WO2018214872A1 - Substituted pyrimidinetrione compound, composition comprising compound, and use thereof - Google Patents

Abstract

The present invention provides a substituted pyrimidinetrione compound, a composition comprising the compound, and a use thereof. The present invention provides a pyrimidinetrione compound as shown in formula (I) or a crystal form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate, or solvate thereof. The pyrimidinetrione compound and composition comprising the compound described in the present invention can regulate hypoxia-inducible factors (HIF) and/or endogenous erythropoietin (EPO) and can be used in preparing a drug regulating human anemia.

Description

Substituted pyrimidinone compound and composition comprising the same and use thereof Technical field

The invention belongs to the technical field of medicine, in particular to a substituted pyrimidinone compound and a composition comprising the same, and capable of regulating the stability of a hypoxia inducible factor (HIF) subunit and increasing endogenous red blood cells in vitro and in vivo. Methods and compounds for the production of hormones.

Background technique

Hypoxia-inducible factor (HIF) is a basic helix-loop-helix (bHLH) PAS (Per/Arnt/Sim) transcriptional activator that regulates changes in gene expression as a function of cellular oxygen concentration. HIF is a heterodimer containing an oxygen-regulated alpha subunit (HIF-alpha) and a constitutively expressed beta subunit (HIF-beta), also known as the aryl hydrocarbon receptor nuclear transporter (ARNT). In oxygenated (normally) cells, the HIF-[alpha] subunit is rapidly degraded by a mechanism involving ubiquitination of the retinal hemangiostatin (pVHL) E3 ligase complex. Under hypoxic conditions, HIF-α does not degrade, and an active HIF-α/β complex accumulates in the nucleus and activates the expression of several genes, including glycolytic enzymes, glucose transporter (GLUT)-1, and Erythropoietin (EPO) and vascular endothelial growth factor (VEGF). (Maxwell et al., Nature, 1999, 399, 271-275).

Erythropoietin (EPO) is a naturally occurring hormone produced by HIF-α that stimulates the production of red blood cells that carry oxygen throughout the body. EPO is usually secreted by the kidney, and endogenous EPO is increased under conditions of reduced oxygen (anoxia). All types of anemia are characterized by a reduced ability of the blood to carry oxygen and are thus accompanied by similar signs and symptoms, including pale and weak skin, mucous membranes, dizziness, fatigue, and lethargy, leading to a decline in quality of life. Subjects with severe anemia showed difficulty breathing and cardiac malformations. Anemia is usually associated with a lack of blood in red blood cells or hemoglobin.

Ischemia and hypoxia are the leading causes of morbidity and mortality. Cardiovascular disease causes at least 15 million deaths each year and is responsible for 30% of deaths worldwide. Among various cardiovascular diseases, ischemic heart disease and cerebrovascular disease cause about 17% of deaths. A total of 1.3 million cases of non-fatal acute myocardial infarction are reported each year, constituting an incidence of approximately 300 out of every 100,000 people. On the other hand, it is estimated that five million Americans suffer from venous thrombosis each year, and about 600,000 of these cases lead to pulmonary embolism. About one-third of patients with pulmonary embolism eventually die, making pulmonary embolism the third most common cause of death in the United States.

Currently, the treatment of ischemic and hypoxic conditions focuses on the relief of symptoms and the treatment of pathogenic conditions. For example, treatment of myocardial infarction includes nitroglycerin and analgesics to control pain and reduce cardiac workload. Use other drugs, including digoxin, diuretics, amrinone, beta-blockers, lipid lowering agents, and angiotensin-converting enzyme inhibitors to stabilize the condition, but none of these therapies Directly acts on tissue damage caused by ischemia and hypoxia.

Daprodustat (chemical name N-[(1,3-dicyclohexyl-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine, which has the following structural formula) is developed by GlaxoSmithKline An oral HIF-α-prolyl hydroxylase inhibitor for the treatment of chronic kidney disease-related anemia, currently in Phase III clinical trials.

Poor absorption, distribution, metabolism, and/or excretion (ADME) properties are known to be the primary cause of clinical trial failure in many drug candidates. Many of the drugs currently on the market also limit their range of applications due to poor ADME properties. The rapid metabolism of drugs can lead to the inability of many drugs that could be effectively treated to treat diseases because they are too quickly removed from the body. Frequent or high-dose medications may solve the problem of rapid drug clearance, but this approach can lead to problems such as poor patient compliance, side effects caused by high-dose medications, and increased treatment costs. In addition, rapidly metabolizing drugs may also expose patients to undesirable toxic or reactive metabolites.

There is still a serious clinical unmet need in this area, and it has been found that having novel compounds that treat HIF-related and EPO-related diseases with good oral bioavailability and drug-forming properties is a challenging task.

Summary of the invention

In response to the above technical problems, the present invention discloses a compound and a composition comprising the same, which can be used to modulate hypoxia-inducible factor (HIF) and/or endogenous erythropoietin (EPO) and/or have better Pharmacodynamics / pharmacokinetic properties.

In this regard, the technical solution adopted by the present invention is:

It is an object of the present invention to provide a novel class of compounds which can be used to modulate hypoxia-inducible factor (HIF) and/or endogenous erythropoietin (EPO) and/or have better pharmacodynamic/pharmacokinetic properties. .

In a first aspect of the invention, there is provided a compound of formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound.

Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And at least one of R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ is deuterated or deuterated.

In another preferred embodiment, R ²⁴ and R ²⁵ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ and R ¹¹ are each independently hydrazine or hydrogen.

In another preferred embodiment, R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ and R ²² are each independently hydrazine or hydrogen.

In another preferred embodiment, the cerium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, and even more preferably greater than 75%. Preferably, the ground is greater than 95%, more preferably greater than 99%.

Specifically, in the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , The cesium isotope content of each of the R ⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ is at least 5%, preferably greater than 10 More preferably, more than 15%, more preferably more than 20%, more preferably more than 25%, more preferably more than 30%, more preferably more than 35%, more preferably more than 40%, more preferably more than 45% More preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, and even more preferably greater than 80%, More preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, and even more preferably greater than 99%.

In another preferred embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² of the compound of formula (I), R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ , at least one of which contains ruthenium, more preferably two R contains 氘, more preferably three R contains 氘, more preferably four R contains 氘, more preferably five R contains 氘, more preferably six R contains 氘, more preferably seven R contains 氘, More preferably, eight R contain ruthenium, more preferably nine R 氘, more preferably ten R 氘, more preferably eleven R 氘, more preferably twelve R 氘, more preferably Thirteen R 氘, more preferably fourteen R 氘, more preferably fifteen R 氘, more preferably sixteen R 氘, more preferably seventeen R 氘, more preferably Eighteen R containing strontium, more preferably nineteen R containing strontium, more preferably twenty R containing strontium, more preferably twenty-one R containing hydrazine, more preferably twenty-two R containing hydrazine, more preferably twenty-two R containing hydrazine, more Twenty-three R contains 氘, preferably twenty-four R contains 氘, and more preferably twenty-five R contains 氘.

In another preferred embodiment, the compound does not include a non-deuterated compound.

In a second aspect of the invention, there is provided a method of preparing a pharmaceutical composition comprising the steps of: pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, pharmaceutically acceptable The accepted salt, hydrate or solvate is mixed to form a pharmaceutical composition.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, a pharmaceutically acceptable salt, hydrated Or a solvate.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, any glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents A dispersing agent, a disintegrating agent, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.

The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, coagulation Glues, microspheres and aerosols.

Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Herein, "halogen" means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.

As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".

As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).

The invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. Examples of isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, respectively. , ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. a compound, or an enantiomer, a diastereomer, an isomer, or a pharmaceutically acceptable salt or solvate of the present invention, wherein an isotope or other isotopic atom containing the above compound is within the scope of the present invention . Certain isotopically-labeled compounds of the present invention, such as the radioisotopes of ³ H and ¹⁴ C, are also among them, useful in tissue distribution experiments of drugs and substrates.氚, ie ³ H and carbon-14, ie ¹⁴ C, are easier to prepare and detect and are preferred in isotopes. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium). Salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl A base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed of morpholine, piperazine, and lysine, respectively.

The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.

The present invention provides a method of modulating HIF and/or EPO which stabilizes HIF and activates expression of a HIF regulatory gene by inhibiting hydroxylation of HIFα. The methods are also applicable to the prevention, pretreatment or treatment of HIF and/or EPO related conditions, including anemia, ischemia and anoxic conditions.

Ischemia and hypoxia are two conditions associated with HIF and include, but are not limited to, myocardial infarction, hepatic ischemia, renal ischemia and stroke; peripheral vascular disorders, ulcers, burns and chronic wounds; pulmonary embolism; And ischemia-reperfusion injury, including, for example, ischemia-reperfusion injury associated with surgery and organ transplantation.

One aspect of the invention provides methods for treating a variety of ischemic and anoxic conditions, particularly using the compounds described herein. In one embodiment, the methods of the invention produce a therapeutic benefit when administered after ischemia or hypoxia. For example, after myocardial infarction, the methods of the present invention result in a dramatic reduction in morbidity and mortality and a significant improvement in cardiac structure and performance. On the other hand, the method of the present invention improves liver function when administered after hepatotoxic-ischemic injury. Hypoxia is an important component of liver disease, especially in chronic liver diseases associated with hepatotoxic compounds such as ethanol. In addition, it is known that gene expression induced by HIFα is increased in alcoholic liver disease, such as nitric oxide synthase and glucose transporter-1.

Accordingly, the present invention provides a method of treating a condition associated with ischemia or hypoxia, the method comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient, Tester. In one embodiment, the compound is administered immediately following the development of an ischemic condition, such as myocardial infarction, pulmonary embolism, intestinal infarction, ischemic stroke, and renal ischemia-reperfusion injury. In another embodiment, the compound is administered to a patient diagnosed as a condition associated with the development of chronic ischemia, such as cardiogenic cirrhosis, macular degeneration, pulmonary embolism, acute respiratory failure, neonatal respiratory distress synthesis Symptoms and congestive heart failure.

Another aspect of the invention provides a method of treating a patient at risk of developing an ischemic or hypoxic condition, such as an atherosclerotic high risk individual, using a compound described herein. Risk factors for atherosclerosis include, for example, hyperlipidemia, smoking, hypertension, diabetes, hyperinsulinemia, and abdominal obesity. Accordingly, the present invention provides a method of preventing ischemic tissue damage, comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient, to a patient in need thereof . In one embodiment, the compound can be administered based on a predisposition to a condition such as hypertension, diabetes, arterial occlusive disease, chronic venous insufficiency, Raynaud's disease, chronic skin ulcer, sclerosis, congestive heart failure, and systemic hardening.

In a particular embodiment, the method is used to increase angiogenesis and/or granulation tissue formation in damaged tissues, wounds, and ulcers. For example, the compounds of the invention have been shown to be effective in stimulating granulation tissue formation during wound healing. Granulation tissue contains newly formed leaky blood vessels and temporary plasma protein matrices such as fibrinogen and plasma fibronectin. The release of growth factors from inflammatory cells, platelets and activated endothelium stimulates the migration and proliferation of fibroblasts and endothelial cells in granulation tissue. If angiogenesis or nerve stimulation is weakened, ulceration can occur. The method of the invention effectively promotes the formation of granulation tissue. Accordingly, the present invention provides methods for treating a patient having a tissue injury due to, for example, an infarction, having a wound induced by, for example, trauma or injury, or having a chronic wound or ulcer resulting from a condition such as diabetes. The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, alone or in combination with a pharmaceutically acceptable excipient.

Another aspect of the invention provides a method of pre-treating a subject with the compound to reduce or prevent the occurrence of tissue damage associated with ischemia or hypoxia. The methods of the invention produce a therapeutic benefit when administered immediately prior to a condition involving ischemia or hypoxia. For example, the application of the method of the invention prior to induction of myocardial infarction showed a statistically significant improvement in cardiac structure and performance. On the other hand, when administered immediately before and between ischemia-reperfusion injury, the methods of the invention produce a therapeutic benefit that significantly reduces diagnostic parameters associated with renal failure.

Accordingly, the present invention provides a method of pretreating a subject to reduce or prevent tissue damage associated with ischemia or hypoxia, the method comprising administering a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof, alone or in combination with a medicament The combination of acceptable excipients is administered to a patient having a history of ischemic conditions, such as a myocardial infarction, or a patient having impending ischemic symptoms, such as angina pectoris. In another embodiment, the compound can be administered based on physical parameters suggesting possible ischemia, such as for individuals under general anesthesia or temporarily working at high altitudes. In yet another embodiment, the compound can be used in an organ transplant to pre-treat an organ donor or to maintain an organ removed by the body prior to implantation into the recipient.

Previous studies have shown that certain compounds used in the methods of the invention are potent inhibitors of procollagen prolyl 4-hydroxylase. While recognizing that the initial infarction or recovery of the wound requires deposition of connective tissue in the necrotic area, the present invention demonstrates no side effects for the treatment of scar formation. Thus, based on the benefits provided by certain compounds of the invention in the treatment and prevention of hypoxic tissue damage and fibrosis, the present invention encompasses a "dual therapy" method for treating or preventing a condition involving ischemia or hypoxia, including Ischemia or hypoxia associated with concurrent reactive fibrosis, such as myocardial infarction and consequent congestive heart failure. The method may use a compound which inhibits more than one 2-ketoglutarate dioxygenase having the same specificity or different specificity, such as HIF prolyl hydroxylase and sol prolyl 4- Hydroxylase. Alternatively, the method may employ a combination of compounds wherein each compound specifically inhibits only one 2-ketoglutarate dioxygenase, for example, one compound specifically inhibits HIF prolyl hydroxylase and the second compound is specific Inhibition of sol-prolyl-hydroxylase.

In one aspect, the compounds of the invention inhibit one or more 2-ketoglutarate dioxygenases. In one embodiment, the compound inhibits at least two members of the 2-ketoglutarate dioxygenase family having the same specificity or different specificities, such as HIF prolyl hydroxylase and HIF asparagine-hydroxyl Chemical enzyme (FIH-1). In another embodiment, the compound is specific for a 2-ketoglutarate dioxygenase, such as HIF prolyl hydroxylase, and exhibits little or no specificity for other family members. Display specificity.

The compounds can be administered in combination with a variety of other therapeutic methods. In one embodiment, the compound is administered with another 2-ketoglutarate dioxygenase inhibitor, wherein the two compounds have an individual member of the 2-ketoglutarate dioxygenase family. Different specificities. The two compounds can be administered simultaneously in a ratio relative to the other. Determination of the proportions suitable for a given therapeutic procedure or a particular subject is within the skill of the art. Alternatively, the two compounds can be administered continuously over the course of treatment, for example after myocardial infarction. In a particular embodiment, a compound specifically inhibits the activity of HIF prolyl hydroxylase, and the second compound specifically inhibits the activity of the procollagen prolyl 4-hydroxylase. In another specific embodiment, one compound specifically inhibits the activity of HIF prolyl hydroxylase, and the second compound specifically inhibits the activity of HIF asparaginyl-hydroxylase. In another embodiment, the compound is administered with another therapeutic agent having a different mode of action, such as an ACE inhibitor (ACEI), an angiotensin-II receptor blocker (ARB), statin, diuretic Agent, digoxin, carnitine, etc.

The present invention provides a method of increasing endogenous erythropoietin (EPO). These methods can be applied in vivo, such as in plasma, or in vitro, such as in a conditioned cell culture medium. The invention further provides methods of increasing endogenous EPO levels for preventing, pre-treating or treating EPO-related conditions, including, for example, conditions associated with anemia and nervous system disorders. Anemia-related conditions include conditions such as acute or chronic kidney disease, diabetes, cancer, ulcers, viral infections (eg, HIV, bacteria or parasites), inflammation, and the like. Anemia conditions may further include conditions associated with a procedure or treatment including, for example, radiation therapy, chemotherapy, dialysis, and surgery. Anemia-related disorders additionally include abnormal hemoglobin and/or red blood cells, such as found in conditions such as small red cell anemia, hypohemoglobinemia, aplastic anemia, and the like.

The invention may be used to prophylactically or simultaneously increase endogenous EPO in a subject undergoing a particular treatment or procedure, such as HIV anemia being treated with azidothymidine (Zidovudine) or other reverse transcriptase inhibitors Patients, anemia cancer patients receiving cyclic chemotherapy with cyclohexyl cisplatin or cisplatin, or anemia or non-anemic patients scheduled to undergo surgery. Methods of increasing endogenous EPO can also be used to prevent, pre-treat, or treat EPO-related conditions associated with neurological damage or neuronal degeneration, including, but not limited to, stroke, trauma, epilepsy, spinal cord injury, and neurodegenerative disorders.

Additionally, the method can be used to increase endogenous EPO levels in anemia or non-anemic patients scheduled to undergo surgery to reduce the need for exogenous blood transfusion or to facilitate pre-operative blood storage. A small reduction in blood hematocrit that usually occurs after autologous blood supply before surgery does not stimulate an increase in endogenous EPO or compensatory red blood cell production. However, pre-surgical stimulation of endogenous EPO will effectively increase red blood cell mass and autologous blood supply volume while maintaining higher hematocrit levels, and the method specificity is encompassed herein. In some surgical populations, particularly individuals with surgical blood loss of more than 2 liters, the methods of the present invention can be applied to reduce heterologous blood exposure.

The methods of the invention can also be used to enhance athletic performance, improve exercise performance, and promote or enhance aerobic conditioning. For example, an athlete can use the method to facilitate training and the soldier can use the method to improve, for example, stamina and endurance.

The methods of the present invention have been shown to increase endogenous erythropoietin levels in the culture medium and in vivo treated animal plasma in vitro for treatment of cultured cells. Although the kidney is the main source of erythropoietin in the body, other organs, including the brain, liver and bone marrow, can and do synthesize erythropoietin once properly stimulated. The expression of endogenous erythropoietin in a plurality of body organs, including the brain, kidney and liver, can be increased using the method of the invention. In fact, the method of the invention even increases the content of endogenous erythropoietin in animals undergoing double nephrectomy.

The method of the present invention demonstrates that the content of erythropoietin can be increased even when renal function is impaired. Although the present invention is not limited by the mechanism of erythropoietin production, the reduction in erythropoietin secretion normally seen during renal failure can be attributed to hyperoxemia caused by increased flow/perfusion in renal tissue.

In another aspect, the methods of the invention increase hematocrit and blood hemoglobin levels in an animal treated in vivo. As the increase in plasma EPO, hematocrit, and hemoglobin produced by the use of the compound in the methods of the invention is dose sensitive, the dosage regimen can be determined to produce a constant, controllable response level of the compound of the invention. In another aspect, treatment with a compound of the invention can treat anemia, such as anemia induced by a toxic compound, such as the chemotherapeutic agent cisplatin, or anemia due to blood loss, such as trauma, injury, parasites or surgery.

In animals treated with the compounds of the invention, the percentage of circulating immature red blood cells (reticulocytes) in the blood is increased before hematocrit and hemoglobin increase. Thus, the present invention encompasses methods of increasing the amount of reticulocytes in the blood of an animal to produce a cell-free reticulocyte lysate by the compounds of the invention (e.g., Pelham and Jackson in Eur. J. Biochem. 67). Use in: 247-256 (described in 1976). The amount of circulating reticulocytes in an animal (e.g., rabbit, etc.) is increased by treatment with a compound of the invention alone or in combination with another compound, such as acetophenone or the like.

In contrast to the prior art, the beneficial effects of the present invention are that the compounds of the invention are useful for modulating hypoxia inducible factor (HIF) and/or endogenous erythropoietin (EPO). By deuteration this technique changes the metabolism of the compound in the organism, giving the compound a better pharmacokinetic parameter characteristic. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability. Replacing a hydrogen atom in a compound with hydrazine can increase the drug concentration of the compound in an animal to improve the efficacy of the drug due to its strontium isotope effect. Substitution of a hydrogen atom in a compound with hydrazine may increase the safety of the compound due to inhibition of certain metabolites.

detailed description

Compound

The present invention provides a compound of the formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound.

Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And at least one of R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ is deuterated or deuterated.

In a specific embodiment, "R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ are each independently hydrogen, deuterium, halogen or trifluoromethyl" including R ¹ selected From hydrogen, deuterium, halogen or trifluoromethyl, R ^{2 is} selected from hydrogen, deuterium, halogen or trifluoromethyl, R ^{3 is} selected from hydrogen, deuterium, halogen or trifluoromethyl, and so on, up to R ²⁵ From hydrogen, hydrazine, halogen or trifluoromethyl, R ^{3 is} selected from the group consisting of hydrogen, hydrazine, halogen or trifluoromethyl. More specifically, R ¹ is hydrogen, R ¹ is deuterium, R ¹ is halogen (F, Cl, Br or I) or R ¹ is trifluoromethyl, R ² is hydrogen, R ² is deuterium, and R ² is Halogen (F, Cl, Br or I) or R ² is trifluoromethyl, R ³ is hydrogen, R ³ is deuterium, R ³ is halogen (F, Cl, Br or I) or R ³ is trifluoromethyl And so on, until R ²⁵ is hydrogen, R ²⁵ is deuterium, R ²⁵ is halogen (F, Cl, Br or I) or R ²⁵ is trifluoromethyl.

In a preferred embodiment, to a formula (I) compounds of the present invention, or a polymorph, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein, R ²³ is hydrogen and R ¹ -R ²² And R ²⁴ -R ²⁵ are as defined above, with the proviso that the compound contains at least one ruthenium atom.

In a preferred embodiment, the invention relates to a compound of formula (I), or a crystalline form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R ²³ is hydrogen and R ¹ -R ²² And R ²⁴ -R ²⁵ are each independently selected from hydrogen or deuterium with the proviso that the compound contains at least one deuterium atom.

In a preferred embodiment, the invention relates to a compound of formula (I), or a crystalline form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R ²³ is hydrogen, R ¹ -R ²² and R ^{24 -} R ²⁵ are each independently selected from hydrogen or deuterium, and R ¹ and R ² are the same, with the proviso that the compound contains at least one deuterium atom.

In a preferred embodiment, the invention relates to a compound of formula (I), or a crystalline form, pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R ¹ -R ⁶ , R ¹⁴ , R ¹⁵ And R ¹⁷ , R ¹⁸ , R ²¹ -R ²³ are hydrogen, and R ⁷ -R ¹¹ , R ¹² , R ¹³ , R ¹⁶ , R ¹⁹ , R ²⁰ and R ²⁴ , R ²⁵ are each independently selected from hydrogen or deuterium. An additional condition is that the compound contains at least one ruthenium atom.

In a preferred embodiment, R ^{7 -} R ¹⁰ are the same.

In a preferred embodiment, R ¹² , R ¹³ , R ¹⁹ , R ²⁰ are the same.

In a preferred embodiment, R ²⁴ and R ²⁵ are the same.

In a preferred embodiment, R ¹ and R ² are the same.

As a preferred embodiment of the present invention, the compound is any of the following structures, or a pharmaceutically acceptable salt thereof, but is not limited to the following structures:

Example

The preparation of the structural compound of the formula (I) of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

Example 1 N-[(1,3-Dicyclohexyl-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine-2,2-d ₂ , a compound T-1, the molecular formula is as follows:

Use the following synthetic route:

Step 1: Synthesis of Compound 2.

Under a nitrogen atmosphere, a solution of malonyl chloride (1.30 mL, 13.39 mmol) in anhydrous chloroform (20 mL) was added dropwise to 1,3-bis(cyclohexyl)urea (3.00 g, 13.39 mmol) in anhydrous chloroform (80 mL) After the completion of the dropwise addition in the solution, the reaction solution was heated to 50 ° C for 4.5 hrs. After cooling to room temperature, the reaction was quenched with EtOAc EtOAc (EtOAc)EtOAc. v) = 10: 1) to give a white solid 1.50g, yield: 38.4%, LC-MS ( APCI): m / z = 293.2 (m + 1) +.

Step 2: Synthesis of Compound 3.

Ethyl isocyanate (0.70 g, 5.50 mmol) was added to a solution of compound 2 (1.50 g, 5.50 mmol) and DIPEA (1.40 g, 11.00 mmol) in anhydrous dichloromethane (80 mL). The solution was stirred at room temperature overnight. EtOAc EtOAc m. LC-MS (APCI): m / z = 422.3 (M + 1) +.

Step 3: Synthesis of Compound 4.

The above crude compound 3 was dissolved in ethanol (10 mL) under a greenhouse, and then 1M sodium hydroxide (5 mL) was added, and the reaction was stirred at room temperature for 3 hrs, and the reaction mixture was acidified with 1M diluted hydrochloric acid. The mixture was extracted with EtOAc (EtOAc)EtOAc. The filter cake was washed with a solvent and dried under vacuum to give a white solid (yield: 2.0 g). LC-MS (APCI): m / z = 394.2 (M + 1) +. Purity 99.21% (HPLC). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.18 (t, J = 5.7 Hz, 1H), 4.64 (t, J = 12.0 Hz, 2H), 4.08 (d, J = 5.6 Hz, 2H), 2.28 (q, J = 12.3 Hz, 4H), 1.78 (d, J = 12.5 Hz, 4H), 1.69-1.49 (m, 6H), 1.27 (q, J = 12.9 Hz, 4H), 1.18-1.04 (m, 2H).

Step 4: Synthesis of Compound T-1.

40% sodium bismuth oxide (0.31 mL, 3.05 mmol) was added to a solution of compound 4 (300 mg, 0.76 mmol) in water (15 mL), and the reaction mixture was sealed at 140 ° C overnight. Cool to room temperature. The pH was adjusted to about 3 with 1M of diluted hydrochloric acid, and extracted with ethyl acetate (30 mL×3). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The filtrate was concentrated under reduced pressure. Methylene chloride/methanol (v/v) = 10:1) gave 104 mg in a yield: 34.6%. Purity: 98.15%. LC-MS (APCI): m / z = 396.2 (M + 1) +. ^{1 H NMR (400MHz, DMSO-} d 6) δ12.99 (br, 1H), 10.17 (s, 1H), 4.62 (t, J = 12.2Hz, 2H), 2.26 (dd, J = 22.4,12.0Hz, 4H), 1.77 (d, J = 12.7 Hz, 4H), 1.64-1.51 (m, 6H), 1.31-1.22 (m, 4H), 1.7-1.07 (m, 2H).

Example 2 N-[(1,3-Di(cyclohexyl-2,2,6,6-d ₄ )-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine, ie Chemical Compound T-2, the molecular formula is as follows:

Use the following synthetic route:

Step 1: Synthesis of Compound 7.

Cyclohexanone (1.0 g, 10.00 mmol) and 40% NaOD (0.27 mL) were added to 10 ml of D ₂ O, and the mixture was refluxed for 2 days. After cooling to room temperature, hydroxylamine hydrochloride (2.0 g) and sodium acetate (4.0 g) were added at 0 ° C, and the mixture was heated to 70 ° C and stirred for 20 min. Then, it was cooled, and a solid was precipitated, filtered, and the filter cake was dried and dissolved in 160 mL of diethyl ether. Lithium aluminum hydride (2.0 g) was slowly added, and then the mixture was heated and refluxed for 45 minutes. After cooling to room temperature, a saturated sodium bicarbonate solution was added dropwise, a large amount of solid was evaporated, filtered, and the filter cake was rinsed twice with diethyl ether. Yield: 40%. LC-MS (APCI): m / z = 104.2 (M + 1) +.

Step 2: Synthesis of Compound 8.

Compound 8 (3.90 g, 37.86 mmol) and ethylene carbonate (1.67 g, 18.93 mmol) and 1,5,7-triazidebicyclo(4.4.0)indole-5-ene (53 mg) were added to 50 mL In a single-mouth bottle, heat to 120 ° C for 2 hrs, and solids are formed. After cooling to room temperature, 10 mL of water was added, stirred for ten minutes, then filtered, and the filter cake was washed three times with DCM, then dried to give a white solid. Yield: 13.6%. LC-MS (APCI): m / z = 233.2 (M + 1) +.

Step 3: Synthesis of Compound 9.

Under a nitrogen atmosphere, a solution of malonyl chloride (365 mg, 2.59 mmol) in anhydrous chloroform (0.3 mL) was added dropwise to a solution of Compound 8 (600 mg, 2.59 mmol) in anhydrous chloroform (16 mL). The liquid was heated to 50 ° C for 4.5 hrs. After cooling to room temperature, the reaction was quenched with EtOAc EtOAc (EtOAc)EtOAc. v) = 10: 1) to give 190mg white solid, yield: 24.5%, LC-MS ( APCI): m / z = 301.2 (m + 1) +.

Step 4: Synthesis of Compound 10.

Ethyl isocyanate (82 mg, 0.63 mmol) was added to a solution of compound 9 (190 mg, 0.63 mmol) and DIPEA (164 mg, 1.26 mmol) in anhydrous dichloromethane (10 mL). After stirring overnight, the reaction mixture was quenched with EtOAc EtOAc (EtOAc) LC-MS (APCI): m / z = 430.3 (M + 1) +.

Step 5: Synthesis of compound T-2.

The above crude product was dissolved in ethanol (10 mL) in a br., and then 1M sodium hydroxide (5 mL) was added, and the reaction was stirred at room temperature for 3 days, and the reaction mixture was acidified with 1M diluted hydrochloric acid. The organic layer was extracted with EtOAc (EtOAc)EtOAc. The filter cake was washed and dried in vacuo to give 159 mg of white solid. LC-MS (APCI): m/z = 402.2 (M + 1) ⁺ ; purity: 99.77% (HPLC). ^{1 H NMR (400MHz, DMSO-} d 6): δ10.18 (t, J = 5.6Hz, 1H), 4.60 (s, 2H), 4.11 (d, J = 5.7Hz, 2H), 1.76 (d, J = 12.9 Hz, 4H), 1.62 (d, J = 12.4 Hz, 2H), 1.25 (t, J = 12.7 Hz, 4H), 1.12 (t, J = 12.9 Hz, 2H).

Example 3 N-[(1,3-Di(cyclohexyl-2,2,6,6-d ₄ )-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine-2 , 2-d ₂ , That is, the compound T-3 has the following formula:

Use the following synthetic route:

40% sodium cerium oxide (0.1 mL) was added to a mixed solution of T-2 (90 mg, 0.22 mmol) in heavy water (1 mL) and deuterated ethanol (2 mL). The reaction mixture was stirred at room temperature for 2 days and then warmed to 60. The reaction was carried out at ° C for 4 days. Cool to room temperature. The reaction mixture was acidified with EtOAc (EtOAc m. The filter cake was washed with a small amount of a mixed solvent of diethyl ether and n-hexane, and dried in vacuo to give 50 mg of white solid. LC-MS (APCI): m / z = 404.1 (M + 1) +, ^{purity: 97.12% (HPLC) 1 H} NMR (400MHz, DMSO-d 6):. Δ12.92 (br, 1H), 10.17 ( s, 1H), 4.60 (s, 2H), 1.83-1.55 (m, 6H), 1.-1.21 (m, 4H), 1.16-1.08 (m, 2H).

Example 4 N-[(1,3-Di(cyclohexyl-1-d)-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine, a compound T-4, the molecular formula is as follows:

Use the following synthetic route:

Step 1: Synthesis of Compound 11.

Freshly activated at room temperature

Molecular sieves (5.0 g) were added to a solution of cyclohexanone (2.5 mL, 23.9 mmol) and benzylamine (2.6 mL, 23.9 mmol) in anhydrous dichloromethane (24 mL). Concentration was used for the next reaction. LC-MS (APCI): m / z = 188.2 (M + 1) +.

Step 2: Synthesis of Compound 12.

The above crude product was dissolved in anhydrous methanol (24 mL), and sodium borohydride (1.0 g, 23.9 mmol) was added portionwise at 0 ° C. The reaction was stirred at room temperature for 1.5 hrs. The ether was dissolved and filtered. The filtrate was concentrated under reduced pressure. EtOAc (EtOAc:MeOH:MeOH: +1) ⁺ .

Step 3: Synthesis of Compound 13.

Under the greenhouse, Pd/C (10%, 250 mg) and Pd(OH) ₂ (250 mg) were added to a solution of compound 12 in anhydrous methanol (50 mL). The reaction was stirred at room temperature for 72 hrs, filtered over Celite, filtrate concentrated under reduced pressure to give a colorless liquid 1.2g, overall yield: 50%, LC-MS ( APCI): m / z = 101.2 (m + 1) +.

Step 4: Synthesis of Compound 14.

Compound 13 (2.0 g, 20 mmol) and ethylene carbonate (0.88 g, 10 mmol) and 1,5,7-triazidebicyclo(4.4.0) 癸-5-ene (28 mg) were added to a 50 mL single port. In the bottle, it was heated to 120 ° C and stirred for 2 hrs, and solids were formed. After cooling to room temperature, 5 mL of water was added, stirred for ten minutes, then filtered, and the filter cake was washed three times with DCM and then dried to give 185 mg of white solid. Yield: 4.07%. LC-MS (APCI): m / z = 227.1 (M + 1) +.

Step 5: Synthesis of Compound 15.

Under a nitrogen atmosphere, a solution of malonyl chloride (115 mg, 0.82 mmol) in anhydrous chloroform (0.2 mL) was added dropwise to a solution of compound 14 (185 mg, 0.82 mmol) in anhydrous chloroform (10 mL). The liquid was heated to 50 ° C for 4.5 hrs. After cooling to room temperature, the reaction was quenched with EtOAc EtOAc (EtOAc)EtOAc. v) = 10: 1) to give 135mg white solid, yield: 56.0%, LC-MS ( APCI): m / z = 295.2 (m + 1) +.

Step 6: Synthesis of Compound 16.

Ethyl isocyanate (61 mg, 0.48 mmol) was added to a solution of compound 15 (135 mg, 0.48 mmol) and DIPEA (122 mg, 0.96 mmol) in anhydrous dichloromethane (10 mL) After stirring overnight, the reaction mixture was quenched with EtOAc EtOAc (EtOAc) LC-MS (APCI): m / z = 424.1 (M + 1) +.

Step 7: Synthesis of compound T-4.

The above crude product was dissolved in ethanol (5 mL) in a br., and then 1M sodium hydroxide (2 mL) was added, and the reaction was stirred at room temperature for 3 days, and the reaction mixture was acidified with 1M diluted hydrochloric acid. Ethyl acetate (20 mL, EtOAc) was evaporated.jjjjjjjjjjjjjjjjj The filter cake was washed with a solvent, and the solid was dried in vacuo to give 120 mg of white solid. LC-MS (APCI): m / z = 396.0 (M + 1) +, ^{purity: 94.46% (HPLC) 1 H} NMR (400MHz, DMSO-d 6):. Δ10.16 (t, J = 5.5Hz, 1H), 4.04 (d, J = 5.6 Hz, 2H), 2.27 (t, J = 11.2 Hz, 4H), 1.77 (d, J = 12.6 Hz, 4H), 1.65-1.49 (m, 6H), 1.30- 1.22 (m, 4H), 1.17.1.05 (m, 2H).

Example 5 N-[(1-Cyclohexyl-3-(cyclohexyl-2,2,6,6-d ₄ )-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine , That is, compound T-5, the molecular formula is as follows:

Use the following synthetic route:

Step 1: Synthesis of compounds 18 and 19.

Add cyclohexylamine (500 mg, 5.04 mmol) to ethylene carbonate (443 mg, 5.04 mmol) and 1,5,7-triazidebicyclo(4.4.0) 癸-5-ene (10 mg) to 50 mL In a single-mouth flask, the reaction was heated at 120 ° C for 4 hrs. Then, it was cooled to room temperature, and then Compound 7 (519 mg, 5.04 mmol) was added, and the mixture was further heated to 120 ° C for 4.5 hrs. After cooling to room temperature, 5 mL of water was added, a solid was precipitated, filtered, and the filter cake was washed twice with a small portion of DCM and dried in vacuo to afford 293 g of white solid. LC-MS (APCI): m / z = 229.1 (M + 1) +; Yield: 25.4%.

Step 2: Synthesis of Compound 20.

Under a nitrogen atmosphere, a solution of malonyl chloride (181 mg, 1.29 mmol) in anhydrous chloroform (0.4 mL) was added dropwise to a solution of compound 19 (293 mg, 1.29 mmol) in anhydrous chloroform (16 mL). The liquid was heated to 50 ° C for 4.5 hrs. After cooling to room temperature, the reaction was quenched with EtOAc EtOAc (EtOAc)EtOAc. v) = 10: 1) to give 238mg white solid, yield: 56.0%, LC-MS ( APCI): m / z = 297.2 (m + 1) +.

Step 3: Synthesis of Compound 21.

Ethyl isocyanate (104 mg, 0.80 mmol) was added to a solution of compound 20 (238 mg, 0.80 mmol) and DIPEA (207 mg, 1.60 mmol) in anhydrous dichloromethane (10 mL). After stirring overnight, the reaction mixture was quenched with EtOAc EtOAc (EtOAc) LC-MS (APCI): m/z =426.1 (M+1) ⁺ .

Step 4: Synthesis of compound T-5.

The above crude product was dissolved in ethanol (10 mL) in a br., and then 1M sodium hydroxide (5 mL) was added, and the reaction was stirred at room temperature for 3 days, and the reaction mixture was acidified with 1M diluted hydrochloric acid. Ethyl acetate (20 mL, EtOAc) was evaporated.jjjjjjjjjjjjjjjjj The filter cake was washed with a solvent and dried under vacuum to give 218 g of white solid. LC-MS (APCI): m / z = 398.2 (M + 1) +; ^{purity: 98.98% (HPLC), 1} H NMR (400MHz, DMSO-d 6) δ10.16 (t, J = 5.4Hz, 1H ), 4.70-4.47 (m, 2H), 4.07 (d, J = 5.7 Hz, 2H), 2.25 (q, J = 11.8 Hz, 2H), 1.75 (d, J = 10.1 Hz, 4H), 1.58 (t , J = 14.2 Hz, 4H), 1.24 (t, J = 12.7 Hz, 4H), 1.11 (t, J = 12.7 Hz, 2H).

Example 6 N-[(1-Cyclohexyl-3-(cyclohexyl-2,2,6,6-d ₄ )-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine -2,2-d ₂ , that is, compound T-6, the molecular formula is as follows:

Use the following synthetic route:

40% sodium cerium oxide (0.1 mL) was added to heavy water (1 mL) and deuterated ethanol (2 mL) added to the compound T-5 (100 mg), and the reaction mixture was stirred at room temperature for 2 days and then heated to 60 ° C. The reaction was 4 days. Cool to room temperature. The reaction mixture was acidified with EtOAc (EtOAc m. The filter cake was washed with a small amount of a mixed solvent of diethyl ether and n-hexane, and the solid was dried in vacuo to give a pale yellow solid (yield: 79.8%). LC-MS (APCI): m/z = 400.2 (M + 1) ⁺ ; purity: 99.17% (HPLC), ¹ H NMR (400 MHz, DMSO-d6): δ 13.03 (br, 1H), 10.15 (s , 1H), 4.73–4.44 (m, 2H), 2.30–2.18 (m, 2H), 1.82–1.68 (m, 4H), 1.64–1.52 (m, 4H), 1.26–1.18 (m, 4H), 1.15 –1.05 (m, 2H).

Example 7 N-[(1,3-bis(cyclohexyl-1-d)-2,4,6-trioxo-5-hexahydropyrimidinyl)carbonyl]glycine-2,2-d ₂ , ie Chemical Compound T-7, the molecular formula is as follows:

Use the following synthetic route:

40% sodium cerium oxide (0.1 mL) was added to heavy water (1 mL) and deuterated ethanol (2 mL) added to the compound T-4 (80 mg), and the reaction mixture was stirred at room temperature for 2 days and then heated to 60 ° C. The reaction was 4 days. Cool to room temperature. The reaction mixture was acidified with EtOAc (EtOAc m. The filter cake was washed with a small amount of a mixed solvent of diethyl ether and n-hexane, and dried in vacuo to give 70 mg of a yellow solid.

LC-MS (APCI): m / z = 398.2 (M + 1) +; ^{purity: 94.35% (HPLC), 1} H NMR (400MHz, DMSO-d 6) δ10.15 (s, 1H), 2.24 (t , J = 11.3 Hz, 4H), 1.76 (d, J = 12.6 Hz, 4H), 1.63 - 1.53 (m, 6H), 1.25 - 1.19 (m, 4H), 1.13 - 1.04 (m, 2H).

Biological activity test.

(1) Mouse tissue HIF Western blot analysis.

The mouse tissues stored at -80 ° C were ground into a powder using a mortar and pestle frozen under liquid nitrogen. Nuclear extracts were prepared using NE-PER kit (Pierce Biotechnology). For immunoprecipitation, the nuclear extract was added to the HIF-1α monoclonal antibody at a ratio of tissue to antibody of 200:1. The suspension was incubated for 4 hours at 4 ° C in a conical microcentrifuge tube. Protein A/G coupled agarose beads (40 [mu]L of 50% suspension) were then added to the tube. After rotating at 4 ° C overnight, the beads were washed 3 times with ice-cold phosphate buffer. The beads were then prepared for SDS-PAGE using 40 [mu]L of Laemmli sample buffer solution. Proteins separated from SDS-PAGE were transferred to nitrocellulose plates with the XCell-II Blot Module system. The blot was blocked with 5% BSA and then incubated with a 1:100 dilution of HIF-1α rabbit antibody. The blots were then washed with Tris buffered saline/Tween-20 buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. The blot was visualized with ECL reagent. The blot image was captured with an Epson Expression 1600 scanner.

(2) Mouse serum EPO test.

Mouse serum EPO was detected using R&D Systems' mouse Quantikine erythropoietin ELISA kit according to the manufacturer's instructions.

The experimental results show that the compound of the present invention is responsive to the detection of serum EPO in mice, indicating that the compound of the present invention can be used for the preparation of a medicament for regulating anemia in humans.

(3) Evaluation of metabolic stability.

Microsomal experiments: rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer (pH 7.4).

Preparation of stock solution: Weigh a certain amount of the test compound powder and dissolve it to 5 mM with DMSO.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

A solution of NADPH regeneration system (containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride) was prepared and placed on wet ice before use.

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of rat liver microsome dilution was added to a 96-well incubation plate (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

The results of metabolic stability experiments are shown in Table 2 below:

Table 2 Metabolic stability in rat liver microsomes

Compound	T 1/2 (min)	Extension rate
Darprodustat	267.2	--
T-1	315.1	17.9%
T-3	479.8	79.5%
T-4	449	68.0%
T-5	1151.2	330.8%
T-6	483.5	80.9%
T-7	485.7	81.7%

The experimental results show that the compound of the present invention can significantly prolong the half-life and the metabolism more stably than the original drug Daprodustat, especially the compound T-3 to the compound T-7, and the half-life is prolonged by more than 60%.

(4) Pharmacokinetic evaluation in rats.

Six male Sprague-Dawley rats, 7-8 weeks old, weighing 210 g, divided into 2 groups, 3 in each group, intravenously or orally administered a single dose of compound (3 mg/kg intravenously, 10 mg/kg orally). Its pharmacokinetic differences.

Rats were fed a standard diet and given water. Fasting began 16 hours before the test. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the eyelids at a time point of 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

Rats were briefly anesthetized after inhalation of ether, and 300 μL of blood samples were collected from the eyelids in test tubes. There was 30 μL of 1% heparin salt solution in the test tube. The tubes were dried overnight at 60 ° C before use. After the blood sample collection was completed at a later time point, the rats were anesthetized with ether and sacrificed.

Immediately after the blood sample was collected, gently invert the tube to 5 times to ensure that the mixture was adequately placed on ice. Blood samples were centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate plasma from red blood cells. Pipette 100 μL of plasma into a clean plastic centrifuge tube to indicate the name and time point of the compound. Plasma was stored at -80 °C prior to analysis. The concentration of the compound of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

The experimental results show that the compound of the present invention has better pharmacokinetics in animals than the control compound AKB-6548, and thus has better pharmacodynamics and therapeutic effects.

It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples, usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (7)

1. A substituted pyrimidinone compound characterized by a pyrimidinetrione compound represented by formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvent Compound,

   

   Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ are each independently hydrogen, deuterium, halogen or trifluoromethyl;

   Additional conditions are R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ And at least one of R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ is deuterated or deuterated.
2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ²⁴ and R ²⁵ are each independently hydrazine or hydrogen.
3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ And R ¹¹ are each independently hydrazine or hydrogen.
4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ And R ²² are each independently hydrazine or hydrogen.
5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of the following compounds:

   

   
6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a substituted pyrimidinone compound according to any one of claims 1 to 5, or a crystalline form thereof, a pharmaceutically acceptable salt, A pharmaceutical composition of a hydrate or solvate, stereoisomer, prodrug or isotopic variation.
7. Use of a compound according to claim 1, or a crystalline form, pharmaceutically acceptable salt, hydrate or solvate thereof, for the preparation of a prophylactic and chronic anemia, glucose intolerance and/or Or use in kidney disease-related anemia, as well as drugs for cancer anemia or blood cell related disorders.