WO2017181924A1 - Substituted pyrimidinedione and pharmaceutical composition thereof - Google Patents

Abstract

The present invention discloses a substituted pyrimidinedione and a pharmaceutical composition thereof. The substituted pyrimidinedione is a compound represented by formula (I), or a crystalline form, pharmaceutically acceptable salt, prodrug, tautomer, stereoisomer, isotopic isomer, hydrate, or solvate thereof. The compound disclosed by the invention has improved inhibition against a dipeptidyl peptidase, improved pharmacodynamics/pharmacokinetics, good compound usability, and high levels of safety. The compound can be used to prepare a drug for treating a disease related to the dipeptidyl peptidase.

Description

Substituted pyrimidinedione compound and pharmaceutical composition thereof Technical field

The invention belongs to the technical field of medicine, and in particular relates to a substituted pyrimidinedione compound and a pharmaceutical composition thereof, which are useful for treating a dipeptidyl peptidase-mediated related disease.

Background technique

Dipeptidyl peptidase IV (DPP-IV) is a type II membrane protein that acts as a non-classical serine aminodipeptidase that removes the Xaa-Pro dipeptide from the amino terminus (N-terminus) of the polypeptide and protein. Some naturally occurring peptides have also been reported to have DPP-IV dependent slow release of X-Gly or X-Ser type dipeptides.

DPP-IV is constitutively expressed on epithelial and endothelial cells of a variety of different tissues (intestine, liver, kidney, and placenta) and is also found in body fluids. DPP-IV is also expressed on circulating T-lymphocytes and has been shown to be synonymous with the cell-surface antigen CD-26. DPP-IV is responsible for the metabolic cleavage of certain endogenous peptides (GLP-1 (7-36), glucagon) in vivo and has been shown to be resistant to a variety of other peptides in vitro (GHRH, NPY, GLP-2, VIP). ) proteolytic activity.

GLP-1 (7-36) is a 29 amino acid peptide derived from post-translational processing of proglucagon in the small intestine. GLP-1 (7-36) has a variety of in vivo effects, including stimulation of insulin secretion, inhibition of glucagon secretion, promotion of satiety, and delay in gastric emptying. Based on its physiological behavior, it is believed that the role of GLP-1 (7-36) is beneficial for the prevention and treatment of type 2 diabetes, and possibly also obesity. For example, exogenous administration (continuous infusion) of GLP-1 (7-36) in diabetic patients has been found to be effective for this patient population. Unfortunately, GLP-1 (7-36) rapidly degrades in vivo and has been shown to have a short in vivo half-life (t _1/2 ).

Studies based on genetically engineered DPP-IV knockout mice and in vivo/in vitro studies of selective DPP-IV inhibitors have shown that DPP-IV is the major degrading enzyme of GLP-1 (7-36) in vivo. GLP-1 (7-36) is efficiently degraded by DPP-IV to GLP-1 (9-36), which is presumed to act as a physiological antagonist of GLP-1 (7-36). It is therefore believed that inhibition of DPP-IV in vivo can be used to potentiate endogenous GLP-1 (7-36) levels and attenuate the production of its antagonist GLP-1 (9-36). Thus, it is believed that DPP-IV inhibitors are drugs that can be used to prevent, delay their progression and/or treat DPP-IV mediated disorders, in particular diabetes, more specifically type II diabetes, diabetic lipemia Abnormalities, impaired glucose tolerance (IGT), fasting plasma glucose reduction (IFG), metabolic acidosis, ketosis, appetite regulation, and obesity.

Therefore, there is still a need in the art to develop DPP-IV inhibitors that have inhibitory activity or better pharmacodynamic properties for dipeptidyl peptidases.

Summary of the invention

In view of the above technical problems, the present invention discloses a dipeptidyl peptidase inhibitor, a pharmaceutical composition and use thereof, which have better dipeptidyl peptidase inhibitory activity and/or have better pharmacodynamics/pharmacokinetics Kinetic performance.

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

A dipeptidyl peptidase inhibitor, such as a substituted pyrimidinedione compound represented by formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a prodrug, a tautomer, a stereoisomer, Isotope variant, hydrate or 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 and R ¹⁸ 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 At least one of R ¹⁷ and R ¹⁸ is deuterated or deuterated.

With this technical solution, the shape and volume of the ruthenium in the drug molecule are substantially the same as those of the hydrogen. If the hydrogen in the drug molecule is selectively replaced with hydrazine, the deuterated drug generally retains the original biological activity and selectivity. At the same time, the inventors have confirmed through experiments that the binding of carbon-germanium bonds is more stable than the combination of carbon-hydrogen bonds, which can directly affect the absorption, distribution, metabolism and excretion of some drugs, thereby improving the efficacy, safety and tolerability of the drugs.

Preferably, the strontium 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%, more preferably greater than 75%, and even more preferably 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 strontium isotope content of each of the R ⁹ , R ¹⁶ , R ¹⁷ and R ¹⁸ deuterated sites is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, and even more preferably greater than 25. More preferably, more than 30%, more preferably more than 35%, more preferably more than 40%, more preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60% More preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, and even more preferably greater than 95%, More preferably greater than 99%.

Preferably, 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 of the compound of formula (I) ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ and R ¹⁸ , at least one of R contains ruthenium, more preferably two R contains ruthenium, more preferably three R contains ruthenium, more preferably four R contains ruthenium, more preferably The ground five R contains 氘, more preferably six R contains 氘, more preferably seven R contains 氘, more preferably eight R contains 氘, more preferably nine R contains 氘, more preferably ten R contains氘, preferably, eleven R contains 氘, more preferably twelve R 氘, more preferably thirteen R 氘, more preferably fourteen R 氘, more preferably fifteen R氘, preferably, sixteen R 氘, more preferably seventeen R 氘, more preferably eighteen R 氘.

As a further improvement of the present invention, R ¹ , R ² , and R ³ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁴ and R ⁵ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁶ , R ⁷ and R ⁸ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁹ is hydrazine.

As a further improvement of the present invention, R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ and R ¹⁸ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, the compound may be selected from the following compounds or a pharmaceutically acceptable salt thereof, but is not limited to the following compounds:

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

The invention also discloses a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the dipeptidyl peptidase inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or A pharmaceutical composition of a solvate, stereoisomer, prodrug or isotopic variation.

As a further improvement of the present invention, the pharmaceutically acceptable carrier includes a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersant At least one of a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.

As a further improvement of the present invention, the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, an ointment, an emulsion, a suspension, a solution, a suppository, an injection, an inhalant, a gel, a microsphere or Aerosol.

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.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: pharmaceutically acceptable carrier with a dipeptidyl peptidase inhibitor as described above, or a crystalline form thereof, a pharmaceutically acceptable salt, hydrated The solvates or solvates are mixed to form a pharmaceutical composition.

The compound of the present invention has dipeptidyl peptidase enzyme inhibitory activity, and thus it is expected to be useful as a therapeutic agent for treating a patient suffering from a disease or condition which is treated by inhibiting dipeptidyl peptidase or by increasing its peptide substrate content. Accordingly, one aspect of the invention relates to a method of treating a patient suffering from a disease or condition treated by inhibition of a dipeptidyl peptidase comprising administering to the patient a therapeutically effective amount of a compound of the invention. Another aspect of the invention relates to a method of treating a cardiovascular disease comprising administering to a patient a therapeutically effective amount of a compound of the invention. Another aspect of the invention In one aspect, the invention relates to a method of treating hyperglycemia involving inhibition of a dipeptidyl peptidase in a mammal comprising administering to the mammal a dipeptidyl peptidase inhibitory amount of a compound of the invention.

The present invention also discloses the use of a substituted pyrimidinedione compound as described above as a dipeptidyl peptidase inhibitor, i.e., the compound of the present invention can be advantageously used as a therapeutic agent for treating a condition such as type II diabetes.

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. , 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 salt), ammonium salts (such as lower alkanolammonium salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine Salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trishydroxyethylamine salt, and morpholine, piperazine, respectively An amine salt formed by lysine.

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.

Compared with the prior art, the beneficial effects of the present invention are: the compound of the present invention has excellent inhibition to dipeptidyl peptidase; the technology of deuteration is used to change the metabolism of the compound in the organism, so that the compound has better Pharmacokinetic parameter characteristics. In this case, the dosage can be changed and a long-acting preparation can be formed to improve the applicability; the substitution of a hydrogen atom in the compound with hydrazine increases the drug concentration of the compound in the animal due to its strontium isotope effect, and improves the therapeutic effect of the drug; Substituting a hydrogen atom in a compound inhibits certain metabolites and increases the safety of the compound.

detailed description

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.

Example 1 Preparation of 2-{(6-[(3R)-3-aminopiperidin-1-yl]-3-d3-methyl-2,4-dioxo-3,4-dipyrimidine -1(2H)-yl)methyl}-4-fluorobenzonitrile (Compound T-1)

The specific synthesis steps are as follows:

Step 1: Synthesis of 4-fluoro-2-methylbenzonitrile (Compound 2).

Compound 1 (3.5 g, 18.5 mmol), copper cyanide (2 g, 22 mmol), 100 mL of DMF were added, the temperature was raised to 140 ° C, and the reaction was stirred for 24 hours. The reaction of the starting material was completely confirmed by TLC, and quenched by adding 60 mL of water. the reaction was extracted three times with methyl tert-butyl ether, and the combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated, purified by column chromatography to give compound 2 1.16g, yield 46.4%, ¹ H _{NMR (300MHz, CDCl 3) δ7.62-7.59} (m, 1H), 7.04-7.02 (m, 1H), 7.00-6.96 (m, 1H).

Step 2: Synthesis of 2-bromomethyl-4-fluorobenzonitrile (Compound 3).

To the reaction flask was added compound 2 (100 mg, 0.74 mmol), N-bromosuccinimide (NBS, 131 g, 0.74 mmol), azobisisobutyronitrile (AIBN, 5 mg, 0.029 mmol), tetrachlorobenzene 5 mL of carbon was reacted for 3 hours under reflux, and the reaction of the starting material was completely confirmed by TLC, cooled to room temperature, and solid impurities were removed by filtration, and the filtrate was concentrated to obtain 260 mg of the yellow solid compound 3, which was directly used for the next reaction.

Step 3: Synthesis of 2-{(2,4-dioxo-3,4-dipyrimidin-1(2H)-yl)methyl}-4-fluorobenzonitrile (Compound 5).

Add compound 4 (600 mg, 4.08 mmol) to the reaction flask under nitrogen atmosphere. Add DMF: DMSO = 6:120 mL, cool to 0 ° C, add sodium hydrogen (165 mg, 4.08 mmol), stir for 0.5 hour, add lithium bromide (240 mg, 0.276) After stirring for 0.5 hours, 3 mL of a solution of compound 3 (750 mg, 4.08 mmol) in DMF was added to the reaction mixture, and the mixture was reacted at 0 ° C for 1 hour, and then stirred at room temperature overnight, and the reaction was completed by TLC, quenched with water and acetic acid. The ethyl ester was taken three times, and the combined organic layers were washed with saturated sodium chloride, dried over anhydrous sodium sulfate.

Step 4: 2-{(3-d3-methyl-2,4-dioxo-3,4-dipyrimidin-1(2H)-yl)methyl}-4-fluorobenzonitrile (Compound 6) Synthesis.

Compound 5 (120 mg, 0.429 mmol) was added to the reaction flask, DMF: THF = 1:1 (7 mL) was added, the mixture was cooled to 0 ° C, sodium hydrogen (18 mg, 0.45 mmol) was added, and lithium bromide (22 mg, 0.253 mmol) was added. After stirring for 20 minutes at room temperature, d3-iodomethane (120 mg, 0.83 mmol) was added, and the reaction was stirred at room temperature for 2 hours, and the reaction was carried out at 35 ° C overnight. The reaction was completed by TLC. washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated, purified by column chromatography to give 170mg of compound 6, yield ~ 100%, LC-MS ( APCI): m / z = 297 (m + 1) + .

Step 5: 2-{(6-[(3R)-3-tert-Butoxycarbonylaminopiperidin-1-yl]-3-d3-methyl-2,4-dioxo-3,4-dipyrimidine Synthesis of -1(2H)-yl)methyl}-4-fluorobenzonitrile (Compound 8).

Add compound 6 (170 mg, 0.572 mmol), compound 7 (275 mg, 1.38 mmol), sodium hydrogencarbonate (134 mg, 1.6 mmol), add 16 mL of ethanol, and warm to 100 ° C for 2 hours. TLC detection of TLC detection materials The reaction was completed, and the residue was evaporated to ethyl ether. EtOAc was evaporated. The yield was 60.8%.

Step 6: 2-{(6-[(3R)-3-Aminopiperidin-1-yl]-3-d3-methyl-2,4-dioxo-3,4-dipyrimidine-1 (2H Synthesis of -yl}methyl)-4-fluorobenzonitrile (Compound T-1).

Compound 8 (160 mg, 0.347 mmol) was added to the reaction flask, and dissolved in 5 mL of ethanol. 5 mL of dioxane hydrochloride was added to the reaction flask, and the reaction was carried out for 5 hours at room temperature. The reaction of the starting material was completely confirmed by TLC. 10, yield 49.6%, LC-MS (APCI): m / z = 361 (M + 1) ⁺ , ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.97 - 7.94 (m, 1H), 7.73 - 7.33 ( m, 1H), 7.19-7.16 (m, 1H), 5.32 (s, 1H), 5.17 (s, 2H), 3.32 (s, 2H), 2.99 (d, 2H), 2.90 (d, 2H), 2.69 (m, 1H), 2.58 (m, 1H), 2.33 (m, 1H), 1.77-1.75 (m, 1H), 1.67-1.64 (m, 1H).

Compound biological activity test

The protease inhibitory activity of the DPP-IV inhibitor can be readily determined by methods known to those of ordinary skill in the art, as in vitro assays suitable for measuring protease activity and inhibition of test compounds are known. Examples of assays that can be used to measure protease inhibitory activity and selectivity are described below.

1. Protease inhibitory activity test

(1) DPP-IV assay

Preparation of test compound solutions of different concentrations (final concentration ≤ 10 mM) in dimethyl sulfoxide, followed by dilution In the assay buffer, it contained: 20 mM Tris, pH 7.4, 20 mM KCl and 0.1 mg/mL BSA. Human DPP-IV (final concentration 0.1 nM) was added to the dilution, pre-incubated for 10 minutes at ambient temperature, and then AP-7-carboxamido-4-trifluoromethylcoumarin (AP-AFC; final The reaction was initiated at a concentration of 10 μM. The total volume of the reaction mixture is 10-100 μL depending on the assay format used (384 or 96-well plates). The reaction was dynamically monitored (excitation λ = 400 nm; emission λ = 505 nm) for 5-10 minutes, or the endpoint was measured after 10 minutes. The inhibition constant was calculated from the enzymatic process curve using a standard mathematical model.

(2) FAPa assay

Different concentrations (final concentration ≤ 10 mM) of the test compound solution were prepared in dimethyl sulfoxide and then diluted in assay buffer containing: 20 mM Tris, pH 7.4; 20 mM KCl and 0.1 mg/mL BSA. Human FAPα (final concentration 2 nM) was added to the dilution, pre-incubated for 10 minutes at ambient temperature, and then AP-7-carboxamido-4-trifluoromethylcoumarin (AP-AFC; final concentration 40 μM) Initiate the reaction. The total volume of the reaction mixture is 10-100 μL depending on the assay format used (384 or 96-well plates). The reaction was dynamically monitored (excitation λ = 400 nm; emission λ = 505 nm) for 5-10 minutes, or the endpoint was measured after 10 minutes. The inhibition constant was calculated from the enzymatic process curve using a standard mathematical model.

(3) PREP assay

Prepare test compound solutions of different concentrations (final concentration ≤ 10 mM) in dimethyl sulfoxide and then dilute in assay buffer containing: 20 mM sodium phosphate, pH 7.4, 0.5 mM EDTA, 0.5 mM DTT and 0.1 Mg/mL BSA. PREP (EC 3.4.21.26 from G. septicum, final concentration 0.2 nM) was added to the dilution. The PRE and compound were pre-incubated for 10 minutes at ambient temperature and then primed with Z-G-P-AMC (final concentration 10 [mu]M). The total volume of the reaction mixture is 10-100 μL depending on the assay format used (384 or 96-well plates). The reaction was monitored dynamically (excitation λ = 375 emission λ = 460 for 5-10 minutes, or the endpoint was measured after 10 minutes. The inhibition constant was calculated from the enzymatic process curve using a standard mathematical model.

(4) Tryptase assay

Test solutions of different concentrations (final concentration ≤ 10 mM) were prepared in dimethyl sulfoxide and then diluted in assay buffer containing: 100 mM Hepes, pH 7.4, 0.01% Brij 35 and 10% glycerol. Trypsin (rhLungβ, final concentration 0.1 nM) was added to the dilution and pre-incubated with the compound for 10 minutes at ambient temperature. The enzymatic reaction was initiated with 25 μM Z-lys-SBzl and 400 μM DTNB. The total volume of the reaction mixture was 100 μL and was carried out in a Costar A/296 well plate. The colorimetric reaction was monitored (λ = 405 nm) for 10 minutes. The inhibition constant was calculated from the enzymatic process curve using a standard mathematical model.

The protease inhibition of the compounds of the present invention was tested according to the above assay, and it was observed to exhibit selective DPP-IV inhibitory activity. The apparent inhibition constant (Ki) of the compounds of the invention for DPP-IV is in the range of from about 10 ^-9 M to about 10 ^-5 M.

2. Metabolic stability evaluation

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

Preparation of the stock solution: A certain amount of the powder of Example 1 was accurately weighed and dissolved to 5 mM with DMSO, respectively.

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). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 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 human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (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. Take 100 μL of the supernatant to pre-add 100 μL of steam The 96-well plates of the distilled water were mixed and analyzed 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 compound of Example 1 was analyzed according to the above procedure, and the results are shown in Table 2.

Table 1 Results of determination of metabolic stability of the compound of Example 1

As shown in Table 1, the compounds of the present invention exhibited excellent metabolic stability in both human liver microsomes and rat liver microsome experiments.

3. Rat pharmacokinetic experiments

EXPERIMENTAL OBJECTIVE: To investigate the pharmacokinetic behavior of the compounds of the present invention after administration of Trelagliptin, an example compound, in rats.

Experimental animals:

Type and strain: SD rat grade: SPF grade

Gender and quantity: male, 6

Weight range: 180 ~ 220g (actual weight range is 187 ~ 197g)

Source: Shanghai Xipuer Bikai Experimental Animal Co., Ltd.

Experimental and animal certificate number: SCXK (Shanghai) 2013-0016

experiment procedure:

Before the blood sample was collected, 20 L of 2M sodium fluoride solution (esterase inhibitor) was previously added to the EDTA-K2 anticoagulation tube, dried in an 80 degree oven, and stored in a 4 degree refrigerator.

Rats, males, weighing 187-197 g, were randomly divided into 2 groups. They were fasted overnight in the afternoon before the experiment but were free to drink water. Food was given 4 h after administration. Group A was given Trelagliptin 3 mg/kg, and group B was given 3 mg/kg of the compound of the example, respectively, from the rat eyelid at 15 min, 30 min, 1, 2, 3, 5, 8, 10 h after administration. Intravenous blood collection of about 100-200L, placed in EDTA-K2 anticoagulated 0.5mL Eppendorf tube, mix immediately, anticoagulation, as soon as possible, gently invert the test tube 5-6 times, after the blood is taken Place in an ice box, centrifuge the blood sample at 4000 rpm, 10 min, 4 ° C for 30 min, collect all plasma and store at -20 ° C immediately. Plasma concentrations in plasma at each time point were determined after sample collection at all time points.

According to the average blood drug concentration-time data obtained after the above, the male SD rats were subjected to ig-administered Trelagliptin (3 mg/kg) and the compound of the example (3 mg/kg) according to the non-compartmental statistical moment theory using Winnonin software. Post-pharmacokinetic related parameters.

Experiments show that compared with Trelagliptin, the compound of the present invention has better activity than Trelagliptin and has excellent pharmacokinetic properties, so it is more suitable as a compound for inhibiting dipeptidyl peptidase, and is suitable for preparing type II diabetes. drug.

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 (9)

1. A dipeptidyl peptidase inhibitor characterized by a pyrimidinedione compound represented by formula (I), or a crystal form thereof, a pharmaceutically acceptable salt, a prodrug, a tautomer, a stereoisomer a conformation, an isotope variant, 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 and R ¹⁸ 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 At least one of R ¹⁷ and R ¹⁸ is deuterated or deuterated.
2. The dipeptidyl peptidase inhibitor according to claim 1, wherein each of R ¹ , R ² and R ^{3 is} independently hydrazine or hydrogen.
3. The dipeptidyl peptidase inhibitor according to claim 1, wherein each of R ⁴ and R ^{5 is} independently hydrazine or hydrogen.
4. The dipeptidyl peptidase inhibitor according to claim 1, wherein each of R ⁶ , R ⁷ and R ^{8 is} independently hydrazine or hydrogen.
5. The dipeptidyl peptidase inhibitor according to claim 1, wherein R ⁹ is hydrazine.
6. The dipeptidyl peptidase inhibitor according to claim 1, wherein R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ and R ¹⁸ are each independently 氘Or hydrogen.
7. The dipeptidyl peptidase inhibitor according to claim 1, wherein the compound is selected from the group consisting of Or a pharmaceutically acceptable salt thereof:

   

   
8. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the dipeptidyl peptidase inhibitor according to any one of claims 1 to 7, or a crystalline form thereof, a pharmaceutically acceptable salt thereof A pharmaceutical composition of a hydrate or solvate, tautomer, stereoisomer, prodrug or isotopic variation.
9. Use of a dipeptidyl peptidase inhibitor according to any one of claims 1 to 8 for the preparation of a medicament for treating a dipeptidyl peptidase-mediated disease, such as type II diabetes.