CN116283927A - Pyrimidine amino aryl alanine derivatives and application thereof as leucine-rich repeat kinase 2 inhibitor - Google Patents

Abstract

The invention discloses a pyrimidine amino aryl alanine derivative and application thereof as a leucine-rich repeat kinase 2 inhibitor, the derivative not only has a good inhibition effect on LRRK2 activity, but also has the structural characteristic of being actively transported by amino acid transport proteins on BBB, and the capability of increasing the drug entering the center, thus being a potential therapeutic drug for treating central nervous degenerative diseases (such as Parkinson's disease).

Description

Pyrimidine amino aryl alanine derivatives and application thereof as leucine-rich repeat kinase 2 inhibitor

Technical Field

The invention belongs to the technical field of chemical medicaments, and relates to pyrimidine amino aryl alanine derivatives and application thereof as leucine-rich repeat kinase 2 inhibitors.

Background

Parkinson's Disease (PD) is a second most chronic neurodegenerative disease that is highly advanced in middle-aged and elderly, second only to alzheimer's disease. At present, people have low cognition, low diagnosis rate and low diagnosis rate on the disease, and can not cure the disease, and patients can show motor nervous system disorders such as tremors, limb stiffness, hypokinesia, gait abnormality and the like, and non-motor symptoms such as hyposmia, sleep disorder, constipation and the like for life. The existing medicines can only relieve symptoms per se to different degrees, and cannot control disease progression. The current clinical commonly used medicines can not meet the requirements of the existing patients with parkinsonism middle and late stages, and a medicine capable of preventing parkinsonism and biochemical degeneration is needed. Disease modification therapy is the current mainstay of developing therapeutic drugs for parkinson's disease, which can affect the initial trigger of neuronal degeneration and promote neuronal compensatory responses or reduce pathological transmission and progression. Currently, the main current research considers that the aggregation of alpha-Syn in Louis's small body (LB) is an important cause of the pathogenesis of Parkinson's disease, and the reduction of the aggregation of alpha-Syn is a potential method for treating Parkinson's disease.

Leucine-rich repeat kinase 2 (Leucine-rich repeat kinase 2, LRRK2) blocks chaperone-mediated autophagy, resulting in alpha-syn not being degraded and resulting in toxicity. LRRK2 is involved in α -syn mediated neurotoxicity, LRRK2 induces mitochondrial damage, endolysosomal dysfunction through an oxidative mechanism, inducing parkinson's disease progression. LRRK2 kinase inhibitors can alleviate pathological lesions in parkinson's disease models, improving motor dysfunction in patients. The safety and tolerability of two novel LRRK2 kinase inhibitors, DNL201 and DNL151, phase IB clinical trials were successful, and DNL151 has developed a phase IIb/III registration clinical study. Therefore, the development of the LRRK2 small molecule inhibitor is one of the research directions which have the highest potential for developing the Parkinson therapeutic drugs at present.

Therefore, the development of potent inhibitors of LRRK2 kinase as well as mutated LRRK2 kinase is an important approach to the current treatment of neurodegenerative diseases. The invention aims to invent a compound which can highly inhibit LRRK2 kinase, thereby further inventing a medicament which can well treat neurodegenerative diseases.

Patent US8802674B2 discloses that pyrimidine aminobenzamide compounds of Gentech company are inhibitors of LRRK2, and the chemical structural general formula is shown as follows. And J.Med. Chem.2012, 55, 9416-9433 discloses a preferred compound GNE-7915 of the general structure of the company Gentech, the chemical structure of which is shown below,

although GNE-7915 has better LRRK2 inhibition activity and certain blood brain barrier permeability, animal experiments still find that the GNE-7915 has obvious toxic and side effects on peripheral tissues (such as kidneys and lungs) and stops in phase 1 clinical development, which indicates that a better blood brain barrier permeability compound is required to be searched for aiming at the target, so that effective administration of the brain can be ensured, and meanwhile, the drug concentration and toxic and side effects of the peripheral tissues can be reduced.

The blood-brain barrier (BBB) is composed of components such as brain microvascular endothelial cells, endothelial cell tight junctions, glial cells, astrocytes, pericytes, basal membranes, etc., and generally only allows gas molecules and fat-soluble molecules with smaller relative molecular mass to pass through, so that most drugs for treating central nervous system diseases cannot pass through the BBB to achieve effective therapeutic concentration in the brain. Yet another important feature of the BBB is the inclusion of a variety of substance transporters on the brain capillary endothelial cell membrane, associated with transmembrane transport of brain nutrients, one of which is the L-type amino acid transporter (L-type aminotransporters, LATs) in the main member, which is closely related to drug transport, wherein the most studied LAT1 can transport amino acid central nervous drugs similar in structure to its substrate, such as: anti-parkinsonism levodopa; the structural characteristics of dopamine limit the blood brain barrier penetration rate, and the levodopa modified by the amino acid structure is a LAT1 substrate, and can be transported through the blood brain barrier by the LAT1 mediation to enter the center to exert curative effect. Gabapentin, a drug used in the treatment of neuralgia and epilepsy, is also a drug possessing an amino acid structure that is transported into brain tissue by LAT 1.

Disclosure of Invention

Accordingly, the present invention is directed to pyrimidine amino aryl alanine derivatives and their use as leucine-rich repeat kinase 2 inhibitors.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. pyrimidine amino aryl alanine derivatives, or optical isomers thereof, or prodrugs thereof, or pharmaceutically acceptable salts thereof, or hydrates, solvates, N-oxides and deuterated matters thereof, wherein the structures of the derivatives are shown as a general formula (1) or a general formula (2):

in the general formula (1),

R ₁ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 An alkyl group;

R ₂ selected from H, F, cl, br, I;

n is selected from 0,1,2;

y is selected from-O-or-N-;

R ₃ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl and the usual protecting groups for various O or N;

R ₄ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl and the various common N protecting groups;

alternatively, R ₃ And R is R ₄ Is a group taken together selected from-C (=O) -, -CH ₂ -C(＝O)-、

-C(＝O)-CH ₂ -、-(CH ₂ -CH ₂ )-、-C(＝O)-C(＝O)-；

In the general formula (2),

R ₅ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 An alkyl group;

x is selected from-C-, or-N-;

R ₆ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl and various common O protecting groups;

R ₇ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl groups and the various N protecting groups commonly found.

Preferably, the derivative is

7 '-fluoro-4' -methoxy-5'- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -1',3 '-dihydro-spiro [ imidazole-4, 2' -indene ] -2,5-dione,

4' -methoxy-5' - ((4- (methylimine) -5- (trifluoromethyl) pyrimid-2-yl) amino) -1',3' -dihydropipiro [ imidozolidine-4, 2' -indene ] -2,5-dione, having the following chemical formula:

preferably, the derivative is

(S) -2-amino-3- (4- ((4- ((2-hydroxyethyl) amino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -3-methoxyphenyl) propionic acid, (S) -2-amino-3- (4- ((2-hydroxyyethyl) amino) -5- (trifluormethyl) pyrimid-2-yl) amino) -3-methox yphenyl) propionic acid having the formula:

preferably, the derivative is

(S) -2-amino-3- (5- ((4- ((2-hydroxyethyl) amino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -4-methoxypyridin-2-yl) propanoic acid,

(S) -2-amino-3- (5- ((4- ((2-hydroxyyl) amino) -5- (trifluoromethyl) pyrimid-2-yl) amino) -4-methox pyridin-2-yl) propanic acid, the chemical structural formula of which is as follows:

2. pyrimidine amino aryl alanine derivatives, or optical isomers thereof, or prodrugs thereof, or pharmaceutically acceptable salts thereof, or hydrates, solvates, N-oxides and deuterated substances thereof are used as leucine-rich repeat kinase 2 inhibitors.

3. The pyrimidine amino aryl alanine derivative, or an optical isomer thereof, or a prodrug thereof, or pharmaceutically acceptable salt thereof, or hydrate, solvate, N-oxide and deuterated compound thereof are applied to the preparation of medicines for treating or preventing parkinsonism.

4. The pyrimidine amino aryl alanine derivative, or an optical isomer thereof, or a prodrug thereof, or pharmaceutically acceptable salt thereof, or hydrate, solvate, N-oxide and deuterated compound thereof are applied to the preparation of medicines for treating or preventing chronic neurodegenerative diseases.

5. Use of a pyrimidine amino aryl alanine derivative, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated product thereof, in the preparation of a medicament for inhibiting the activity of leucine-rich repeat kinase 2 to prevent and/or treat diseases.

6. A pharmaceutical composition or formulation comprising the foregoing pyrimidine amino aryl alanine derivative, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated thereof.

Preferably, the composition further comprises pharmaceutically acceptable auxiliary materials, auxiliary agents or carriers.

The invention has the beneficial effects that:

applicants have sought potential therapeutic agents for parkinson's disease by introducing amino acid fragments into the structure of existing leptin compounds, which aim to increase the active transport of the compound into the hub through amino acid transporters on the BBB while maintaining inhibitory activity against LRRK 2.

The invention provides an amino acid derivative with a pyrimidine amino aryl structure, which not only has a good inhibition effect on the activity of LRRK2, but also has the structural characteristic of active transportation by amino acid transport proteins on BBB, and can increase the capacity of drug entering the center, thus being a potential therapeutic drug for treating central nervous degenerative diseases (such as Parkinson's disease).

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention is illustrated in the following drawings.

FIG. 1 is a synthetic route for F001 in example 1;

FIG. 2 is a synthetic route for F002 in example 2;

FIG. 3 is a synthetic route for F003 in example 3.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Definition and description

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure solution or in a suitable inert solvent. Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts.

Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of 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 both.

The term "therapeutically effective amount" refers to an amount of a compound of formula (la) sufficient to be therapeutically effective when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending on the particular activity of the therapeutic agent used, the age of the patient, the physiological condition, the presence of other disease states, and the nutritional condition. In addition, other medications that a patient may be receiving will affect the determination of a therapeutically effective amount of the therapeutic agent to be administered.

The term "treatment" means any treatment for a disease in a mammal, including: (i) Preventing the disease, i.e. causing no development of clinical symptoms of the disease; (ii) inhibiting the disease, i.e., arresting the development of clinical symptoms; and/or (iii) alleviating the disease, i.e., causing regression of the clinical symptoms.

The term "pharmaceutically acceptable adjuvants, adjuvants or vehicles" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Such media and agents are well known in the art for use with pharmaceutically active substances. The use thereof in therapeutic compositions is contemplated, except that any conventional medium or agent is incompatible with the active ingredient. Supplementary active ingredients may also be incorporated into the compositions.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention. Any intermediate or compound 1 that can be obtained in the synthetic route by other reaction conditions is considered an alternative to the invention.

Example 1: synthesis of F-001

The synthetic route is shown in FIG. 1.

The method comprises the following specific steps:

step 1 preparation of intermediate 2

2, 4-dichloro-5-trifluoromethylpyrimidine (5 g,23.04 mmol), triethylamine (4.66 g,46.08 mmol) were added to methanol (50 mL) and cooled to-80 ℃. After completion of the cooling, methylamine hydrochloride (1.56 g,23.04 mmol) was added to the reaction system in portions, and the mixture was reacted at-80℃for 2 hours. TLC monitored the progress of the reaction and after completion of the reaction was warmed to room temperature. The solvent was removed by concentration under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=10/1)Volume ratio) to give intermediate 2 (1.34 g,27.5%, white solid), [ M+H ]] ⁺ ＝212。

Step 2 preparation of intermediate 4

To a 250mL single-necked flask, intermediate 3 (10.0 g,42.0 mmol) and 50mL of methanol were added, and the solution was stirred at room temperature. Sodium methoxide (2.5 g,46.0 mmol) was then weighed and dissolved in 50mL of methanol (exothermic), sodium methoxide solution was added to the reaction solution of intermediate 3, which was then washed with 30mL of methanol and added to the reaction flask, stirred at room temperature for 1.5 hours, solids were precipitated, TLC monitored that the starting material was not reacted, the reaction flask was placed in 45℃oil bath and stirred for 1 hour, TLC monitored that the starting material was reacted. The reaction solution was concentrated to dryness under reduced pressure, and extracted with ethyl acetate. The extracted organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. Petroleum ether was added to the concentrated residue and beaten for 2 hours. Filtration and drying gave intermediate 4 (9.65 g,91.8% as an off-white solid).

Step 3 preparation of intermediate 5

120mL of the reaction mixture was capped, intermediate 4 (4.0 g,16.0 mmol), palladium acetate (150 mg,0.67 mmol), N, N-Dimethylformamide (DMF)) 12mL, triethylamine (TEA) (6.67 mL,48.0 mmol) and methyl acrylate (2.88 mL,32.0 mmol) were added, and the mixture was warmed to 100℃and capped for 18 hours. LC-MS and TLC monitoring, reaction was completed. The mixture was filtered through celite, and the filtrate was concentrated to dryness at 50℃under reduced pressure. To the concentrated residue was added 200mL of water, and extracted with ethyl acetate (100 mL. Times.3). The extracted organic phase was washed with saturated NaCl, dried over anhydrous sodium sulfate, and concentrated by filtration to give 4.9g of a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=5/1, volume ratio) to give intermediate 5 (3.2 g,78.3%, yellow solid), [ m+h] ⁺ ＝256。

Step 4 preparation of intermediate 6

To a 250mL single-necked flask was added intermediate 5 (3.2 g,12.5 mmol), 10% Pd/C (350 mg), tetrahydrofuran (65 mL), and the mixture was reacted at 40℃for 72 hours by replacing three times with hydrogen. LC-MS monitoring, reaction was complete. The mixture is filtered by diatomite, and the filtrate is concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=3/1, volume ratio) to give intermediate 6 (2.3 g, 80.7%)Yellow solid) [ m+h ]] ⁺ ＝228。

Step 5 preparation of intermediate 7

To the reaction tube was added intermediate 6 (2.3 g,10.1 mmol) and 15mL of methanol, and the solution was stirred at room temperature. Lithium hydroxide (720 mg,30.3 mmol) was weighed and dissolved in 15mL of water, and added to the above reaction tube, and reacted at room temperature after the addition. LC-MS and TLC monitoring, adding formic acid water solution with mass concentration of 5% to adjust pH to 2, extracting the reaction solution with dichloromethane (50 mL×5), and adding small amount of methanol each time. The extracted organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to afford intermediate 7 (2.1 g,97.3%, gray solid) which was directly taken to the next step. [ M+H ]] ⁺ ＝214。

Step 6 preparation of intermediate 8

48mL of a tube was capped, intermediate 7 (2.1 g,9.8 mmol) and trifluoromethanesulfonic acid (10 mL) were added, and the tube capped was reacted at 100℃for 5 hours. TLC and LC-MS monitoring, reaction was completed. Cooling in ice water bath to room temperature, adding saturated NaHCO ₃ The pH was adjusted to neutral, and the reaction solution was extracted 4 times with methylene chloride. The extracted organic phase was washed with saturated NaCl, dried over anhydrous sodium sulfate, filtered, and concentrated to give intermediate 8 (900 mg,46.8%, black solid), [ M+H ]] ⁺ ＝196。

Step 7 preparation of intermediate 9

A100 mL single-necked flask was taken, intermediate 8 (900 mg,4.6 mmol) and 20mL of methanol were added, and the solution was stirred at room temperature. Weighing NaBH ₄ (520 mg,13.8 mmol) was slowly added to the above reaction flask, and a large amount of gas was evolved, and the reaction was completed at room temperature for 2 hours. LC-MS and TLC monitoring, reaction was completed. Adding formic acid water solution with mass concentration of 5% to adjust pH to 2, and then using saturated NaHCO ₃ The pH was adjusted to neutral, and the reaction mixture was extracted with EA (50 mL. Times.4). The extracted organic phase was washed with saturated NaCl, dried over anhydrous sodium sulfate, filtered and concentrated to give crude intermediate 9 (805 mg,88.5%, black oil) which was directly taken into the next step. [ M+H ]] ⁺ ＝198，[M+H-18] ⁺ ＝180。

Step 8 preparation of intermediate 10

To a mixture of intermediate 9 (805 mg,4.1 mmol) and toluene (20 mL) was added p-toluenesulfonic acid monohydrateThe mixture (1.5 g,8.16 mmol) was reacted at 100℃for 2h after the addition. LC-MS and TLC monitoring, reaction was completed. 50mL of EA was added to the reaction solution followed by saturated NaHCO ₃ The pH was adjusted to neutral, the mixture was separated, and the aqueous phase was extracted with EA (50 mL. Times.3). The extracted organic layer was washed with saturated NaCl, dried over anhydrous sodium sulfate, filtered, and concentrated to give intermediate 10 (460 mg,62.8%, black oil) which was directly taken into the next step. [ M+H ]] ⁺ ＝180。

Step 9 preparation of intermediate 11

Intermediate 10 (460 mg,2.6 mmol) was added to a mixture of dioxane (10 mL) and water (6 mL) and stirred at room temperature. Sequentially adding NaHCO into the mixed solution ₃ (650 mg,7.7 mmol) and CbzCl (benzyloxycarbide chloride, 550. Mu.l, 3.8 mmol) were reacted at room temperature for 3h after the addition. LC-MS and TLC monitoring, reaction was completed. To the reaction solution was added 20mL of water, and the product was extracted with EA (30 mL. Times.2). The extracted organic layer was washed with saturated NaCl, dried over anhydrous sodium sulfate, filtered, and concentrated to give 880mg of crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=10/1, volume ratio) and dried to give intermediate 11 (330 mg,41.0%, yellow oil). [ M+H ]] ⁺ ＝314，[M+H-44] ⁺ ＝270。

Step 10 preparation of intermediate 12

Intermediate 11 (240 mg,0.76 mmol) was dissolved in DCM (5 mL) followed by the sequential addition of m-CPBA (m-chloroperoxybenzoic acid, 198mg,1.15 mmol) and NaHCO ₃ (193 mg,2.3 mmol) was added and reacted at room temperature for 18h. LC-MS and TLC monitoring, after the reaction was completed, 20mL of DCM and 20mL of an aqueous solution were added to the reaction solution. The separated DCM layer was washed successively with 20mL of 10% strength by mass sodium thiosulfate, then with saturated NaCl, dried over anhydrous sodium sulfate, filtered and concentrated to give 420mg of the crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=3/1, volume ratio) and dried to give intermediate 12 (217 mg,86.0%, white solid as bubbles). [ M+H ]] ⁺ ＝330，[M+H-44] ⁺ ＝286。

Step 11 preparation of intermediate 13

Intermediate 12 (200 mg,0.6 mmol) and ZnI ₂ (775mg,2.4 mmol) was added to 5mL toluene, N ₂ Heating to 50 ℃ under protection, and reacting for 60h. LC-MS and TLC were used for monitoring, and after the reaction was completed, EA was added to the reaction solution for extraction. The extracted organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=3/1, volume ratio) and dried to give intermediate 13 (50 mg,25.0%, oil). [ M+H ]] ⁺ ＝330，[M+H-44] ⁺ ＝286。

Step 12 preparation of intermediate 14

Intermediate 13 (48 mg,0.146 mmol) was dissolved in a mixture of ethanol (2 mL) and water (2 mL), followed by the sequential addition of ammonium bicarbonate (230 mg,2.91 mmol), cesium fluoride (67 mg,0.44 mmol), and trimethylsilicon cyanide (55 ul,0.44 mmol), and the addition was completed and warmed to 50℃for 24h. LC-MS and TLC monitoring, reaction was completed. To the reaction solution was added 5mL of water, and the product was extracted with EA (5 mL. Times.3). The extracted organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give 40mg of a crude product. The crude product was isolated by preparative plate to afford intermediate 14 (15 mg,25.7% oil). [ M+H ]] ⁺ ＝400，[M+H-44] ⁺ ＝356。

Step 13 preparation of intermediate 15

Intermediate 14 (19 mg,0.048 mmol) and palladium on carbon (5 mg, palladium content 10%) were added to 2mL of methanol, replaced with hydrogen three times, and reacted at room temperature for 2h. LC-MS and TLC monitoring, reaction was completed. The reaction solution was filtered and concentrated to give intermediate 15 (17 mg,134.7% oil). [ M+H ]] ⁺ ＝266。

Preparation of Step 14-preparation of target Compound F-001

To a solution of intermediate 15 (9 mg,0.034 mmol) and intermediate 2 (7.2 mg,0.034 mmol) in t-butanol (1.5 mL) was added glacial acetic acid (15 μl), and the mixture was heated to 90 ℃ and refluxed for 20h. LC-MS and TLC monitoring, reaction was completed. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was isolated by preparative plate to give the title compound F-001 (6 mg,40.1% as an off-white solid). [ M+H ]] ⁺ ＝441。1H NMR(400MHz,DMSO-d6)δ10.84(s,1H),8.48(s,1H),8.30(s,1H),8.20(s,1H),8.00(d,J＝11.2Hz,1H),7.28(s,1H),3.76(s,3H),3.46(d,J＝16.8Hz,1H),3.31(d,J＝16.5Hz,1H),3.20(d,J＝16.8Hz,1H),3.07(d,J＝16.5Hz,1H),2.92(d,J＝4.4Hz,3H).

Example 2: synthesis of target Compound F-002

The synthesis is shown in FIG. 2.

The method comprises the following specific steps:

step 1 preparation of intermediate 2

4-bromo-2-methoxyaniline (8 g,39.80 mmol) and sodium bicarbonate (8.69 g,103.48 mmol) were added to a mixture of 1, 4-dioxane (80 mL)/water (48 mL), and the addition was completed and cooled to 0 ℃. Benzyl chloroformate (10.86 g,63.68 mmol) was slowly added dropwise to the above reaction system, and reacted at room temperature for 0.5h after the addition, and the progress of the reaction was monitored by TLC plate. After the reaction, the reaction mixture was extracted with ethyl acetate, and the extracted organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=3/1, volume ratio) to give intermediate 2 (12.6 g,94.7%, white solid). [ M+H ]] ⁺ ＝336；[M+H] ⁺ ＝338；[M-44] ⁺ ＝292；[M-44] ⁺ ＝294。

Step 2 preparation of intermediate 3

Zn powder (9.37 g,143.28 mmol) and dried DMF (10 mL) were added to the first three-necked flask (100 mL) dried under nitrogen, and the temperature was lowered to 0 ℃. Elemental iodine (4.55 g,17.91 mmol) was weighed and dissolved in ultra-dry DMF (2 mL), the iodine solution was slowly added dropwise to the Zn powder solution using a syringe, and the mixture was stirred at 0deg.C for 0.5h. To a second, dried three-necked flask (100 mL) under nitrogen protection were added methyl (R) -N-t-butoxycarbonyl-3-iodoalaninate (17.68 g,53.73 mmol) and 10mL of ultra-dry DMF, and the mixed solution was pumped into the first three-necked flask by syringe and reacted at room temperature for 2 hours. During the reaction, TLC plate was used to examine whether the reaction of (R) -N-t-butoxycarbonyl-3-iodoalanine methyl ester was completed. 2-dicyclohexylphosphine-2 ',6' -dimethoxybiphenyl (734.85 mg,1.79 mmol), tris (dibenzylideneacetone) dipalladium (824.12 mg,0.90 mmol), intermediate 2 (6 g,17.91 mmol) and 20mL of ultra-dry DMF were added to a third oven-dried three-neck flask (100 mL) under nitrogen atmosphere, and the temperature was raised to 60 ℃. Slowly dripping the reaction solution in the first three-mouth flask into the third three-mouth flask, and carrying out reaction for 2h at the temperature of 60 ℃.

After the reaction is finished, the reaction solution is placed in an ice-water bath, and a water quenching Zn reagent is slowly added. The mixture was filtered to remove excess zinc powder, and the filtrate was extracted with ethyl acetate. The extracted organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The crude product was purified by column chromatography on silica gel (elution gradient dichloromethane/methanol=10/1, volume ratio) to give intermediate 3 (5.2 g,63.4%, pale yellow oil). [ M+H ]] ⁺ ＝459；[M-56] ⁺ ＝403；[M-Boc] ⁺ ＝359；[M-44] ⁺ ＝415。

Step 3 preparation of intermediate 4

Intermediate 3 (2.5 g,5.36 mmol), 10% palladium on carbon (5.7 g,5.36 mmol) and tetrahydrofuran (25 mL) were added to a 100mL round bottom flask and reacted at room temperature for 2h after hydrogen displacement. After 2 hours, the reaction was not completed, and 0.1 equivalent of glacial acetic acid was added for catalysis. After completion of the reaction, the reaction mixture was filtered and concentrated under reduced pressure to give intermediate 4 (1.5 g, 88.2%) as a pale red oil. [ M+H ]] ⁺ ＝325；[M-56] ⁺ ＝269；[M-Boc] ⁺ ＝225。

Step 4 preparation of intermediate 7

2, 4-dichloro-5-trifluoromethylpyrimidine (3 g,13.83 mmol) and TEA (2.80 g,27.66 mmol) were added to EtOH (30 mL) and cooled to-80 ℃. To the reaction mixture was added ethanolamine (844.74 mg,13.83 mmol), and the mixture was reacted at-80℃for 2 hours. TLC monitored the progress of the reaction and after completion of the reaction, the temperature was raised to room temperature. Concentrating under reduced pressure to obtain crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=10/1, volume ratio) to give intermediate 7 (1.16 g,34.8%, white solid), [ m+h] ⁺ ＝242。

Step 5 preparation of intermediate 8

Intermediate 4 (200 mg,0.62 mmol), intermediate 7 (178.35 mg,0.74 mmol) and acetic acid (18.62 mg,0.31 mmol) were added to t-BuOH (2 mL) and reacted for 16h at 80℃under nitrogen. After the completion of the reaction, the reaction mixture was extracted with ethyl acetate. The extracted organic phase is washed by saturated saline water, dried by anhydrous sodium sulfate and concentrated under reduced pressure to obtainTo the crude product. The crude product was purified by column chromatography on silica gel (elution gradient dichloromethane/methanol=10/1, volume ratio) to give intermediate 8 (300 mg,92.0%, brown oil). [ M+H ]] ⁺ ＝530；[M-56] ⁺ ＝474；[M-Boc] ⁺ ＝430。

Step 6: preparation of intermediate 9

Intermediate 8 (300 mg,0.57 mmol) and lithium hydroxide (27.06 mg,1.13 mmol) were added to MeOH (4 mL)/H ₂ O (1 mL) was added and reacted at room temperature for 2 hours. After the completion of the reaction, the pH of the reaction solution was adjusted to be weakly acidic with an aqueous formic acid solution (1:10), followed by extraction with ethyl acetate. The extracted organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give intermediate 9 (252 mg, 86.30%) as a brown oil. [ M+H ]] ⁺ ＝516；[M-56] ⁺ ＝460；[M-Boc] ⁺ ＝416。

Preparation of Step 7-preparation of target Compound F-002

Intermediate 9 (250 mg,0.49 mmol) was added to 3mL of 1, 4-dioxane solution (3.0M) of hydrochloric acid and reacted at room temperature for 2h. And after the reaction is finished, filtering to obtain a crude product.

1mL of isopropyl alcohol was added to the crude product, the temperature was raised to 80℃to dissolve the product, the product was transferred to room temperature and stirred slowly for 1 hour after dissolution, the product was precipitated, filtered and dried to give the target compound F-002 (141.3 mg, 64.55%) as a white solid with a purity of 97.11%. [ M+H ]] ⁺ ＝416。1H NMR(400MHz,DMSO-d6)δ9.85(s,1H),8.81–8.13(m,5H),7.86(s,1H),7.17(s,1H),6.88(dd,J＝8.1,1.7Hz,1H),4.24–4.12(m,1H),3.86(s,3H),3.61–3.46(m,4H),3.28–3.06(m,2H).

Example 3: synthesis of target Compound F-003

The synthetic route is shown in FIG. 3.

The method comprises the following specific steps:

step 1 preparation of intermediate 2

Zn powder (1.57 g,24 mmol) and super dry DMF (10 mL) were added to the first three-necked flask (100 mL) dried under nitrogen protection, and the temperature was lowered to 0 ℃. Elemental iodine (761 mg,3 mmol) was weighed and dissolved in ultra-dry DMF (5 mL), the iodine solution was slowly added dropwise to the Zn powder solution, and the mixture was stirred at 0deg.C for 0.5h. Subsequently, a DMF solution (5 mL, overdry) of (R) -N-t-butoxycarbonyl-3-iodoalanine methyl ester (2.96 g,9 mmol) was added to the reaction system, and the mixture was reacted at room temperature for 2 hours. During the reaction, TLC plate was used to examine whether the reaction of (R) -N-t-butoxycarbonyl-3-iodoalanine methyl ester was completed.

Bis (triphenylphosphine) palladium dichloride (60 mg,0.15 mmol), intermediate 1 (566 mg,3 mmol) and 10mL of ultra-dry DMF were added to another three-necked flask (100 mL) under nitrogen and warmed to 70 ℃. Slowly dripping the organic zinc reagent prepared in the first three-mouth bottle into the mixed solution of the intermediate 2, and preserving the temperature of 70 ℃ for reaction for 2 hours after the addition.

After the reaction is finished, the reaction solution is placed in an ice-water bath, and a saturated ammonium chloride solution is slowly added to quench the zinc reagent. The mixture was filtered to remove excess zinc powder, and the filtrate was extracted with ethyl acetate. The extracted organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=3/1, volume ratio) to give intermediate 2 (496 mg,46.3%, pale yellow oil), [ m+h ]] ⁺ ＝356。

Step 2 preparation of intermediate 3

Intermediate 2 (480 mg,1.35 mmol) was dissolved in 10mL of absolute ethanol, and after the solution was clear, iron powder (277 mg,4.05 mmol), ammonium chloride (360 mg,6.8 mmol) and 3mL of water were added, and the mixture was stirred at 50℃for 6 hours. And after the reaction is finished, cooling to room temperature. Filtering, and concentrating the filtrate under reduced pressure to obtain a crude product. The crude product was purified by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=3/1, volume ratio) to give intermediate 3 (356.9 mg,81.2% as pale yellow oil), [ m+h] ⁺ ＝325。

Step 3 preparation of intermediate 5

2, 4-dichloro-5-trifluoromethylpyrimidine (239.5 mg,1.1 mmol) and DIPEA (N, N-diisopropylethylamine, 237.8mg,1.84 mmol) were added to methanol (10 mL) and stirred at room temperature. Intermediate 3 (300 mg,0.92 mmol) was added to the reaction mixture, and the mixture was heated to 50℃for 4 hours. After completion of the TLC, the reaction was warmed to room temperature. Concentrating under reduced pressure to obtain crude product. Crude product is passed through siliconPurification by column chromatography (elution gradient of petroleum ether/ethyl acetate=3/1, volume ratio) afforded intermediate 5 (167.9 mg,36%, white solid), [ m+h ]] ⁺ ＝506。

Step 4 preparation of intermediate 7

Intermediate 5 (101 mg,0.2 mmol), ethanolamine (50 mg,0.8 mmol) and triethylamine (80 mg,0.8 mmol) were added to acetonitrile (2 mL), and after the addition, the temperature was raised to 50℃for 2h. After completion of the TLC, the reaction was warmed to room temperature. Concentrating under reduced pressure to obtain crude product. Purification of the crude product by column chromatography on silica gel (elution gradient petroleum ether/ethyl acetate=1/1, volume ratio) afforded intermediate 7 (71 mg,67.0%, white solid), [ m+h] ⁺ ＝531。

Step 5: preparation of intermediate 8

Intermediate 7 (58 mg,0.11 mmol) and lithium hydroxide (5.3 mg,0.22 mmol) were added to MeOH (0.5 mL)/H ₂ O (2 mL) was added and reacted at room temperature for 2 hours. After the reaction, the pH of the reaction solution is adjusted to be weak acid by using a formic acid aqueous solution (1:10), and then ethyl acetate is added for extraction for five times (the polarity of the product is large, and multiple times of extraction are needed). The extracted organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give intermediate 8 (32.7 mg,58.1%, white solid), [ M+H ]] ⁺ ＝517。

Preparation of Step 6-preparation of target Compound F-003

To intermediate 8 (30 mg,0.058 mmol) was added 2mL of a 3.0M solution of 1, 4-dioxane hydrochloride and reacted at room temperature for 2h. And after the reaction is finished, filtering to obtain a crude product.

Adding 0.5mL of isopropanol into the crude product, heating to 80 ℃ to dissolve the product, transferring to room temperature after dissolving, slowly stirring for 1H, precipitating the product, filtering, and drying to obtain the target compound F-003 (11 mg,42.3%, white solid) with the purity of 95%, [ M+H ]] ⁺ ＝417。1H NMR(400MHz,DMSO-d6)δ9.37–9.19(m,2H),9.12–8.42(m,2H),8.33(s,1H),7.76(s,1H),7.47(s,1H),4.62(t,J＝7.3Hz,1H),4.11(s,3H),3.70–3.46(m,6H)。

Biological Activity test

Protein binding experiments:

reagent consumable:

LRRK2G2019S enzyme (Sieimerfide), substrate (LRRKtide) (Sieimerfide), ATP (Sieimerfide) TR-FRET dilution (Sieimerfide), pLRRKtide antibody (Sieimerfide), 384 well Plate (PE) DMSO (Soilebao)

The experimental process comprises the following steps:

all test compounds (including positive controls and test samples) were diluted to 1mM with DMSO to give corresponding test compound solutions. 35. Mu.L of positive compound (structural formula shown in Table 1, genntech company in literature (strada AA, liu X, baker-Glenn C, et al.discover of high spot, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK 2) small molecular inhibitors.J. Med chem.2012nov 26;55 (22): 9416-33. Similar structural compounds disclosed therein, and synthesized by reference thereto), 35. Mu.L of test compound solution, 35. Mu.L of blank solution were added to 384 well plates at once, plates were centrifuged at 2500rpm for 1 minute at 1mM as initial concentration, 3-fold gradients were diluted for 10 spots, and 100nL of positive compound, test compound, blank well solution per well were added to another 384 assay plate, 3 multiplex wells, respectively, and the plates were centrifuged at 2500rpm for 1 minute and sealed in foil.

Enzyme reaction: a mixed working solution of LRRKtide substrate and LRRK2G2019S kinase (final concentration of LRRKtide substrate: 400nM and LRRK2G2019S kinase: 580 ng/mL) was diluted with assay buffer (Siemens flight TR-FRET Dilution buffer) and added to all sample wells of 384 assay plates described above, 5. Mu.L per well, and 384 assay plates were incubated at 23℃for 20 minutes. After incubation, 2X ATP working solution (134. Mu.M) was diluted with assay buffer and added to each well, and each well incubated for 60 minutes at 23℃in 5mL 384 assay plates.

And (3) detection: EDTA and pLRRKtide antibody were diluted with assay buffer (TR-FRET Dilution buffer) to give a mixed working solution (final concentration: EDTA:10mM, pLRRKtide antibody: 2 nM). Subsequently, 10. Mu.L of the antibody-mixed working solution was added to each well of the 384 assay plate described above, and incubated at 23℃for 60 minutes. The plate was read in a microplate reader in TE-FRET mode with 340nm excitation light, 520nm fluorescence emission light and 490nm terbium emission light.

The method refers to: j debarkinoformosia et al compounds, compositions and methods are described in CN113939294A [ P ].2022-01-14.

The activity data for each compound are shown in table 1.

TABLE 1 data on compound activity

As can be seen from Table 1, the novel compounds provided in the examples of the present invention have a stronger inhibitory effect on the kinase LRRK2G2019S, compared with the inhibitory activity of the positive structure on LRRK2G2019S, and in particular, the compound F-001 provided in the example 1 and the compound F-003 provided in the example 3 of the present invention both show an inhibitory effect on the mutant kinase LRRK2G2019S which is equivalent or superior to that of the positive structure. Has potential application value in preparing medicines for preventing and/or treating diseases related to the increase of the activity of the gene LRRK2 in vivo.

Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (9)

1. Pyrimidine amino aryl alanine derivatives, or optical isomers thereof, or prodrugs thereof, or pharmaceutically acceptable salts thereof, or hydrates, solvates, N-oxides and deuterated substances thereof, and are characterized in that the structures of the derivatives are shown as a general formula (1) or a general formula (2):

in the general formula (1),

R ₁ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 An alkyl group;

R ₂ selected from H, F, cl, br, I;

n is selected from 0,1,2;

y is selected from-O-or-N-;

R ₃ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl and the usual protecting groups for various O or N;

R ₄ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl and the various common N protecting groups;

alternatively, R ₃ And R is R ₄ Is a group taken together selected from-C (=O) -, -CH ₂ -C(＝O)-、-C(＝O)-CH ₂ -、-(CH ₂ -CH ₂ )-、-C(＝O)-C(＝O)-；

In the general formula (2),

R ₅ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 An alkyl group;

x is selected from-C-, or-N-;

R ₆ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl and various common O protecting groups;

R ₇ selected from H, C _1-3 Alkyl, oxo C _1-3 Alkyl, halogenated C _1-3 Alkyl groups and the various N protecting groups commonly found.

2. The pyrimidine amino-aryl-alanine derivative according to claim 1, wherein the derivative is 7 '-fluoro-4' -methoxy-5'- ((4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -1',3 '-dihydro-spiro [ imidazole-4, 2' -indene ] -2,5-dione, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated thereof, of the formula:

3. the pyrimidine amino aryl alanine derivative, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated thereof according to claim 1, wherein the derivative is (S) -2-amino-3- (4- ((4- ((2-hydroxyethyl) amino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -3-methoxyphenyl) propionic acid having the chemical structural formula:

4. the pyrimidine amino aryl alanine derivative, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated thereof according to claim 1, wherein the derivative is (S) -2-amino-3- (5- ((4- ((2-hydroxyethyl) amino) -5- (trifluoromethyl) pyrimidin-2-yl) amino) -4-methoxypyridin-2-yl) propionic acid having the chemical structural formula:

5. the use of a pyrimidine aminoarylalanine derivative according to any of claims 1 to 4, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuteride thereof as a leucine-rich repeat kinase 2 inhibitor.

6. The use of a pyrimidine amino aryl alanine derivative according to any one of claims 1 to 4, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated thereof, in the manufacture of a medicament for the treatment or prophylaxis of parkinson's disease.

7. The use of a pyrimidine amino aryl alanine derivative according to any one of claims 1 to 4, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterate thereof, in the manufacture of a medicament for the treatment or prevention of chronic neurodegenerative diseases.

8. Use of a pyrimidine amino aryl alanine derivative according to any one of claims 1 to 4, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterated thereof, in the manufacture of a medicament for inhibiting the activity of leucine rich repeat kinase 2 to prevent and/or treat a disease.

9. A pharmaceutical composition or formulation comprising a pyrimidine amino aryl alanine derivative according to any one of claims 1 to 4, or an optical isomer thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate, solvate, N-oxide, deuterate thereof.

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