CN115894456A

CN115894456A - Deuterated pyrazole aminopyrimidine compound, pharmaceutical composition and application

Info

Publication number: CN115894456A
Application number: CN202211416671.7A
Authority: CN
Inventors: 张广永; 蔡家昌; 徐秀秀; 孔亚运
Original assignee: Yaokang Zhongtuo Jiangsu Pharmaceutical Technology Co ltd
Current assignee: Yaokang Zhongtuo Jiangsu Pharmaceutical Technology Co ltd
Priority date: 2022-11-13
Filing date: 2022-11-13
Publication date: 2023-04-04

Abstract

The invention discloses a compound shown in a formula I, or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, a pharmaceutical composition and application. The compound shown in the formula I has good inhibitory activity on LRKK2 kinase and has good therapeutic effect on cancer.

Description

Deuterated pyrazole aminopyrimidine compound, pharmaceutical composition and application

Technical Field

The invention belongs to the field of innovative pharmaceutical chemistry, and relates to a deuterated pyrazolylaminopyrimidine compound, a pharmaceutical composition and application.

Background

Parkinson's Disease (PD) is a degenerative disease of the central nervous system affecting about 5% of the population over the age of 80. It is characterized by the pathological manifestations of gradual loss of dopaminergic neurons and dopamine secretion in the substantia nigra. Currently, approved drugs for the treatment of parkinson's disease, including dopamine agonists and dopamine precursors, only ameliorate dyskinesias in the early stages of the disease, but not the non-motor symptoms of PD, including dementia, depression, sleep disorders, and sometimes psychosis. Therefore, there is an urgent need to develop effective drugs to inhibit the disease progression of PD. Multiple gene studies have shown that familial and spontaneous PD development is associated with multiple genes, with LRRK2 gene mutations being the most common cause of familial PD development. In addition, the LRRK2 gene polymorphism is related to spontaneous Parkinson's disease in terms of onset age, symptoms and pathology. In addition, GWAS findings indicate that LRRK2 is a risk factor for PD development, suggesting that targeting LRRK2 is a potentially effective approach to treating parkinson's disease. Currently, a number of LRRK2 mutations are reported, the most common of which are autosomal dominant G2019S mutations located in the kinase domain activation loop (a-loop), which result in LRRK2 over-activation, closely linked to the development of familial and spontaneous PD. Therefore, LRRK2 is potentially useful for developing treatments or prevention of PD. Some LRRK2 inhibitors have been reported, of which the compound GNE-0877 is in early clinical studies, but no drug is yet on the market. The development of novel LRRK2 inhibitors is of great significance for the prevention or treatment of PD and the study of LRRK2 biological effects.

Deuterated drugs refer to replacement of a portion of the hydrogen atoms in a drug molecule with deuterium. Because deuterium is close to hydrogen in shape and volume in a drug molecule, deuterated drugs generally retain the biological activity and selectivity of the original drug. Because the C-D bond is more stable than the C-H bond, the C-D bond is less prone to break and the half-life period of the deuterated drug is prolonged in the chemical reaction process. Since 2000, deuteration strategies have been widely used in drug research.

Disclosure of Invention

The invention provides a compound shown as a formula I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, wherein the structure of the compound is as follows:

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the invention provides application of a compound shown as a formula I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in preparation of an LRRK2 kinase inhibitor.

The invention provides application of a compound shown as I or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in preparing a medicament for treating and/or preventing Parkinson's disease.

In some embodiments, the parkinson's disease is parkinson's disease associated with an aberrant LRRK2 kinase enzyme activity.

The invention provides a pharmaceutical composition, which contains a compound shown as a formula I, or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or auxiliary material.

In the pharmaceutical composition, the compound shown in formula I, or pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is used in an amount of therapeutically effective amount.

The invention provides application of a pharmaceutical composition in preparing an LRRK2 kinase inhibitor.

The invention provides application of a pharmaceutical composition in preparing a medicament for treating and/or preventing Parkinson's disease.

In some embodiments, the parkinson's disease is parkinson's disease associated with aberrant LRRK2 kinase enzyme activity.

The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, antiadherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

The pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention may also be in a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry preparations which can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free forms of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting free forms of such compounds with a sufficient amount of an acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonates or bicarbonates), phosphoric acid (forming phosphates, monohydrogen phosphates, dihydrogen phosphates, sulfuric acid (forming sulfates or bicarbonates), hydroiodic acid, phosphorous acid, and the like), and salts of organic acids including such acids as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, and the like, and salts of amino acids (such as arginine, and the like), and salts of organic acids such as glucuronic acid.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

The term "isomers" refers to compounds having the same chemical formula but different arrangements of atoms.

The term "metabolite" refers to a pharmaceutically active product produced by the in vivo metabolism of a compound of formula I or a salt thereof. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a method comprising contacting a compound of the present invention with a mammal for a period of time sufficient to obtain a metabolite thereof.

Identification of metabolites typically occurs by preparing a radiolabeled isotope of a compound of the invention, parenterally administering it at a detectable dose (e.g., greater than about 0.5 mg/kg) to an animal, such as a rat, mouse, guinea pig, monkey, or a human, allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolating its conversion products from urine, blood or other biological samples. These products are easy to isolate because they are labelled (others are isolated by using antibodies capable of binding to epitopes present in the metabolite). Metabolite structure is determined in a conventional manner, e.g., by MS, LC/MS or NMR analysis. Typically, analysis of metabolites is performed in the same manner as conventional drug metabolism studies well known to those skilled in the art. Metabolite products are useful in assays for the administration of therapeutic doses of the compounds of the invention, provided that they are not otherwise detectable in vivo. The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compound may be labeled with a radioisotope, such as tritium ( ³ H) Iodine-125 ( ¹²⁵ I) Or C-14 ( ¹⁴ C) In that respect All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

In addition to salt forms, the compounds provided herein also exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula I) is a prodrug within the scope and spirit of the present invention. For example, compounds containing a carboxyl group may form physiologically hydrolyzable esters that act as prodrugs by hydrolyzing in vivo to give the compounds of formula I themselves. The prodrugs are preferably administered orally, since hydrolysis in many cases takes place mainly under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood.

The positive progress effects of the invention are as follows:

(1) The compound has good inhibitory activity and selectivity on LRRK2 kinase.

(2) The compound of the invention has the advantages of obviously improved metabolic stability, higher oral bioavailability, prolonged half-life and capability of reducing the dosage of single administration.

(3) The compound has good therapeutic action on the Parkinson disease.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1: synthesis of Compound 1

The method comprises the following steps: synthesis of Compound b

Starting material a (10g, 78.6mmol) was dissolved in anhydrous DMF (50 mL) at 0 ℃ and NaH (4.72g, 118.0mmol, 60%) was added to the above solution in portions. The reaction was transferred to room temperature and stirred at room temperature for 2h. Then, compound g (21.3 g,118.0 mmol) was added to the above suspension, and the reaction mixture was reacted at room temperature overnight. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give compound b (11.5g, 80%). MS (ESI, M/z): 228 (M) ⁺ +1).

Step two: synthesis of Compound c

To a solution of compound b (107mg, 0.47mmol) in N, N-dimethylformamide (15 mL) were added potassium hydroxide (105.5 mg, 1.88mmol) and iodine (239mg, 0.94mmol), the reaction was carried out at room temperature for 3 hours, TLC was used to monitor completion of the reaction, saturated sodium sulfite solution was added to quench the reaction, the aqueous phase was extracted with ethyl acetate (10 mL. Times.2), washed with water (20 mL. Times.2), washed with saturated common salt (20 mL) and dried over anhydrous sodium sulfate, and concentrated column chromatography was used to isolate and purify iodo compound b (91mg, 55%). MS (ESI, M/z): 354 (M) ⁺ +1).

Step three: synthesis of Compound d

Sodium acetate (97.9 mg, 0.72mmol) was added to a deuterated acetic acid solution (8 mL) of compound c (127mg, 0.36mmol), and the mixture was added dropwise over 2 hours, reacted at room temperature for 24 hours, checked by TLC for completion of the reaction, concentrated under reduced pressure, and purified by column chromatography to give compound d (55mg, 67%). MS (ESI, M/z): 229 (M) ⁺ +1).

Step four: synthesis of Compound e

Compound d (2.29g, 10 mmol) was dissolved in methanol (10 mL), and ammonia/methanol solution (4M, 20mL) was added to the above solution, and the reaction was stirred at 50 ℃ for 24 hours in a sealed tube. After the reaction is completed, the solvent is removed under reduced pressure, and the compound e (1.5g, 70%) is obtained by column chromatography separation and purification. MS (ESI, M/z): 214 (M) ⁺ +1).

Step five: synthesis of Compound 1

Compound e (1.5g, 7.04mmol) was dissolved in methanol (20 mL), pd/C (150 mg) was added, and the temperature was raised to 40 ℃ under hydrogen, and the reaction was allowed to proceed overnight. After the reaction was completed, it was cooled to room temperature, filtered with celite, concentrated under reduced pressure, and purified by column chromatography to give compound 1 (1.1g, 85%). MS (ESI, M/z): 184 (M) ⁺ +1).

Example 2: synthesis of Compound I

The method comprises the following steps: synthesis of Compound 3

Compound 1 (250mg, 1.37mmol) was dissolved in 2-methoxyethanol (5 mL), compound 2 (290mg, 1.37mmol) and TFA (290mg, 1.37mmol) were added to the above solution,the reaction temperature was raised to 70 ℃ and stirred for 30min. Then, the reaction mixture was cooled to room temperature, ice water (10 mL) was added to the reaction mixture to quench the reaction, saturated sodium bicarbonate solution was added to adjust pH to 8, ethyl acetate extraction (10 mL. Times.3) was performed, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to column chromatography to obtain Compound 3 (250mg, 51%). MS (ESI, M/z): 359 (M) ⁺ +1).

Step two: synthesis of Compound I

A solution of compound 3 (250mg, 0.7 mmol) in phosphorus oxychloride (5 mL) was warmed to 90 ℃ and stirred for 1h. The reaction solution is cooled to room temperature, and the residual phosphorus oxychloride is removed under reduced pressure. To the residue was slowly added ice water and saturated sodium bicarbonate adjusted to pH 8. Extraction with ethyl acetate (10 mL. Times.3), combination of the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtration, concentration, and column chromatography purification yielded the title compound I (95mg, 40%). ¹ H NMR(500MHz,DMSO)δ9.18(s,1H),8.14(s,1H),7.10(s,1H),2.91(s,3H),2.22(s,3H),1.94(s,6H).MS(ESI,m/z):341(M ⁺ +1).

Example 3: LRRK2 kinase inhibitory Activity assay

And (3) carrying out LRRK2 kinase inhibition activity test by adopting a microfluidic capillary electrophoresis analysis method. LRRK2 standard enzymatic reaction 5 μ L2 × enzyme contained: 10nM G2019S, 1. Mu.M FAM-LRRKtide (5 FAMGAGRLGRDKYKTLRQIRQ-CONH 2), 130. Mu.M ATP,25mM Tris, pH 8.0,5mM MgCl ₂ 0.01% Triton X-100,2mM DTT,5 mM. Beta. -glycerophosphate, 5mM NaF, 100. Mu.M Na ₃ VO ₄ And 1 Xprotease inhibitor cocktail (Calbiochem), 5. Mu.L of 2 XATP was added to the above enzymatic reaction solution to start the reaction. After incubation for 120 minutes at room temperature, the reaction product and substrate were separated using a 12-sipper microfluidic chip running on a Caliper LC 3000. Separation of product from substrate the voltage and pressure were optimized by selecting the software using Caliper. The separation buffer contained 100mm HEPES, pH 7.2,0.015% Brij-35,0.1% coating reagent 3,10mM EDTA, DMSO solution. The downward voltage used in the separation conditions was-500V, the upward voltage was-2350V, and the screening pressure was-1.4 psi. Fluorescence of the product and substrate was excited at 488nm and detected at 530 nm. Extent of substrate conversion Using HTS WellThe electropherograms drawn by Analyzer software were calculated.

TABLE 1 inhibitory Activity of test Compounds on LRRK2 kinase

Name (R)	LRKK2(Ki nM)
		I	0.035
M-4076	0.100

As shown in table 1, compound I has significant inhibitory activity against LRRK2 kinase and is superior to the positive control GNE-0877.

Example 4: PD mouse neuron injury effect test

1-methyl-4-phenyltetrahydropyridine (MPTP) is a Dopamine (DA) nervotoxin that causes clinical symptoms similar to PD. MPTP can reliably and stably damage DA energy pathways of the nigrostriatal striatum after being systemically administered. 1-MPTP was dissolved in PBS, and male C57 mice (20-25 g in weight) were intraperitoneally injected with 30mg/kg once a day for 5 days. The behavioral phenotype of the mouse is observed, and the mouse has dyskinesia phenotypes such as obvious reduction of overall locomotor activity, impaired balance and coordination ability, bradykinesia, resting tremor, abnormal gait and the like, which indicates that the subacute PD model is successfully modeled. The compounds of the invention were solubilized with PEG400 and dissolved in PBS. 4 to 6h prior to MPTP administration, mice were administered daily for 7 days by subcutaneous injection of Compound I (10 mg/kg,30mg/kg and 60 mg/kg) and M-4076 (60 mg/kg). On the day of the last injection, mice were sacrificed and whole brains were taken, fixed and paraffin-embedded and sectioned, the section position was selected as a black section, and the same position was taken for each mouse. Slicing and dewaxing to water, repairing antigen, permeabilizing and sealing; adding primary anti-dilution solution and incubating overnight; adding a second antibody diluent for incubation, developing with a DAB developing solution, counterstaining with hematoxylin, dehydrating and sealing. Tyrosine Hydroxylase (TH) neurons (in brown-yellow color) were observed and counted under a microscope. Surviving dopaminergic neurons were represented as TH + neurons.

The research result shows that compared with the normal control group, the number of the TH + neurons of the substantia nigra in the PD model group is obviously reduced, and the number of the TH + neurons of the substantia nigra in the high, medium and low dose administration group and the M-4076 administration group of the compound I is obviously higher than that in the PD model group. And when the administration dose is 60mg/kg simultaneously, the number of TH + neurons of substantia nigra of PD mice in the compound I administration group is higher than that in the compound M-4076 administration group, which shows that the compound I has better capability of improving the neuron damage of the PD mice than M-4076.

Example 5: human liver microsome stability test

After constructing 200. Mu.L NADPH-containing generating system, 1. Mu.L inhibitor was added to the system, then 5. Mu.L liver microsomal enzyme was added, mixed well and incubated at 37 ℃. Samples were collected at 5 time points set between 0-60 min. After the reaction was completed, 400. Mu.L of glacial methanol containing an internal standard was added to terminate the reaction. And performing linear regression on the natural logarithm of the percentage residual quantity of each time point to the time to calculate the slope k. Half life T in vitro _1/2 = 0.693/k. Clearance rate in liver microsome (CL) =0.693/T _1/2 * Incubation (mL)/liver microsomes (mg).

Research results show that the metabolic stability of the compound I is obviously improved and is obviously superior to that of M-4076.

Example 6: detection of pharmacokinetic Properties of test Compounds

Male SD rats are selected for oral administration (10 mg/kg) or intravenous injection (2 mg/kg), blood is continuously taken from the fundus venous plexus and placed in an EP tube containing heparin after administration, the blood is centrifuged, the upper layer plasma is taken for LC-MS/MS analysis, and pharmacokinetic parameters are calculated by adopting WinNonlin software according to the blood concentration-time data obtained by testing, so that the oral bioavailability is calculated.

Research results show that the M-4076 has 40 percent oral bioavailability, a half-life of 1h and a short half-life; while the oral bioavailability of the compound I is improved to 78 percent, and the half life is prolonged to 3 hours.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, having the structure:

2. a pharmaceutical composition, which comprises a therapeutically effective amount of the compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or adjuvant.

3. Use of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, or a pharmaceutical composition according to claim 2, in the preparation of an LRRK2 kinase inhibitor.

4. Use of a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, or a pharmaceutical composition as defined in claim 2, for the manufacture of a medicament for the treatment and/or prevention of parkinson's disease.

5. The use of claim 4, wherein the Parkinson's disease is Parkinson's disease associated with an abnormal LRRK2 kinase enzyme activity.