CN108368085B

CN108368085B - Substituted pyrimidinedione compound and pharmaceutical composition thereof

Info

Publication number: CN108368085B
Application number: CN201780004366.8A
Authority: CN
Inventors: 王义汉; 赵九洋
Original assignee: Shenzhen Targetrx Inc
Current assignee: Shenzhen Targetrx Inc
Priority date: 2016-04-20
Filing date: 2017-04-17
Publication date: 2021-05-04
Anticipated expiration: 2037-04-17
Also published as: CN108368085A; WO2017181924A1

Abstract

The invention provides a substituted pyrimidinedione compound and a pharmaceutical composition thereof, wherein the substituted pyrimidinedione compound is a compound shown as a formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a tautomer, a stereoisomer, an isotopic variant, a hydrate or a solvate compound thereof. The compound has better dipeptidyl peptidase inhibitory activity, better pharmacodynamics/pharmacokinetics performance, good applicability and high safety, and can be used for preparing and treating diseases related to dipeptidyl peptidase.

Description

Substituted pyrimidinedione compound and pharmaceutical composition thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a substituted pyrimidinedione compound and a pharmaceutical composition thereof, which can be used for treating related diseases mediated by dipeptidyl peptidase.

Background

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

DPP-IV is constitutively expressed on epithelial and endothelial cells of a variety of different tissues (intestine, liver, kidney and placenta), 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 have proteolytic activity against a variety of other peptides (GHRH, NPY, GLP-2, VIP) in vitro.

GLP-1(7-36) is a 29 amino acid peptide derived from post-translational processing of preproglucagon 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 of gastric emptying. Based on its physiological behavior, the effects of GLP-1(7-36) are believed to be beneficial in the prevention and treatment of type II diabetes, and possibly obesity. For example, exogenous administration (continuous infusion) of GLP-1(7-36) in diabetic patients has been found to be effective in this patient population. Unfortunately, GLP-1(7-36) degrades rapidly in vivo and has been shown to have a very short half-life (t) in vivo_1/2)。

Based on the study of genetically bred DPP-IV knockout mice and in vivo/in vitro studies of selective DPP-IV inhibitors, DPP-IV has been shown to be 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). Thus, in vivo inhibition of DPP-IV is believed to be useful for potentiating endogenous GLP-1(7-36) levels and for attenuating the production of its antagonist GLP-1 (9-36). Thus, DPP-IV inhibitors are believed to be useful drugs for preventing, delaying the progression thereof and/or treating conditions mediated by DPP-IV, in particular diabetes, more in particular type II diabetes, diabetic dyslipidemia, Impaired Glucose Tolerance (IGT) disorder, impaired fasting plasma glucose (IFG) disorder, metabolic acidosis, ketosis, appetite regulation and obesity.

Accordingly, there remains a need in the art to develop DPP-IV inhibitors with inhibitory activity or better pharmacodynamic properties towards dipeptidyl peptidases.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a dipeptidyl peptidase inhibitor, a pharmaceutical composition and use thereof, which has better dipeptidyl peptidase inhibitory activity and/or better pharmacodynamic/pharmacokinetic properties.

In contrast, the technical scheme adopted by the invention is as follows:

a dipeptidyl peptidase inhibitor is a substituted pyrimidinedione compound shown as a formula (I) or a crystal form, a pharmaceutically acceptable salt, a prodrug, a tautomer, a stereoisomer, an isotopic variant, a hydrate or a solvate compound thereof,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸Each independently is hydrogen, deuterium, halogen or trifluoromethyl;

with the proviso that R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸At least one of which is deuterated or deuterium.

By adopting the technical scheme, the shape and the volume of deuterium in a drug molecule are basically the same as those of hydrogen, and if the hydrogen in the drug molecule is selectively replaced by deuterium, the original biological activity and selectivity of the deuterium-substituted drug can be generally kept. Meanwhile, the inventor proves that the combination of carbon and deuterium bonds is more stable than the combination of carbon and hydrogen bonds, and the absorption, distribution, metabolism, excretion and other properties of some medicines can be directly influenced, so that the curative effect, safety and tolerance of the medicines are improved.

Preferably, the deuterium isotope content of deuterium at the deuterated position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷And R¹⁸The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater 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%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

Preferably, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷And R¹⁸Preferably, at least one of R comprises deuterium, more preferably two of R comprises deuterium, more preferably three of R comprises deuterium, more preferably four of R comprises deuterium, more preferably five of R comprises deuterium, more preferably six of R comprises deuterium, more preferably seven of R comprises deuterium, more preferably eight of R comprises deuterium, more preferably nine of R comprises deuterium, more preferably ten of R comprises deuterium, more preferably eleven of R comprises deuterium, more preferably twelve of R comprises deuterium, more preferably thirteen of R comprises deuterium, more preferably fourteen of R comprises deuterium, more preferably fifteen of R comprises deuterium, more preferably sixteen of R comprises deuterium, more preferably seventeen of R comprises deuterium, more preferably eighteen of R comprises deuterium.

As a further improvement of the invention, R¹、R²And R³Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁴And R⁵Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁶、R⁷And R⁸Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁹Is deuterium.

As a further improvement of the invention, R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷And R¹⁸Each independently is deuterium or hydrogen.

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

in another preferred embodiment, the compound does not include non-deuterated compounds.

The invention also discloses a pharmaceutical composition which contains a pharmaceutically acceptable carrier and the dipeptidyl peptidase inhibitor, or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate, a stereoisomer, a prodrug or an isotopic variant thereof.

As a further improvement of the invention, the pharmaceutically acceptable carrier comprises at least one of a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersing agent, 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, pill, capsule, powder, granule, paste, emulsion, suspension, solution, suppository, injection, inhalant, gel, microsphere or aerosol.

Typical routes of administration of the pharmaceutical compositions of the present 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 compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: mixing a pharmaceutically acceptable carrier and the dipeptidyl peptidase inhibitor, or a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate thereof to form the pharmaceutical composition.

The compounds of the present invention have dipeptidyl peptidase enzyme inhibitory activity and are therefore expected to be useful as therapeutic agents for treating patients suffering from diseases or conditions that are treated by inhibiting dipeptidyl peptidase or by increasing the peptide substrate content thereof. Accordingly, one aspect of the present invention relates to a method of treating a patient suffering from a disease or disorder that is treated by inhibition of dipeptidyl peptidase, comprising administering to the patient a therapeutically effective amount of a compound of the present invention. Another aspect of the invention relates to a method of treating cardiovascular disease comprising administering to a patient a therapeutically effective amount of a compound of the invention. Another aspect of the present invention relates to a method of inhibiting dipeptidyl peptidase in a mammal in the treatment of hyperglycemia comprising administering to the mammal a dipeptidyl peptidase inhibiting amount of a compound of the present invention.

The present invention also discloses the use of a substituted pyrimidinedione compound as described above as a dipeptidyl peptidase inhibitor, i.e. the compounds of the present invention may be advantageously used as therapeutic agents for the treatment of conditions such as type II diabetes.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

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

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The invention also includes isotopically-labeled compounds, equivalent to those disclosed herein as the original compound. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，¹⁸F and³⁶and (4) Cl. The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, wherein isotopes or other isotopic atoms containing such compounds are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, 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 and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

Compared with the prior art, the invention has the beneficial effects that: the compound of the present invention has excellent inhibitory activity against dipeptidyl peptidase; the deuteration technology changes the metabolism of the compound in organisms, so that the compound has better pharmacokinetic parameter characteristics. In this case, the dosage can be varied and a depot formulation formed, improving the applicability; deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body is improved, and the medicine curative effect is improved; deuterium is used for replacing hydrogen atoms in the compound, so that certain metabolites can be inhibited, and the safety of the compound is improved.

Detailed Description

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

EXAMPLE 1 preparation of 2- { (6- [ (3R) -3-Aminopiperidin-1-yl)]-3-d 3-methyl-2, 4-dioxo-3, 4-di Pyrimidin-1 (2H) -yl) methyl } -4-fluorobenzonitrile (compound T-1)

The specific synthesis steps are as follows:

the method comprises the following steps: synthesis of 4-fluoro-2-methylbenzonitrile (Compound 2).

Adding compound 1(3.5g, 18.5mmol) and cuprous cyanide (2g, 22mmol) into a reaction bottle, adding DMF 100mL, heating to 140 ℃, stirring for 24 hours, detecting by TLC that the raw materials are completely reacted, adding water 60mL to quench the reaction, extracting three times by methyl tert-butyl ether, combining organic phases, washing by saturated sodium chloride, drying by anhydrous sodium sulfate, concentrating, purifying by column chromatography to obtain 1.16g of compound 2 with the yield of 46.4%,¹H NMR(300MHz，CDCl₃)δ7.62-7.59(m，1H)，7.04-7.02(m，1H)，7.00-6.96(m，1H)。

step two: synthesis of 2-bromomethyl-4-fluorobenzonitrile (Compound 3).

Adding compound 2(100mg, 0.74mmol), N-bromosuccinimide (NBS, 131g, 0.74mmol) and azobisisobutyronitrile (AIBN, 5mg, 0.029mmol) into a reaction flask, adding 5mL of carbon tetrachloride, carrying out reflux reaction for 3 hours, detecting that the raw materials are completely reacted by TLC, cooling to room temperature, filtering to remove solid impurities, and concentrating the filtrate to obtain 260mg of yellow solid compound 3 which is directly used for the next reaction.

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

Adding compound 4(600mg, 4.08mmol) into a reaction flask under the protection of nitrogen, adding DMF and DMSO to be 6: 120mL, cooling to 0 ℃, adding sodium hydrogen (165mg, 4.08mmol), stirring for 0.5 hour, adding lithium bromide (240mg, 0.276mmol), stirring for 0.5 hour, adding 3mL of DMF solution of compound 3(750mg, 4.08mmol) into the reaction solution, reacting for 1 hour at 0 ℃, stirring overnight at room temperature, detecting the reaction completion of the raw material by TLC, adding water for quenching, taking three times by using ethyl acetate, combining organic phases, washing by using saturated sodium chloride, drying by using anhydrous sodium sulfate, concentrating, purifying by column chromatography to obtain 120mg of compound 5, wherein the yield is 12.2%.

Step four: synthesis of 2- { (3-d 3-methyl-2, 4-dioxo-3, 4-dipyrimidin-1 (2H) -yl) methyl } -4-fluorobenzonitrile (Compound 6).

Adding compound 5(120mg, 0.429mmol), adding DFM: THF 1: 1(7mL), cooling to 0 ℃, adding sodium hydride (18mg, 0.45mmol), adding lithium bromide (22mg, 0.253mmol), stirring at room temperature for 20 minutes, adding d 3-iodomethane (120mg, 0.83mmol), stirring at room temperature for 2 hours, reacting overnight at 35 ℃, detecting by TLC that the raw material is completely reacted, adding water to quench, taking three times with ethyl acetate, combining organic phases, washing with saturated sodium chloride, drying with anhydrous sodium sulfate, concentrating, purifying by column chromatography to obtain 170mg of compound 6, yield-100%, LC-ms (apci): m/z 297(M +1)⁺。

Step five: synthesis of 2- { (6- [ (3R) -3-tert-Butoxycarbonylaminopiperidin-1-yl ] -3-d 3-methyl-2, 4-dioxo-3, 4-dipyrimidin-1 (2H) -yl) methyl } -4-fluorobenzonitrile (Compound 8).

Compound 6(170mg, 0.572mmol), compound 7(275mg, 1.38mmol), sodium bicarbonate (134mg, 1.6mmol) were added to a reaction flask in 16mL ethanol, the temperature was raised to 100 ℃ for 2 hours, TLC detection showed complete reaction of the starting materials, the ethanol was removed by concentration, water and ethyl acetate were added, extraction was performed three times with ethyl acetate, the organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to give 160mg of compound 8 with a yield of 60.8%.

Step six: synthesis of 2- { (6- [ (3R) -3-aminopiperidin-1-yl ] -3-d 3-methyl-2, 4-dioxo-3, 4-dipyrimidin-1 (2H) -yl } methyl) -4-fluorobenzonitrile (Compound T-1).

Adding the compound 8(160mg, 0.347mmol) into a reaction bottle, adding 5mL of ethanol for dissolving, adding 5mL of dioxane hydrochloride into the reaction bottle, reacting at room temperature for 5 hours, detecting by TLC that the raw material is completely reacted, and obtaining 62mg of compound 10 after column chromatography purification, wherein the yield is 49.6%, LC-MS (APCI): m/z 361(M +1)⁺，¹H NMR(300MHz，CDCl₃)δ7.97-7.94(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 bioactivity test

The protease inhibitory activity of DPP-IV inhibitors 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 by 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 assay

(1) DPP-IV assay

Solutions of test compounds were prepared in dimethylsulfoxide at various concentrations (final concentration ≦ 10mM) and then diluted in assay buffer containing: 20mM Tris, pH7.4, 20mM KCl and 0.1mg/mL BSA. Human DPP-IV (final concentration 0.1nM) was added to the dilution, pre-incubated for 10min at ambient temperature, and then the reaction was initiated with A-P-7-carboxamido-4-trifluoromethylcoumarin (AP-AFC; final concentration 10. mu.M). The total volume of the reaction mixture was 10-100. mu.L, depending on the assay format used (384 or 96 well plates). The reaction was monitored dynamically (excitation. lamda. 400 nm; emission. lamda. 505nm) for 5-10 minutes, or the endpoint was measured after 10 minutes. Inhibition constants were calculated from the enzyme process curves using standard mathematical models.

(2) FAP alpha assay

Solutions of test compounds were prepared in dimethylsulfoxide at various concentrations (final concentration ≦ 10mM) and then diluted in assay buffer containing: 20mM Tris, pH 7.4; 20mM KCl and 0.1mg/mL BSA. Human FAP α (final concentration 2nM) was added to the dilution, pre-incubated for 10min at ambient temperature, and then the reaction was initiated with A-P-7-carboxamido-4-trifluoromethylcoumarin (AP-AFC; final concentration 40 μ M). The total volume of the reaction mixture was 10-100. mu.L, depending on the assay format used (384 or 96 well plates). The reaction was monitored dynamically (excitation. lamda. 400 nm; emission. lamda. 505nm) for 5-10 minutes, or the endpoint was measured after 10 minutes. Inhibition constants were calculated from the enzyme process curves using standard mathematical models.

(3) PREP assay

Solutions of test compounds were prepared in dimethylsulfoxide at various concentrations (final concentration ≦ 10mM) and then diluted in assay buffer containing: 20mM sodium phosphate, pH7.4, 0.5mM EDTA, 0.5mM DTT, and 0.1mg/mL BSA. PREP (EC 3.4.21.26, from F.meningitidis, final concentration 0.2nM) was added to the dilutions. PRE and compound were preincubated for 10min at ambient temperature and then the reaction was initiated with Z-G-P-AMC (final concentration 10. mu.M). The total volume of the reaction mixture was 10-100. mu.L, 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. inhibition constants were calculated from the enzyme process curves using standard mathematical models.

(4) Tryptase assay

Solutions of test compounds were prepared in dimethylsulfoxide at various concentrations (final concentration ≦ 10mM) and then diluted in assay buffer containing: 100mM Hepes, pH7.4, 0.01% Brij35, and 10% glycerol. Tryptase (rhLung. beta. at a final concentration of 0.1nM) was added to the dilutions and preincubated with compound for 10min at ambient temperature. The enzymatic reaction was initiated with 25. mu.M MZ-lys-SBzl and 400. mu.M DTNB. The reaction mixture was made in a total volume of 100. mu.L in Costar A/296 well plates. The reaction was monitored colorimetrically (λ 405nm) for 10 minutes. Inhibition constants were calculated from the enzyme process curves using standard mathematical models.

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

2. Metabolic stability evaluation

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

Preparing a stock solution: an amount of the powder of example 1 was weighed out precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

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

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L 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 are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere 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 measurement results of metabolic stability of the compound of example 1

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

3. Pharmacokinetic experiment of rat

Purpose of the experiment: study after administration of Trelagliptin, the example compound, to rats, the pharmacokinetic behavior of the compound of the invention was examined.

Experimental animals:

species and strain: SD rat grade: SPF stage

Sex and amount: male, 6

Body weight range: 180 to 220g (actual weight range 187 to 197g)

The source is as follows: shanghai Xipulbikai laboratory animals Co., Ltd

Experimental and animal certification numbers: SCXK (Shanghai) 2013-0016

The experimental process comprises the following steps:

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

Rats, males, weighing 187-197 g, were randomized into 2 groups, fasted overnight but with free access to water starting the afternoon of the day before the experiment, and given food 4h after administration. Group A is given 3mg/kg Trelagliptin, group B is given 3mg/kg compound of the embodiment, respectively, after administration, 15min, 30min, 1, 2, 3, 5, 8, 10h, 100-200L blood is taken from orbital veins of rats, the blood is placed in an Eppendorf tube of 0.5mL anticoagulated by EDTA-K2, the mixture is immediately mixed, after the anticoagulation, the tube is gently inverted and mixed uniformly for 5-6 times as soon as possible, the blood is taken and placed in an ice box, a blood sample is centrifugally separated from plasma at 4000rpm, 10min and 4 ℃ within 30min, and the plasma is immediately stored at-20 ℃ after all the plasma is collected. Plasma concentrations were determined in plasma at each time point after sample collection at all time points.

According to the obtained mean plasma concentration-time data after administration, Winnonin software is adopted to calculate pharmacokinetic related parameters of i.g. Trelagliptin (3mg/kg) and the example compound (3mg/kg) after administration of the i.g. Trelagliptin and the example compound to male SD rats respectively according to a non-atrioventricular statistical moment theory.

Experiments show that compared with Trelagliptin, the compound has better activity than Trelagliptin and excellent pharmacokinetic property, so the compound is more suitable to be used as a compound for inhibiting dipeptidyl peptidase and is further suitable to be used for preparing medicines for treating type II diabetes.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A compound, or a pharmaceutically acceptable salt, tautomer thereof:

2. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of claim 1, or a pharmaceutically acceptable salt, tautomer thereof.

3. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of dipeptidyl peptidase mediated diseases.

4. The use according to claim 3, wherein the disease is type II diabetes.