WO2015142218A1 - Pyrimidines bicycliques et leur utilisation comme antioxydants et cyto-protecteurs - Google Patents

Pyrimidines bicycliques et leur utilisation comme antioxydants et cyto-protecteurs Download PDF

Info

Publication number
WO2015142218A1
WO2015142218A1 PCT/RU2015/000122 RU2015000122W WO2015142218A1 WO 2015142218 A1 WO2015142218 A1 WO 2015142218A1 RU 2015000122 W RU2015000122 W RU 2015000122W WO 2015142218 A1 WO2015142218 A1 WO 2015142218A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
compounds
nrf2
stroke
formula
Prior art date
Application number
PCT/RU2015/000122
Other languages
English (en)
Russian (ru)
Inventor
Александр Александрович ШУЛЬЖЕНКО
Original Assignee
Общество с ограниченной ответственностью "Алион"
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Общество с ограниченной ответственностью "Алион" filed Critical Общество с ограниченной ответственностью "Алион"
Publication of WO2015142218A1 publication Critical patent/WO2015142218A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the invention relates to medicine, specifically to neurology, cardiology, as well as surgery and traumatology, namely, drugs with cytoprotective activity in conditions of ischemia-reperfusion, chronic oxidative stress and chemical intoxication.
  • Stroke is a clinical syndrome characterized by sudden onset of clinical symptoms, indicating focal and / or cerebral neurological deficiency, developing as a result of cerebral ischemia or hemorrhage. Every year in highly developed countries among every 10,000 people, 25-30 cases of stroke occur. The economic loss from a stroke in the United States is about $ 30 billion a year. Over 400 thousand strokes occur in Russia, which is equal in number to the population of the average regional city. Among the survivors, the majority have various functional disorders: by the end of the acute period, almost 80% of patients have motor disorders (most often these are paresis of varying severity), more than a third of patients have speech disorders.
  • ischemic cerebral infarction, softening of the brain
  • hemorrhagic cerebral hemorrhage, intracerebral hematoma
  • ischemic strokes account for 80% of all strokes, intracerebral hemorrhages - 17%, subarachnoid hemorrhages - 3%.
  • ischemic penumbra penumbra
  • the formation of 50% of the final volume of a heart attack occurs within the first 90 minutes from the moment of the development of a stroke, 70-80% - within 360 minutes, in connection with which the first 3-6 hours of the disease are called the “therapeutic window”, inside which therapeutic measures can to be most effective by saving the penumbra zone.
  • oxidative damage (changes in glutamate and calcium metabolism, free radical reactions, lipid peroxidation, excessive formation of nitric oxide, etc.) continues further, leading to "long-term consequences of ischemia.” Delayed damage to cerebral tissue is realized through the mechanisms of programmed cell death - apoptosis and aponecrosis. “Long-term consequences of ischemia” cause “deformation” of cerebral infarction due to the spread of damage from the peri-infarction zone to the periphery of the penumbra. The time of "deformation” of heart attacks in each case is individual, ranging from 3 to 7 days after cerebrovascular accident.
  • ischemic stroke The basis of specific therapy for ischemic stroke are two strategic directions: reperfusion and neuroprotection, aimed at protecting weakly or almost non-functioning, but still viable neurons located around the hearth site (ischemic penumbra).
  • the main cerebral thrombolytics are recognized as urokinase, streptokinase and their derivatives, as well as tissue plasminogen activator (tPA). The latter is used only in 4% of cases, due to the possible threat of cerebral hemorrhage.
  • Neuroprotective properties have: postsynaptic glutamate receptor antagonists (not yet available in clinical practice); presynaptic glutamate inhibitors (lubeluzole); antioxidants (alpha-tocopherol, carnosine, mexidol, mildronate, vitamin E, ascorbic acid); calcium channel blockers (nimodipine); drugs of predominantly neurotrophic action (piracetam, cerebrolysin, glycine, picamilon) and others.
  • Nrf2 nuclear factor (erythroid-derived 2) -like 2) is the main regulator of the antioxidant response at the cellular level. Nrf2 binds to the so-called an antioxidant response element (ARE) in the promoter region of the genes of the antioxidant response program responsible for the detoxification of xenobiotics and the neutralization of reactive oxygen species (ROS).
  • ARE antioxidant response element
  • ROS reactive oxygen species
  • Nrf2 s the most active thiols of the Keapl dimer
  • the complex decomposes, Nrf2 stabilizes and is sent to the nucleus, where it interacts with ARE-containing promoters and thereby includes protein synthesis with antioxidant protection.
  • the beneficial effects of Nrf2 activation have been demonstrated in various pathologies such as diabetes [Zheng N., Whitman S. A., Wu W. et al. / Therapeutic potential of Nrf2 activators in streptozotocin-induced diabetic nephropathy // Diabetes.
  • Nrf2 activation protects brain ischemia-reperfusion [Wang B., Cao W., Biswal S., Dore S. .. / Carbon monoxide-activated Nrf2 pathway leads to protection against permanent focal cerebral ischemia. // Stroke.
  • Nrf2 activation has been recently shown [Shen X. D., Ke B., Ji N. et al. / Disruption of type-l I FN pathway ameliorates preservation damage in mouse orthotopic liver transplantation via HO-1 dependent mechanism // The American Journal of Transplantation. -2012, -v.12, -N.7, - p.1730-1739; Mohammadzadeh M., Halabian R., Gharehbaghian A. et al. / Nrf-2 overexpression in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity // Cell Stress and Chaperones.
  • Nrf2 Liver from old donors has a lower level of Nrf2, which worsens the clinical performance of a liver transplant [Zaman M.V., Leonard M.O., Ryan EJ, Nolan NP, Hoti E., Maguire D., et al. / Lower expression of Nrf2 mRNA in older donor livers: a possible contributor to increased ischemia-reperfusion injury? // Transplantation. -2007, -84, -p.1272-1278].
  • Nrf2 The activation of Nrf2, as it has been found out relatively recently, antagonizes the inflammatory cascades initiated by TGF i [Oh C. J., Kim J. Y., Min A. K. et al. / Sulforaphane attenuates hepatic fibrosis via NF-E2-related factor 2-mediated inhibition of transforming growth factor-p / Smad signaling. // Free Radical Biology and Medicine, -2012, -v.52, - N.3, -p.671-682) and NF- ⁇ (Jiang J., Mo Z. C, Yin K. et al.
  • Nrf2 activators both compounds have a pronounced ability to alkylate (covalent modification of active thiol groups in proteins).
  • dimethyl fumarate developed by BioGen for the treatment of multiple sclerosis, BG-12
  • bardoxolone developed by Reata Pharmaceuticals and sold by Abbott Laboratories
  • patent application EP1813269 (A1) - 2007-08-01 describing pharmaceutical agents capable of activating the Nrf2 transcription factor for use in the treatment, prevention, or alleviation of a disease or condition that can be treated, prevented, or improve upon activation of Nrf2 transcription factors (e.g., atherosclerosis, hypertension, diabetes, nervous degenerative diseases, skin diseases, eye diseases, asthma and cancer). Agents increase the potential for protection against oxidative stress.
  • the objective of the present invention is to develop new agents that activate the antioxidant program and that can be used as effective cytoprotectors in a variety of adverse effects on body cells.
  • Ri is a substituent: methoxy, ethoxy, linear or branched alkyl (C G C), halogen, mono-, di-, tri- (C 4 ) alkylamino;
  • R 2 represents a halogen
  • Halogen can be selected from chlorine, bromine, iodine, fluorine. Most preferably represents chlorine.
  • EFFECT absence of suppression of activation of the antioxidant program induced by compounds of formula (1) with the addition of thiol reducing agents such as glutathione and cysteine, and the possibility of combining the drug with stoichiometric antioxidants without compromising the main effect.
  • the compounds of general formula I are stabilizers of the transcription factor Nrf2 and thus activators of the antioxidant program, the trigger of which is Nrf2.
  • the compounds of formula I induce the synthesis of proteins and phase II antioxidant defense enzymes such as hemoxygenase-1 and NADPH: XHHOH oxidoreductase and the rest, leading to a strong increase in the content of reduced glutathione in the tissues.
  • Compounds I have the ability to increase tissue viability in conditions of ischemia-reperfusion (transplantation of organs, stem cells, stroke, heart attack), chronic degeneration associated with oxidative stress (neurodegenerative diseases, renal failure); chemical intoxication of various nature, including the protection of normal cells during chemotherapy for cancer and thereby reducing the side effects of chemotherapy (for example, doxirubicin on the heart).
  • Compounds I can be used proactively to increase the body's defenses before working with toxic chemicals and high doses of radiation.
  • the compounds of formula I are commercially available (for example, from Alfa Aesar, Oakwood, http://www.alfa.com/en/catalog/H50537) or can be obtained by the following methods.
  • 4-chloro-3- (4-methoxyphenyl) -1 H-pyrazolo [3,4-c1] pyrimidine or 4-chloro-3-phenyl-1 H-pyrazolo [3,4-c1 can be mentioned ] pyrimidine.
  • 4-chloro-5- (4-methoxyphenyl) -7H-pyrrolo [2,3-!] Pyrimidine or 4-chloro-5-phenyl-7H-pyrrolo [2,3-c1] pyrimidine can be mentioned .
  • Another object is a cytoprotective and antioxidant pharmaceutical composition containing, as an active principle, an activator of the transcription factor Nrf2, which is a derivative of bicyclic pyrimidines of the general formula (I), or a pharmaceutically acceptable salt thereof in an effective amount and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable salt is intended to indicate salts that are not harmful to the patient.
  • Such salts include salts of pharmaceutically acceptable acids, for example, salts of inorganic and organic acids, and amino acids. Examples of inorganic acids include acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, nitric.
  • a representative of suitable organic acids include acetic, trichloroacetic, benzoic, cinnamon, citric, fumaric, glycolic, lactic, maleic, malic, salicylic succinic, and glutathione, cysteine or its derivatives, and the like can be used.
  • an excipient or substances are selected depending on the appropriate form of administration (tablets, injections, solutions, emulsions, suppositories, capsules): water, lactose, sugar alcohols, cellulose derivatives, phosphates, stearates, talc, surfactant, PEG, PVP, alcohols, etc.
  • Fig. 1 Activation of the ARE-luciferase reporter by Compounds of formula (1).
  • Fig. 2 Increased level of messenger RNA genes when treated with 0.5 ⁇ M compounds of the formula 1: chloro-5- (4-methoxyphenyl) thieno [2,3-c1] pyrimidine (X1) and 4-chloro-5- (4-phenyl) thieno [2,3- C.] pyrimidine (X2).
  • Fig. 3 Antioxidant and hepatoprotective activity of Compound X2 in the model of acute toxic liver damage, a) a significant concentration-dependent decrease in the area of liver necrosis during administration of the compound of formula 1; b) a concentration-dependent decrease in lipid peroxidation in the presence of a compound of formula 1; C) - concentration-dependent restoration of the level of superoxide dismutase to normal during administration of the compounds of formula 1.
  • Fig. 4 The increase in cell viability when treated with a compound of formula (1) against the toxic effects of the main lipid peroxidation product, HNE: a) control pretreatment, 20 ⁇ M HNE; b) pretreatment control, 30 ⁇ m HNE;
  • Fig.5. The effect of compound X (X1) on apoptosis of cardiomyocytes in rats treated with doxorubicin.
  • the apoptotic index is expressed as the ratio of TUNEL-positive cardiomyocyte nuclei to their total number. Data are presented as mean (n> 10), * p ⁇ 0.01 of the control group, ** p ⁇ 0.01 DOX of the group.
  • Fig. 6 Normalization of the level of apoptotic (AP-1) and inflammatory (NF-KB) transcription factors during the treatment of X1 (Compound X) in case of kidney damage with cisplatin. Immunoblots (above) and their quantitative processing on a densitometer (below).
  • FIG. 7 A reliable concentration-dependent decrease in the area of myocardial infarction during ischemic stroke during administration of the Compound of formula (1) - Compound X1.
  • Fig. 8 Comparison of the neuroprotective activity of compounds X1 and X2 in a model of ischemic stroke at a dose of administration of 1 mg / kg: a decrease in the activation of NF-KB transcription factor, a trigger of the inflammatory response of the body.
  • ARE-luciferase reporter was reproduced according to the protocol [Wang XJ, Hayes JD, Wolf CRI Generation of a Stable Antioxidant Response Element-Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents // Cancer Res - 2006; - 66: p. 10983-10994].
  • the pGL3 vector of the company Promega (Great Britain) containing the SV40 promoter in front of the firefly luciferase gene was used. Before the luciferase promoter, an insert was made at the Nhel and Xhol restriction sites of eight mouse ARE sequences:
  • a linker with the sequence 5-CCC-3 and 5-GGG-3 on the opposite chain was placed between the individual cis-elements.
  • the MCF7 cell line (human breast carcinoma) was transformed with the obtained construct in the presence of an orderly lower concentration of plasmid pcDNA3.1 containing a neomycin selection marker (antibiotic geneticin) using lipofectamine.
  • Transformed cells were selected on DMEM-glutamax medium with 10% FBS and antibiotics (including 0.8 mg / ml geneticin). Cells were cultured at 37 ° C in an incubator (95% air and 5% C0 2 ), reseeding every 3-4 days.
  • Compound X1 is the best activator (Fig. 1 A, 1 B), the main structural requirement is the presence of an aromatic substituent at position 5 of the bicycle, while the presence of a phenyl ring at position 7 of the bicycle (R3 in pyrrolopyrimidines) leads to a suppression of activation in the submicromolar range.
  • the combination of compounds of formula (I) with non-specific intracellular thiol reducing agents such as cysteine, acetylcysteine, or glutathione does not affect the activation parameters (Fig. 1 B), which is explained by the inability of the Compounds of formula (I) to alkylate these thiols in aqueous solutions of in vitro.
  • the compounds of formula 1 are activators of the ARE-luciferase reporter and therefore activators of the antioxidant genetic program on molecular level.
  • Example 2 we measured the activation effect of the best compounds on the content of messenger RNA of the two most significant genes regulated by Nrf2, namely hemoxygenase-1 and NADPH: XHHOH oxidoreductase-1.
  • NADPH XHHOH oxidoreductase 1 (NQ01, 5'-AGCGTTCGGTATTACGATCC-3 'and 5'-AGTAC AATCAGG G CTCTTCTCG-3') using Fast SYBR_ Green Master Mix (Invitrogen, USA).
  • PCR cycle parameters 10 sec at 95 ° C, then 1 min at 60 ° C. The values obtained were normalized to the level of beta-actin expression (primers for amplification of beta-actin: 5'-CTAAGGCCAACCGTGAAAAG-3 'and 5'-ACCAGAGGCATACAGGGACA-3').
  • the compounds of formula 1 activate the synthesis of hemoxygenase-1 and NADPH: XHHOH oxidoreductase-1, the main targets of Nrf2.
  • the degree of activation corresponds to the activity of the compounds in the reporter analysis (Fig. 1), namely, compound X1 shows better reporter activation than compound X2 (Example 1). In further experiments, these two compounds were used.
  • Antioxidant activity was tested in an in vivo experiment on a model of acute toxic liver damage with carbon tetrachloride (SSC).
  • SSC carbon tetrachloride
  • the experiment was conducted on 50 outbred male rats with an initial weight of 190-200 g contained in a standard diet of vivarium. There were 10 animals in each group.
  • Liver damage (experimental hepatitis) in animals was caused by the administration of SCC intragastrically as a 50% solution in liquid paraffin in a volume of 0.25 ml per 100 g of body weight for 3 days [Vengerovsky A. I., Chuchalin BC, Pauls O. V., Saratikov A.S. / Effect of hepatoprotectors on lipid metabolism in SSC - hepatitis. // Bull.
  • liver homogenates were homogenized in 50 mM sodium phosphate buffer containing 0.15 M sodium chloride, pH 7.4. The supernatant was separated by centrifugation at 15,000 g for 20 min, at 4 ° C. The level of lipid peroxidation in liver homogenates was determined according to the test with thiobarbituric acid (TAC) [Steel ID, Garishvili TG / Method for determination of malondialdehyde using thiobarbituric acid. // In: Modern methods in biochemistry. -M. -The medicine. -1977. -from. 66-69], conducting preliminary extraction of lipids according to Folch [Cates M. Technique of lipidology.- M.- Mir.- 1975.- p.
  • TAC thiobarbituric acid
  • Superoxide dismutase (SOD) activity was determined at 438 nm by reducing the absorption of the formazan product of the WST-1 oxidation by the superoxide radical generated by the xanthine / xanthine oxidase system in accordance with the protocol of BioLabs (USA). Protein in homogenates was determined by the Bradford method. The experimental results were processed statistically using Student's t-criterion [E. Gubler. Computational methods of analysis and recognition of pathological processes. -M. -The science. -1978. -from. 365]. Hepatoprotectonic activity was determined as follows. Liver samples were placed in plastic cassettes and filled with 4% paraformaldehyde for 48 hours.
  • liver damage was evaluated by measuring the area of necrosis section by section when stained with hematoxylin and eosin under a microscope. (Olympus BX51, Japan). Necrotic zones were determined visually and the percentage of necrosis was calculated using the Cell F v3.1 program, Olympus Soft Imaging Solutions, Munster, Germany.
  • Compound X2 has a hepatoprotective effect, which manifests itself in a significant decrease in the area of necrotic damage to the liver during acute carbon tetrachloride poisoning.
  • Fig. 36 The results presented in Fig. 36 indicate that administration of SSC to rats led to the accumulation of LPO products in the liver. In animals of the experimental groups, which were administered one of the studied compounds, a significant decrease in the content of primary lipid peroxidation products in the liver was noted.
  • Fig. Sv indicate that administration of SSC to rats led to a drop in the level of superoxide dismutase. In animals of the experimental groups, which were injected with one of the studied compounds, a significant increase in enzyme activity was noted up to the normal level.
  • the compound of general formula (1) has a pronounced antioxidant effect in the model of acute toxic liver damage.
  • ROS Active oxygen species
  • Neonatal ventricular myocytes from 1-2 day old outbred male rats were subjected to gradient centrifugation and differential seeding to enrich the culture with cardiomyocytes.
  • Cell viability was determined using a commercial kit (LIVE / DEAD Viability / Cytotoxicity Assay Kit, LifeTechnologies Corp., USA), the principle of which is based on the simultaneous determination of living and dead cells using calcein AM and ethidium homodimer-1, specific for intracellular esterase activity and membrane integrity, respectively. Fluorescence micrographs (living cells are stained green and dead cell nuclei are red) were obtained with a BZ-9000 fluorescence microscope (Keuepse, Japan).
  • cardiomyocytes were treated with 0.25 ⁇ M of compound X1 and a control that did not contain this substance, incubated for 14 hours and then treated with a cytotoxic concentration of HNE for 24 hours.
  • the viability of the control cardiomyocytes was 24.6% ⁇ 4.3% for 20 ⁇ M HNE and 15.4% ⁇ 1.8% for 30 ⁇ M HNE.
  • Pretreatment with compound X1 (0.25 ⁇ M) significantly increased cardiomyocyte viability to 72.0% ⁇ 5.5% (20 ⁇ M HNE) and 38.6% ⁇ 4.1% (30 ⁇ M HNE), respectively (Fig. 4a, b )
  • the Compound of general formula (1) has cardioprotective properties: pretreatment with it significantly increases the survival of cardiomyocytes during subsequent treatment with 4-oxinonenal, the main damage factor in ischemia-reperfusion.
  • DOX Doxorubicin
  • an anthracycline antibiotic widely used in chemotherapy of tumors and malignant blood diseases has significant limitations due to cumulative dose-dependent cardiotoxicity.
  • the mechanism of DOX cardiotoxicity is still unclear, but the induction of DOXOM apoptosis in cardiomyocytes has been reliably established.
  • no counterparties have been developed that could have a preventive effect and prevent high cardiotoxicity of DOXa during joint administration.
  • Outbred male rats 220-240 g were kept in cages with a controlled temperature (25 ° C) and a day-night cycle (12/12) and free access to water and food.
  • the males were randomly divided into 4 groups of 10 rats: control group, X group, DOX group, and DOX + X group.
  • the rats of groups X and DOX + X were given one of the test compounds (namely, X1) intragastrically at a dose of 1 mg / kg for 3 days, the animals of the first two groups received oral saline in an equivalent amount.
  • mice received every second day (7 times, 14 days) intraperitoneally: saline (control group), DOX 2.5 mg per kg in the DOX group; 1 mg / kg of compound X1 intragastrically one hour before DOX injection group DOX + X; 1 mg / kg of compound X1 intragastrically in the X group.
  • echocardiographic measurements (17.5-Hz RMV 707 nozzle) were performed in rats under anesthesia with sodium pentabarbital.
  • Left ventricular (LV) end diastolic and systolic diameters were determined in the M-modal mode.
  • Left ventricular end diastolic volume (LVEDV), stroke volume (SV), and LV global contractility (ejection fraction) (EF) were calculated according to the program. Data were averaged over three consecutive cardiac cycles.
  • LVEDV left ventricular end diastolic volume
  • SV stroke volume
  • the Compound of formula (1) increases the life expectancy of the graft from 10 to 13 or more days.
  • the compound of formula (1) has the ability to increase the viability of the transplants.
  • Kidney damage was caused by a single intraperitoneal injection of cisplatin (Sigma Chemical Co., USA) (7 mg / kg).
  • 30 outbred male rats were divided into 4 groups: 1 group control (physiological saline), group 2 1 mg / kg Compounds X1 daily for 12 days, starting 2 days before cisplatin injection, Group 3 one injection of cisplatin, Group 4 injection cisplatin and administration of Compound X1 according to the same scheme as group 2.
  • rats were decapitated. Blood samples were taken for analysis and kidneys were taken for histological studies.
  • Biochemical analysis blood samples were centrifuged at 3000 g for 10 min to obtain serum. Serum nitrogen (urea- ⁇ ) and creatinine were measured on a Flexor Junior biochemistry analyzer (Vital Scientific BV, Netherlands). The protein was extracted by homogenization of rat kidneys in 1 ml of ice-cold hypotonic buffer A containing 10 mM HEPES (pH 7.8), 10 mM KCI, 2 mM MgCI2, 1 mM dithiothreitol (DTT), 0.1 mM ethylene diaminetetraacetic acid (EDTA), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF).
  • NP-40 Nonidet P-40
  • the pellet was washed with 500 ⁇ l of buffer A, in which was added 40 ⁇ l of 10% NP-40, centrifuged, resuspended in 200 ⁇ l of buffer C (50 mm HEPES (pH 7.8), 50 mm KCI, 300 mm NaCI, 0.1 mm EDTA, 1 mm DTT, 0.1 mM PMSF, 20% glycerol), centrifuged for 5 min at 14800 d. The supernatant was collected for immunoblotting. Protein was determined by Lowry kit company Sigma (St. Louis, Missouri, USA).
  • Blots were washed and incubated with HRP (horseradish root peroxidase) -conjugated anti-mouse goat antibodies (Abeam, UK). Staining was performed with hydrogen peroxide and diaminobenzidine. Monoclonal antibodies against ⁇ -actin were used as a control of protein content (A5316; Sigma). The staining intensity was measured on a densitometer.
  • HRP horseradish root peroxidase
  • the compound of formula (1) has the ability to reduce the nephrotoxicity of cisplatin as follows from the data in table 4, namely it reduces the levels of nitrogen (3 times) and creatinine (2 times), which show a strong increase in kidney damage with cisplatin, and significantly reduce the level of transcription factors , activation of which is characteristic of the inflammatory process (NF- ⁇ ) and apoptosis (AR-1).
  • the compound was administered intraperitoneally immediately after a stroke.
  • the volume of a heart attack was measured 24 hours after a stroke.
  • Immunohistochemistry and biochemical analyzes were performed as described in the Example (see above).
  • CMA middle cerebral artery
  • CMA occlusion was performed by introducing a nylon-coated monofilament into the internal carotid artery up to the onset of CMA (Kuge Y., Minematsu K., Yamaguchi T., Miyake Y. Nylon monofilament for intraluminal middle cerebral artery occlusion in rats. // Stroke.- 1995. - 26.— P. 1655—1658), in which the blood flow was recorded during the entire experiment on a moorVMS-LDF monitor from Moor Instruments Ltd, (Great Britain), using an optical fiber attached to the skull in the region of the MCA (6 mm laterally and 2 mm behind Bregma) before and after clamping the right SMA.
  • Rats in which blood flow through the MCA decreased by less than 70% was excluded from the experiment. Rectal temperature was controlled at 37 +/- 0.5 ° ⁇ . In the control group without a heart attack, the same surgery was performed with the exception of the introduction of a filament.
  • the area of infarction after MCA occlusion was determined using 2,3,5-triphenyltetrazolium chloride (TFC) 24 hours after occlusion.
  • TFC 2,3,5-triphenyltetrazolium chloride
  • the animals were decapitated and the brains were quickly isolated and cut into 5 sections of 3 mm each, stained with a 2% TPC solution at 37 ° C for 20 minutes, and fixed with 4% paraformaldehyde. Normal tissue was stained dark red, and the infarct area was pale gray. Sections were photographed and the image was processed using Image-ProPlus 5.1. The damage area was calculated by multiplying the infarct area by the thickness of the section.
  • Volume of damage,% ⁇ [total volume of a heart attack - (difference between the volumes of the intact ipsilateral and contralateral hemisphere) / volume of the contralateral hemisphere ⁇ x100% (Tatlisumak T., Carano RA, Takano K. , Opgenorth TJ, Sotak S.N., Fisher M. / A novel endothelin antagonist, A-127722, attenuates ischemic lesion size in rats with temporary middle cerebral artery occlusion: a diffusion and perfusion MRI study. // Stroke. -1998, -N29, -p.850-857).
  • the compounds of formula (1) possess neuroprotective properties in ischemic stroke, reducing the volume of stroke (Fig. 7) and inflammation (level of activation of NF- ⁇ . FIG. 8), as well as reducing the level of lipid peroxidation products and increasing the level of superoxide dismutase (table 4 )
  • the animals were divided into several groups: falsely operated rats, which were anesthetized and who then underwent trepanation of the skull, but did not destroy the brain tissue; animals with hemorrhagic stroke; animals with hemorrhagic stroke, which were administered Compound X1 at a dose of 1 mg / kg orally (intragastrically, using a special probe) 30 minutes after operations, then once a day for 7 days. Control animals were injected with saline in an equivalent volume intragastrically.
  • Table 5 The effect of Compound X1 (1 mg / kg, by mouth) on neurological deficit (McGrow scale) in rats after hemorrhagic stroke. The number of animals with various neurological symptoms (%) on the 1st day after surgery is given.
  • the compound of formula (1) has neuroprotective properties in hemorrhagic stroke - it reduces mortality (Table 6) and neurological deficiency (Table 5).
  • lymphocytes from the spleen of 6-8 week old BALB / c male mice were carried out as follows.
  • the spleen was aseptically excised and placed in a sterile dish containing RPMI 1640 medium.
  • a single cell suspension was obtained by gently rubbing the spleen on a sterile nylon mesh in the same container.
  • Splenocytes were centrifuged and red blood cells were lysed with short-term hypotonic shock.
  • cells were pretreated with Compound X1 (0.05-0.5 ⁇ M) 2 hours before irradiation.
  • DMSO was used as an in vitro control.
  • Lymphocytes were suspended in the medium and irradiated with a 60 Co ⁇ -source at a dose of 2.93 Gray / min.
  • a Live / Dead Cell Assay kit (LifeTechnologies Corp., USA) was used to count living and dead cells.
  • Compound X1 has radioprotective properties, pre-treatment of cells significantly increases their viability, and this effect has a clear concentration dependence on the added Compound X1 (Fig. 9).
  • the compound of formula (1) protects against neurodegeneration in the toxic Parkinson model
  • the control group instead of Compound X received 3% DMSO in oil according to the same scheme.
  • MPTP at a dose of 10 mg / kg (30 mg / kg cumulative dose) was administered 3 times every 2 hours. Animals were decapitated on the 7th day after the administration of MPTP. Mice were divided into 4 groups (control, X, MPTP, X + MPTP).
  • Compound X1 did not cause any visible changes in the behavior of mice.
  • the level of dopamine and its metabolites - dopamine turns into 3.4- Dioxyphenylacetic acid (DOPUK) and further into homovanillinic acid (HVA) —measured in the striatum using HPLC and electrochemical detection.
  • DOPUK Dioxyphenylacetic acid
  • HVA homovanillinic acid
  • the Compound of formula (1) at 1 mg / kg / day protects against neurodegeneration caused by the administration of MPTP.
  • Compound X1 has neuroprotective properties in the Parkinson's toxic model, practically returning the level of dopamine and its main metabolites to normal (table 7).
  • the compounds of general formula (1) are activators of the transcription factor Nrf2 (Examples 1 and 2), which launches a genetic program of antioxidant response, which can significantly reduce toxic effects and damage in a number of diseases (Examples 3-11).
  • Nrf2 transcription factor 2
  • examples 3-11 a genetic program of antioxidant response, which can significantly reduce toxic effects and damage in a number of diseases.
  • Dosage forms of a number of compounds of the general formula (I) are prepared by a well-known method.
  • Nrf2 - a transcription factor encoded by the NFE2L2 gene (Nuclear factor (erythroid-derived 2) -like 2)
  • Keapl - a protein encoded by the Keapl gene (Kelch-like ECH-associated protein 1)
  • NF-KB - transcription factor NF- ⁇ (nuclear factor "Kappa-bi")

Abstract

L'invention se rapporte au domaine de la médecine et, plus précisément, de la neurologie, de la cardiologie ainsi que de la chirurgie et de la traumatologie, et concerne des agents médicamenteux ayant une activité cyto-protectrice dans des conditions de reperfusion ischémique, de stress oxydant chronique et d'intoxication chimique. L'invention concerne l'utilisation d'un composé consistant en un dérivé de pyrimidines bicycliques en qualité d'activateurs du facteur de transcription Nrf2. Les composés I peuvent être utilisés de manière préventive afin d'augmenter les forces de protection de l'organisme avant un travail avec des produits chimiques toxiques et des doses de radiations élevées.
PCT/RU2015/000122 2014-03-20 2015-02-27 Pyrimidines bicycliques et leur utilisation comme antioxydants et cyto-protecteurs WO2015142218A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2014110647 2014-03-20
RU2014110647/15A RU2545758C1 (ru) 2014-03-20 2014-03-20 Бициклические пиримидины или их фармацевтически приемлемые соли-активаторы антиоксидантной программы и их применение в качестве цитопротекторов

Publications (1)

Publication Number Publication Date
WO2015142218A1 true WO2015142218A1 (fr) 2015-09-24

Family

ID=53295575

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2015/000122 WO2015142218A1 (fr) 2014-03-20 2015-02-27 Pyrimidines bicycliques et leur utilisation comme antioxydants et cyto-protecteurs

Country Status (2)

Country Link
RU (1) RU2545758C1 (fr)
WO (1) WO2015142218A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11479539B2 (en) 2017-12-22 2022-10-25 Medimmune Limited Small molecule modulators of the BTB domain of Keap1

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR4321M (fr) * 1964-01-15 1966-08-01
WO2006124874A2 (fr) * 2005-05-12 2006-11-23 Kalypsys, Inc. Inhibiteurs de la b-raf kinase
WO2008128072A2 (fr) * 2007-04-13 2008-10-23 Supergen, Inc. Inhibiteurs de la kinase axl
WO2013072694A1 (fr) * 2011-11-15 2013-05-23 Xention Limited Thiéno- et furo- pyrimidines et pyridines, convenant comme inhibiteurs du canal potassium

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1388342A1 (fr) * 2002-08-07 2004-02-11 Aventis Pharma Deutschland GmbH Dérivés de cycloalkenylamine condensé avec un hététoaryle et acylé et leur utilisation comme produits pharmaceutiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR4321M (fr) * 1964-01-15 1966-08-01
WO2006124874A2 (fr) * 2005-05-12 2006-11-23 Kalypsys, Inc. Inhibiteurs de la b-raf kinase
WO2008128072A2 (fr) * 2007-04-13 2008-10-23 Supergen, Inc. Inhibiteurs de la kinase axl
WO2013072694A1 (fr) * 2011-11-15 2013-05-23 Xention Limited Thiéno- et furo- pyrimidines et pyridines, convenant comme inhibiteurs du canal potassium

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CALDERON, FELIX ET AL.: "An Invitation to Open Innovation in Malaria Drag Discovery: 47. Quality Starting Points from the TCAMS", MEDICINAL CHEMISTRY LETTERS, vol. 2, no. 10, 2011, pages 741 - 746, XP055039503, ISSN: 1948-5875 *
HOSSAIN M.I. ET AL.: "Synthesis and Antimicrobial Activities of Some New Thieno and Furopyrimidine Derivatives.", J. SCI. RES., vol. 1, no. 2, 2009, pages 317 - 325, XP055226922, ISSN: 2070-0237 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11479539B2 (en) 2017-12-22 2022-10-25 Medimmune Limited Small molecule modulators of the BTB domain of Keap1

Also Published As

Publication number Publication date
RU2545758C1 (ru) 2015-04-10

Similar Documents

Publication Publication Date Title
Ajoolabady et al. Targeting autophagy in ischemic stroke: From molecular mechanisms to clinical therapeutics
Ghavami et al. Airway mesenchymal cell death by mevalonate cascade inhibition: integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins
EP3244909B1 (fr) Inhibiteurs du protéasome pour traiter des troubles de vieillissement prématuré
Wu et al. Succinate-induced neuronal mitochondrial fission and hexokinase II malfunction in ischemic stroke: Therapeutical effects of kaempferol
Cook et al. Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential
Yang et al. SIRT1 activation by curcumin pretreatment attenuates mitochondrial oxidative damage induced by myocardial ischemia reperfusion injury
Blas‐García et al. Inhibition of mitochondrial function by efavirenz increases lipid content in hepatic cells
Gomez et al. Inhibition of GSK3β by postconditioning is required to prevent opening of the mitochondrial permeability transition pore during reperfusion
Yu et al. Paeoniflorin protects human EA. hy926 endothelial cells against gamma-radiation induced oxidative injury by activating the NF-E2-related factor 2/heme oxygenase-1 pathway
Mollereau et al. Adaptive preconditioning in neurological diseases–therapeutic insights from proteostatic perturbations
Lu et al. Isoalantolactone induces apoptosis through reactive oxygen species-dependent upregulation of death receptor 5 in human esophageal cancer cells
Jin et al. Bisdemethoxycurcumin attenuates cisplatin-induced renal injury through anti-apoptosis, anti-oxidant and anti-inflammatory
Huo et al. Betulinic acid derivative B10 inhibits glioma cell proliferation through suppression of SIRT1, acetylation of FOXO3a and upregulation of Bim/PUMA
Deng et al. Scutellarin acts on the AR-NOX axis to remediate oxidative stress injury in a mouse model of cerebral ischemia/reperfusion injury
Zhu et al. HDAC6 alleviates prion peptide-mediated neuronal death via modulating PI3K-Akt-mTOR pathway
Wang et al. A novel biscoumarin compound ameliorates cerebral ischemia reperfusion-induced mitochondrial oxidative injury via Nrf2/Keap1/ARE signaling
Sinha et al. Mahanine inhibits growth and induces apoptosis in prostate cancer cells through the deactivation of Akt and activation of caspases
Feng et al. Apigenin-7-O-β-d-(-6 ″-p-coumaroyl)-glucopyranoside pretreatment attenuates myocardial ischemia/reperfusion injury via activating AMPK signaling
Lee et al. Caspase-independent autophagic cytotoxicity in etoposide-treated CaSki cervical carcinoma cells
Chen et al. IL37 overexpression inhibits autophagy and apoptosis induced by hepatic ischemia reperfusion injury via modulating AMPK/mTOR/ULLK1 signalling pathways
Al-Saleh et al. Inhibition of NADPH oxidase alleviates germ cell apoptosis and ER stress during testicular ischemia reperfusion injury
Kim et al. Korean Red ginseng prevents endothelial senescence by downregulating the HO-1/NF-κB/miRNA-155-5p/eNOS pathway
Chen et al. Anti-tumor activity of N-thiolated β-lactam antibiotics
Singer et al. Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD
Hu et al. Carnosic acid protects against doxorubicin-induced cardiotoxicity through enhancing the Nrf2/HO-1 pathway

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15765482

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 27/01/2017)

122 Ep: pct application non-entry in european phase

Ref document number: 15765482

Country of ref document: EP

Kind code of ref document: A1