.001 vs. (+)Glucose (Student's t-test).
c p<0.001 vs. (+)Glucose (Student's t-test).

Claims

Claims
1. 5-Carboxymethyl-3-mercapto-l,2,4-triazino-[5,6-b]indoles of general formula I,
Figure imgf000016_0001
I
wherein
Ro is hydrogen atom, an alkyl, alkenyl, alkynyl, cykloalkyl, aryl or aralkyl radical, thienyl, or a radical of a general formula -AlkOH, -AlkNXY,
where Alk is a straight or branched chain alkylene radical containing 2 to 10 carbon atoms and X and Y are H atoms or alkyl radicals or together with the adjacent nitrogen atom form a heterocyclic ring which may contain additional hetero atom;
or a group of a general formula -(CH2)n-Q1, (n = 1-8),
wherein Q1 is selected from
cycloalkyl which is optionally substituted with cyano, nitro, fluoro or alkyl;
or phenyl which is substituted in the ortho or para position (relative to the point of attachment to the -CH2- group) by cyano, nitro, methyl, -C02H and tetrazole and optionally further substituted with cyano, nitro, fluoro, or methyl;
or naphthyl which is optionally substituted with cyano, nitro, fluoro or methyl; or thiophene which is optionally substituted with cyano, nitro, fluoro or methyl;
or
Figure imgf000016_0002
alkyl, cycloalkyl, aryl, a carbon-linked heterocyclyl, a carbon-linked heteroaryl or -NR6R7 where R6 and R7 are each independently selected from hydrogen, methyl, ethyl, phenyl, benzyl or R6 and R7 are linked so that, together with the nitrogen atom to which they are attached, they form a 4-7 membered heterocyclic ring optionally comprising one, two or three additional heteroatoms selected from N, O or S;
and wherein Q2 is optionally substituted by one or more substituents selected from halo, trifluoromethyl, trifluoromethoxy, cyano, isocyano, nitro, hydroxy, mercapto, amino, formyl, carboxy, carbamoyl, ureido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,
alkylsulphinyl, alkylsulphonyl, alkylamino, di-alkyl-amino, alkoxycarbonyl, N- alkylcarbamoyl, N,N-di-alkyl-carbamoyl, alkynoyl, alkanoyloxy, alkanoylamino, sulphamoyl, N-alkylsulphamoyl, N,jV-di-alkyl-sulphamoyl or a phenyl ring which is optionally further substituted by halo, methoxy, ethoxy, methyl, ethyl, cyano or hydroxy;
Ri is a protected or unprotected carboxyl group; groups R2, R3, R4 and R5 are each independently hydrogen atom, a halogen atom, a substituted or unsubstituted alkyl, cykloalkyl, alkylene, aralkyl, hydroxy, alkoxy, nitro, amino, trifluoromethyl, thiophene or aryl, wherein the aryl ring is optionally substituted by one or more substituents selected from halo, trifluoromethyl, trifluoromethoxy, cyano, isocyano, nitro, hydroxy, mercapto, amino, formyl, carboxy, carbamoyl, ureido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulphinyl, alkylsulphonyl, alkylamino, di-alkyl- amino, alkoxycarbonyl, N-alkylcarbamoyl, NN-di-alkyl-carbamoyl, alkynoyl,
alkanoyloxy, alkanoylamino, sulphamoyl, N-alkylsulphamoyl, jV,jV-di-alkyl-sulphamoyl or a phenyl ring which is optionally further substituted by halo, methoxy, ethoxy, methyl, ethyl, cyano or hydroxy;
and its pharmaceutically acceptable salts, hydrates and solvates for use as a medicament in treatment of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases such as uveitis, sepsis, periodontitis, asthma and colorectal cancer, abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system such as menstrual disorders and fertility problems, timing of parturition, mood disorders, psychiatric and neurological diseases.
2. A pharmaceutical composition comprising an effective amount of compound of general formula I and a pharmaceutically acceptable carrier for its use in the treatment, control and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases such as uveitis, sepsis, periodontitis, asthma and colorectal cancer, abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system such as menstrual disorders and fertility problems, timing of parturition, mood disorders, psychiatric and neurological diseases.
3. A pharmaceutical composition comprising an effective amount of a compound of general formula I, the formulation of which is a solid oral form or injection form for systemic administration or it is in the form of drops, pastes, gels or ointments for topical administration for its use in the treatment, control and prevention of human and veterinary diseases in which activities of aldo-keto reductases AKRIBI and AKRIBIO are key etiological factors for their development and progress such as the development of diabetic complications (macro-, microangiopathy, atherosclerosis, retinopathy, cataracts, nephropathy, neuropathy, bone mass loss and atherosclerosis), inflammatory diseases such as uveitis, sepsis, periodontitis, asthma and colorectal cancer, abnormal proliferation of vascular smooth muscle cells in atherosclerosis and restenosis, lung carcinoma in smokers, and several types of cancer (lung, breast, hepatic, prostate, pancreatic,endometrial cancer, cervical cancer, and cervical adenocarcinoma), diseases of the female reproductive system such as menstrual disorders and fertility problems, timing of parturition, mood disorders, psychiatric and neurological diseases.
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WO2017079402A1 (en) * 2015-11-04 2017-05-11 Beijing Normal University Hong Kong Baptist University United International College Substrates of akr1b1/akr1b10 as anti-cancer drugs
US11524017B2 (en) 2015-11-04 2022-12-13 Beijing Normal University Hong Kong Baptist University United International College Method of using substrates of AKR1B1/AKR1B10 as anti-cancer drugs
RU2623052C1 (en) * 2016-04-06 2017-06-21 Надежда Сергеевна Кузнецова Method for prevention of complications in patients after surgical treatment of inflammatory periodontal diseases
WO2022132058A1 (en) * 2020-12-14 2022-06-23 Centrum Experimentálnej Medicíny Sav Cemtirestat disulfide, prodrug of aldo-keto reductase inhibitor, preparation, pharmaceutical composition and use thereof

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