KR20160035411A - Pyridopyrimidine derivatives as lrrk2 (leucine rich repeat kinase 2) inhinitor - Google Patents

Abstract

The present invention relates to an agent for preventing or treating arthritis, containing a pyrimidine derivative which has effects as an inhibitor of leucine rich repeat kinase 2 (LRRK2) kinase. The present invention further relates to a production method thereof. More specifically, the present invention relates to an agent for preventing or treating degenerative brain disease such as Parkinson′s disease, which comprises a compound represented by chemical formula I or a pharmaceutically acceptable salt thereof, a hydrate, a solvate, a pro-drug, or an isomer.

Description

PYRIDOPYRIMIDINE DERIVATIVES AS LRRK2 (LEUCINE RICH REPEAT KINASE 2) INHINITOR as LRRK2 (Leucine Rich Repeat Kinase 2)

The present invention relates to a pyridopyrimidine derivative compound having an effect as an LRRK2 kinase inhibitor, a process for producing the same, and a pharmaceutical composition containing the same.

Protein kinase is an enzyme that catalyzes the transfer of terminal phosphate group of adenosine triphosphate (ATP) to specific residues of protein (tyrosine, serine, threonine). It activates, grows, and differentiates cells against changes in extracellular mediator and environment It is involved in the signal to be adjusted.

Protein kinases are generally classified as serine and / or threonine kinase groups and tyrosine kinase groups depending on the substrate phosphorylated [S. K. Hanks and T. Hunter, FASEB. J., 1995, 9, 576-596]. The serine / threonine kinase group is a protein kinase C isoform [A. C. Newton, J. Biol. Chem., 1995, 270, 28495-28498], cyclin-dependent kinase group and cdc2 [J. Pines, Trends in Biochemical Sciences, 1995, 18, 195-197. The tyrosine kinase group is a membrane-spanning growth factor receptor [S. Cellular Signaling, 1992, 4, 123-132) and cytosolic non-receptor kinases including p56tck, p59fYn, ZAP-70, and C-terminal Src kinase [C. Chan et. al., Ann. Rev. Immunol., 1994, 12, 555-592].

Inappropriately high protein kinase activity is directly or indirectly related to a number of diseases resulting from abnormal cellular function. For example, failure of appropriate regulation mechanisms of kinases involved in mutation, over-expression or inappropriate enzyme activity; Or the overproduction or deficiency of factors involved in signal transduction upstream or downstream of cytokines or kinases. Thus, selective inhibition of kinase activity can be a beneficial target for the development of new drugs for the treatment of disease.

LRRK2 (leucin-rich repeat kinase-2) is a protein belonging to the leucine-rich repeat kinase family. It consists of 2527 amino acid sequences with high similarity between species. Characteristically, (GTPase) and serine-threonine kinase (GTPase) activity. The expressed LRRK2 is observed in various organs and tissues including the brain, and is known to exist in the cytoplasm or cell membrane and the mitochondrial outer membrane at the cellular level. Currently, studies on active in vivo functions of LRRK2 have been actively carried out. It has five functionally important domains, and it has been shown that autophosphorylation can regulate self-activity through protein-protein interaction and enzyme action In particular, chaperone machinery, cytoskelon arrangement, protein translational machinery, synaptic vesicle endocytosis, mitogen-activated protein kinase It is known that the ubiquitin / autophage protein degradation pathways of mitogen-activated protein kinases signaling cascades and ubiquitin / autophage protein degradation pathways are regulated by LRRK2.

LRRK2 has been reported to be highly expressed in dopaminergic neurons in the cerebral cortex, black body, striatum, hippocampus, cerebellum and the like related to the pathogenesis of Parkinson's disease. In the postmortem study, Increased specific expression of LRRK2 has been observed in abnormal protein aggregates called Lewy bodies, which are specifically observed in disease states. There are various disease-related LRRK2 mutations known in clinical studies, most of which are gain-of-function mutants with increased LRRK2 activity, late-onset autosomal dominant Parkinson's disease, , And it is known that the increase in the activity of LRRK2 affects the onset and progression of Parkinson's disease. Among the LRRK2 mutations, the highest incidence is the G2019S mutation in the LRRK2 kinase domain, which is observed in 5 to 6% of patients with posterior malformations and 1 to 2% of patients with idiopathic Parkinson's disease This is the most frequent mutation in patients with familial Parkinson's disease. These LRRK2 mutations have been shown to induce abnormal endocytosis, axonal trafficking, and neurite shortening in neurons as evidenced by cell line and animal tests, aggregation and apoptosis are induced. It has been confirmed that normal LRRK2 is overexpressed in neurons, inducing apoptosis. Therefore, the cell physiological change pattern derived from LRRK2 is very similar to that observed in neuronal apoptosis in Parkinson's disease patients do. In animals with LRRK2 and active mutants, dopaminergic neuronal apoptosis, leucosis and movement disorders similar to those of Parkinson's disease have been reported, and in the case of LRRK2 knock-out animals, aging-dependent α -synuclein inhibition was observed, and the possibility of Parkinson's disease treatment by LRRK2 inhibition is now confirmed in animal models. The activation of LRRK2 has a high clinical relevance to the onset and progression of Parkinson's disease, and it may control both neuronal apoptosis and neuronal inflammatory responses, which are the most important pathological changes of Parkinson's disease through the regulation of LRRK2 activity It is expected.

As a result of investigating the unmet medical needs of Parkinson's disease patients in the treatment group of Parkinson's disease, side effects (exercise relief and tolerance to treatment) and insufficient efficacy were found to be the most important unmet medical need according to the use of the main therapeutic agent (dopamine antagonist) have. At present, 40% of all prescribing patients have experienced motor fluctuation. Especially, when a certain period (4 ~ 6 years after onset) has elapsed after prescription, the rate of patient experiencing exercise relief due to prescription drug resistance Is increasing every year by 10%. Therefore, there is a high demand for the development of a neuroprotective agent and a stem cell / gene therapy method which have a therapeutic effect as well as an agent for improving side effects of Parkinson's disease treatment. Unmet needs other than the existing side effects have been recognized as the cost of the treatment and convenience of medication. This is analyzed as unmet needs due to the requirement of long-term administration due to the nature of Parkinson's disease. Due to the characteristics of this market demand, there is a high need for the development of low-molecular synthetic drug which can be administered orally.

Accordingly, the present inventors have made efforts to develop a compound that exhibits selective and excellent activity of LRRK2 kinase among various kinases present in the human body. As a result, pyridopyrimidine derivative derivatives of the following formula (I) were synthesized. Inhibition of the progression of Parkinson's disease, and therapeutic effect, thus completing the present invention.

A basic object of the present invention is to provide a compound of the general formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug or isomer thereof.

(I)

Wherein R ₁ is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-7 cycloalkyl, aryl, benzyl, heteroaryl, hydroxy-C _1-6 alkyl, C _2-14 heterocycloalkyl, -CONR ₇ R _8, or is -COR _9, n is an integer from 0 to _2, R 2 is C _{1 -6} alkyl, C _{3 -7-cycloalkyl,} C _{2 -14} heterocycloalkyl, C _6-14 aryl or heteroaryl to be.

Another object of the present invention is to provide a process for preparing a compound represented by the formula (I): (i) reacting a compound represented by the formula (a) with a malonate derivative compound in the presence of a base to obtain the following compound (ii) chlorinating the compound (b) to obtain the following compound (c); (iii) oxidizing the compound (c) to obtain the following compound (d); (iv) reacting the compound (d) with an aniline derivative using a base and a palladium catalyst to obtain the following compound (e); And (v) reacting said compound (e) with Bredereck's in the presence of ammonia water to obtain a compound of formula (I).

A further object of the present invention is to provide a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable excipient or carrier.

Another object of the present invention is to provide a therapeutic agent for an LRRK2-mediated disease or condition, which comprises a compound of formula (I)

The basic object of the present invention can be achieved by providing a compound of the general formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug or isomer thereof.

(I)

Wherein R ₁ is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-7 cycloalkyl, aryl, benzyl, heteroaryl, hydroxy-C _1-6 alkyl, C _2-14 heterocycloalkyl, -CONR ₇ R _8, or is -COR _9, n is an integer from 0 to _2, R 2 is C _{1 -6} alkyl, C _{3 -7-cycloalkyl,} C _{2 -14} heterocycloalkyl, C _6-14 aryl or heteroaryl to be.

4-yl) pyrido [4,3-d] pyrimidin-5 (6H) -tetrahydro-2H-pyran- 4-yl) pyrido [4,4- d] pyrimidin-5 (6H) -one, 4- (2-isopropoxyphenyl) 4-ylamino) -2- (piperidin-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (l-cyclopropyl- lH-pyrazole Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (3-isopropylamino) -2- Pyrimidin-5 (6H) -one, 4- (3-isopropylamino) -2- (1-isopropylpiperidine Yl) pyrido [4,3-d] pyrimidin-5 (6H) -one and 4- (l-isopropyl-lH- pyrazol- Pyrazol-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) Isopropyl piperidine Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (2- isopropylpyridin- 4- ylamino) -2- (tetrahydro- Yl) pyrido [4,3-d] pyrimidin-5 (6H) -one and 4- (l-isopropyl- lH- pyrazol- Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (l -cyclopropyl- lH- pyrazol- Yl) pyrido [4,3-d] pyrimidin-5 (6H) -one and 4- (3-t- butylphenylamino) -2- (tetrahydro- Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (1-tert- butyl- Pyrimido-5 (6H) -one and 2-isopropyl-4- (3-isopropylphenylamino) pyrido [4,3 4-ylamino) pyrido [4,3-d] pyrimidin-5 (lH-pyrazol- (6H) -one, < / RTI > 2-cyclohexyl- Propyl-4- (3- (2-cyclopropyl-4- (l-isopropyl-lH-pyrazol-4ylamino) pyrido [4,3- d] pyrimidin- , 3-d] pyrimidin-5 (6H) -one and 2-cyclopropyl-4- (l -cyclopropyl- lH- pyrazol-4ylamino) pyrido [ (6H) -one.

The pharmaceutically acceptable salts of the compounds of formula (I) of the present invention may be hydrochloride, sulfate, acetate, formate, succinate, malate, fumarate, toluenesulfonate or methanesulfonate of the compound of formula (I).

A pharmaceutical composition for the prevention and treatment of arthritis, which comprises a compound of formula (I) of the present invention and a pharmaceutically acceptable excipient or carrier, can be provided.

The pharmaceutical compositions may be prepared according to conventional methods using one or more pharmaceutically acceptable adjuvants or excipients. Such adjuvants include, in particular, diluents, sterile aqueous media and various non-toxic organic solvents.

The pharmaceutical composition may be provided in the form of tablets, pills, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs or syrups. In order to obtain pharmaceutically acceptable preparations, sweeteners, Or < / RTI > stabilizers.

The choice of vehicle and the content of active substance in the vehicle will generally be determined according to the solubility and chemical properties of the active compound, the particular mode of administration, and the conditions to be observed in the pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and excipients such as starch, alginic acid and certain complex silicates, in combination with lubricants such as magnesium stearate, sodium lauryl sulfate and talc, Release may be used for the manufacture of tablets.

In order to prepare capsules, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used, they may contain emulsifying agents or agents which facilitate suspension, and diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol or chloroform, or mixtures thereof may be used.

Thus, the composition of the present invention may be administered parenterally or orally, if necessary, and may be administered at a dose of 0.5 mg / kg body weight to 5 mg / kg body weight, preferably 1 mg / kg body weight per day for parenteral administration With an amount of body weight to 4 mg / kg body weight; In the case of oral administration, one or more doses may be administered in an amount of 5 mg / kg body weight to 50 mg / kg body weight, preferably 10 mg / kg body weight to 40 mg / kg body weight. The dosage for a patient may vary depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc. of each patient.

The compound of formula (I) of the present invention can be prepared by reacting a compound of formula (a) with a malonate derivative compound in the presence of a base to obtain the following compound (b): (ii) obtaining the following compound (c) by chlorinating the compound (b); (iii) oxidizing the compound (c) with manganese dioxide to obtain the following compound (d); (iv) subjecting the compound (d) to an amination reaction under acid catalysis to obtain the following compound (e); And (v) reacting said compound (e) with an enamine compound using Bredereck's reagent and immediately reacting in the presence of ammonia water to obtain a compound of formula (I) .

[Reaction Scheme 1]

Wherein R ₁ is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-7 cycloalkyl, aryl, benzyl, heteroaryl, hydroxy-C _1-6 alkyl, C _2-14 heterocycloalkyl, -CONR ₇ R _8, or is -COR _9, n is an integer from 0 to _2, R 2 is C _{1 -6} alkyl, C _{3 -7-cycloalkyl,} C _{2 -14} heterocycloalkyl, C _{6 -14} aryl or heteroaryl to be.

In step (i) of Scheme 1, the amidine compound represented by the formula (a) and the diethyl-2-ethylidene malonate compound are reacted in an organic solvent such as methanol, ethanol or isopropanol with sodium methoxide The compound of formula (b) may be reacted at a reaction temperature of 60 ° C to 80 ° C for 1 to 2 hours.

In step (ii) of Scheme 1, the compound of formula (b) is reacted with POCl ₃ (20-30 equivalents) at a reaction temperature of 90 ° C to 110 ° C to replace the amide group with chloride, Can be obtained.

In step (iii) of Scheme 1, the compound of formula (c) is reacted with manganese dioxide (5-10 eq.) In an organic solvent such as dichloromethane or dichloroethane at 50 ° C to 80 ° C for 6 hours, ) Can be prepared.

In step (iv) of Scheme 1, the compound of formula (d) is reacted with palladium (II) acetate (0.1-0.5 eq.), BINAP (0.5-1.0 eq.) In an organic solvent such as 1,4- The compound of formula (e) may be prepared by adding potassium carbonate (2-3 equivalents) and aniline derivative (1.0-1.5 equivalents) and reacting at 80 to 110 ° C for 10-12 hours.

In step (v) of Scheme 1, the compound of formula (e) is reacted with Bredereck's (2-3 equivalents) or N, N-dimethylformamide dimethylacetal (2- (10-20 equivalents) is added thereto in methanol or an ethanol solvent, and the mixture is reacted at 80 to 100 DEG C for 5-8 hours to obtain a compound of formula I can be prepared.

The pyridine derivatives of formula (I) according to the present invention have excellent selective inhibitory effect on LRRK2, and particularly excellent inhibitory effect on LRRK2 G2019S mutant cell line. Accordingly, the pyridine derivatives of formula (I) according to the present invention are useful as preventive and therapeutic agents for Parkinson's disease by preventing the death of dopaminergic neurons by LRRK2 mutation.

Fig. 1 shows the results of analysis of neuropeptide germination growth using the compounds of the present invention (compounds 1 and 13).

Hereinafter, the present invention will be described in more detail with reference to the following examples or drawings. It is to be understood, however, that the description of the following embodiments or drawings is only intended to illustrate specific embodiments of the present invention and is not intended to limit or limit the scope of the present invention.

Production Example 1. Synthesis of tetrahydro-2H-pyran-4-carboximidamide hydrochloride

9.27 g (173.41 mmol) of ammonium chloride was added to the toluene solvent, the reaction temperature was cooled to 0 ° C, and 16.62 mL (173.41 mmol) of trimethylaluminum chloride was slowly added thereto. The reaction was allowed to take place at room temperature, followed by stirring at room temperature for 30 minutes. Thereto was added 5 g (34.68 mmol) of ethyltetrahydro-2H-pyran-4-carboxylate and the mixture was heated to 100 DEG C and reacted for 12 hours. The reaction solution is cooled using an ice water bath, and methanol is gradually added to terminate the reaction. The resulting inorganic matter is filtered off and the filtrate is reduced in pressure to obtain an unpurified solid compound. This compound was treated with a mixed solvent of dichloromethane: methanol to obtain 5.66 g of a pale yellow crude compound.

Preparation Example 2. Synthesis of ethyl 4-methyl-6-oxo-2- (tetrahydro-2H-pyran-4-yl) -1,4,5,6-tetrahydropyrimidine-5-carboxylate

5.66 g (35 mmol) of the amidine derivative synthesized above and 4.72 g (69 mmol) of sodium ethoxide were added to the ethanol solvent, and the reaction was allowed to proceed at room temperature for 15 minutes. Diethyl-2-ethylidene malonate was added thereto, and the mixture was heated to 65 DEG C and stirred for 1 hour. The reaction solution is cooled to room temperature, and then saturated ammonium chloride solution is added to terminate the reaction. The reaction solution is removed under reduced pressure, and then extracted with ethyl acetate. After drying over anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 6.94 g (yield: 73.9%) of the crude compound. The resulting compound was used as such in the next reaction without further purification.

Production Example 3. Synthesis of ethyl 6-chloro-4-methyl-2- (tetrahydro-2H-pyran-4-yl) -4,5-dihydropyrimidine-5-carboxylate

54 mL of POCl ₃ is added to 5.4 g (20 mmol) of the compound obtained above and refluxed for 24-36 hours. The reaction solution is removed under reduced pressure, then extracted with ethyl acetate, washed with saturated sodium hydrogencarbonate solution and brine. Dried over anhydrous sodium sulfate and concentrated under reduced pressure. The obtained compound was purified by silica gel column chromatography using dichloromethane / methanol (10: 1) as an eluent to obtain 4.32 g (yield 75%) of the desired compound in the form of yellow oil.

Production Example 4. Synthesis of ethyl 4-chloro-6-methyl-2- (tetrahydro-2H-pyran-4-yl) pyrimidine-5-carboxylate (Intermediate 1)

The compound obtained above (4.32 g, 15 mmol) is added to manganese dioxide (6.55 g, 75.3 mmol) in a dichloroethane solvent and refluxed for 6 hours. After cooling the reaction solution to room temperature, it is subjected to Celite filter and the filtrate is concentrated under reduced pressure. The obtained compound was purified by silica gel column chromatography using dichloromethane / methanol (40: 1) as an eluent to obtain 3 g of a yellow oil-like compound (yield 70%).

MS (ESI) m / z 287.1, 289.2 [M + H] < ^{+ &}

Preparation 5. Synthesis of 1-benzylpiperidine-4-carboximidamide hydrochloride

Synthesis of the compound was made using a procedure similar to that described in Preparation 1 using ethyl 1-benzylpiperidine-4-carboxylate reagent as starting material.

Production Example 6. Synthesis of ethyl 2- (1-benzylpiperidin-4-yl) -4-chloro-6-methylpyrimidine-5-carboxylate (Intermediate 2)

Synthesis of the above compound was synthesized by following procedures analogous to those described in 2-4 for the preparation of the amidine compound synthesized in Preparation Example 5.

MS (ESI) m / z 374.2 [M + H] < ^{+ &}

Preparation 7. Synthesis of ethyl 4-chloro-2-isopropyl-6-methylpyrimidine-5-carboxylate (Intermediate 3)

The synthesis of the compound was synthesized using a commercially available isopropylamidine hydrochloride according to a procedure analogous to that described in 2-4 for the preparation.

MS (ESI) m / z 243.1 [M + H] < ^{+ &}

Preparation Example 8. Synthesis of ethyl 4-chloro-2-cyclopropyl-6-methylpyrimidine-5-carboxylate (Intermediate 4)

The synthesis of the compound was synthesized using a commercially available cyclopropylamidine hydrochloride according to a procedure analogous to that described in 2-4 for the preparation.

MS (ESI) m / z 241.2 [M + H] < ^{+ &}

Production Example 9 Synthesis of ethyl 4- (3-isopropylphenylamino) -6-methyl-2- (tetrahydro-2H-pyran-4-yl) pyrimidine-5-carboxylate

To a 1,4-dioxane solvent was added 0.32 g (0.112 mmol) of ethyl 4-chloro-6-methyl-2- (tetrahydro-2H-pyran-4-yl) pyrimidine- 0.013 g (0.058 mmol) of palladium (II) acetate, 0.07 g (0.112 mmol) of BINAP and 0.4 g (2.89 mmol) of potassium carbonate were added and the mixture was heated to 100 DEG C in a nitrogen stream and reacted for 6 hours . Dichloromethane was added to the reaction solution to dilute it, and the undissolved solid was removed. The resulting filtrate was concentrated under reduced pressure. The obtained compound was purified by silica gel column chromatography using ethyl acetate / heptane (1: 3) as an eluent to obtain 0.287 g (yield 67%) of yellow oil-like compound.

MS (ESI) m / z 384.2 [M + H] < ^{+ &}

Example 1 4- (3-Isopropylphenylamino) -2- (tetrahydro-2H-pyran-4-yl) pyrido [4,3- d] pyrimidin-

0.287 g (0.75 mmol) of the compound obtained in the above was dissolved in N, N-dimethylformamide, 0.4 mL (1.88 mmol) of Bredereck's reagent was added, and the mixture was reacted at 100 ° C. for 2 hours. The reaction solution is removed under reduced pressure, then ethanol solvent is added to the residue, 2 mL of ammonia water is added, and the reaction is carried out at 95 ° C for 6 hours. The reaction solution was removed under reduced pressure, and then the residue was slurried by adding acetonitrile and then filtered to obtain 0.15 g (yield 55%) of the desired compound.

MS (ESI) m / z 365.0 [M + H] < ⁺ >

Example 2 4- (3-Isopropylphenylamino) -2- (piperidin-4-yl) pyrido [4,4- d] pyrimidin-5 (6H)

Synthesis of the above compound was obtained using a procedure similar to that described in Preparative Example 9 and Example 1 using Intermediate 2, followed by debenzylation reaction.

MS (ESI) m / z 364.4 [M + H] < ⁺ >.

Example 3 4- (2-Isopropylpyridin-4-ylamino) -2- (piperidin-4-yl) pyrido [4,3- d] pyrimidin-

Synthesis of the above compound was obtained using a procedure similar to that described in Preparative Example 9 and Example 1 using Intermediate 2, followed by debenzylation reaction.

MS (ESI) m / z 365.4 [M + H] < ⁺ >.

(4H-pyrrolo [4,3-d] pyrimidin-5 (6H) -carbamic acid tert- -won

Synthesis of the above compound was obtained using a procedure similar to that described in Preparative Example 9 and Example 1 using Intermediate 2, followed by debenzylation reaction.

MS (ESI) m / z 354.4 [M + H] < ⁺ >.

4-yl) pyrido [4,3-d] pyrimidin-5 (6H) -carbamic acid tert- -won

Synthesis of the above compound was obtained using a procedure similar to that described in Preparative Example 9 and Example 1 using Intermediate 2, followed by debenzylation reaction.

MS (ESI) m / z 352.4 [M + H] < ⁺ >.

Example 6 4- (3-Isopropylamino) -2- (1 -methylpiperidin-4-yl) pyrido [4,3- d] pyrimidin-5 (6H)

The synthesis of the compound was carried out by reacting the compound synthesized in Example 2 with formaldehyde.

MS (ESI) m / z 378.4 [M + H] < ⁺ >.

Example 7 4- (3-Isopropylamino) -2- (1-isopropylpiperidin-4-yl) pyrido [4,3- d] pyrimidin-

This compound was synthesized by reacting the compound synthesized in Example 2 with acetone.

MS (ESI) m / z 406.4 [M + H] < ⁺ >.

Example 8 4- (l-Isopropyl-lH-pyrazol-4-ylamino) -2- (l -methylpiperidin-4- yl) pyrido [4,3- d] pyrimidin- (6H) -one

The synthesis of the compound was carried out by reacting the compound synthesized in Example 4 with formaldehyde.

MS (ESI) m / z 368.4 [M + H] < ⁺ >.

Example 9. Preparation of 4- (l-isopropyl-lH-pyrazol-4-ylamino) -2- (l-isopropylpiperidin- 5 (6H) -one

This compound was synthesized by reacting the compound synthesized in Example 4 with acetone.

MS (ESI) m / z 396.4 [M + H] < ⁺ >.

Pyridin-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) -won

Synthesis of the above compound was synthesized using intermediate 1 using procedures and methods similar to those described in Preparation 9 and Example 1.

MS (ESI) m / z 366.4 [M + H] < ⁺ >.

Pyrazol-4-yl) pyrido [4,3-d] pyrimidin-4-ylamino) 5 (6H) -one

Synthesis of the above compound was synthesized using intermediate 1 using procedures and methods similar to those described in Preparation 9 and Example 1.

MS (ESI) m / z 355.4 [M + H] < ⁺ >.

Pyrazol-4-yl) pyrido [4,3-d] pyrimidin-2-one [ 5 (6H) -one

Synthesis of the above compound was synthesized using intermediate 1 using procedures and methods similar to those described in Preparation 9 and Example 1.

MS (ESI) m / z 353.4 [M + H] < ⁺ >.

Example 13 Synthesis of 4- (3-t-butylphenylamino) -2- (tetrahydro-2H-pyran-4-yl) pyrido [4,3- d] pyrimidin-

Synthesis of the above compound was synthesized using intermediate 1 using procedures and methods similar to those described in Preparation 9 and Example 1.

MS (ESI) m / z 379.4 [M + H] < ⁺ >.

Example 14. Synthesis of 4- (1-t-butyl-1H-pyrazol-4-ylamino) -2- (tetrahydro-2H- -5 (6H) -one

Synthesis of the above compound was synthesized using intermediate 1 using procedures and methods similar to those described in Preparation 9 and Example 1.

MS (ESI) m / z 369.4 [M + H] < ⁺ >.

Example 15. Synthesis of 2-isopropyl-4- (3-isopropylphenylamino) pyrido [4,3-d] pyrimidin-5 (6H)

The synthesis of this compound was synthesized using procedures similar to those described in Preparation 9 and Example 1 using intermediate 3.

MS (ESI) m / z 323.4 [M + H] < ⁺ >.

Example 16. 2-Isopropyl-4- (l-isopropyl-lH-pyrazol-4-ylamino) pyrido [4,3- d] pyrimidin-

The synthesis of this compound was synthesized using procedures similar to those described in Preparation 9 and Example 1 using intermediate 3.

MS (ESI) m / z 313.4 [M + H] < ⁺ >.

Example 17 4- (l-Cyclopropyl-lH-pyrazol-4-ylamino) -2-isopropylpyrido [4,3- d] pyrimidin-

The synthesis of this compound was synthesized using procedures similar to those described in Preparation 9 and Example 1 using intermediate 3.

MS (ESI) m / z 311.4 [M + H] < ⁺ >.

Example 18 2-Cyclopropyl-4- (3-isopropylphenylamino) pyrido [4,3-d] pyrimidin-5 (6H)

The synthesis of this compound was synthesized using procedures analogous to those described in Preparation 9 and Example 1 using intermediate 4.

MS (ESI) m / z 321.4 [M + H] < ⁺ >.

Example 19. Synthesis of 2-cyclopropyl-4- (l-isopropyl-lH-pyrazol-4ylamino) pyrido [4,3- d] pyrimidin-5 (6H)

The synthesis of this compound was synthesized using procedures analogous to those described in Preparation 9 and Example 1 using intermediate 4.

MS (ESI) m / z 311.4 [M + H] < ⁺ >.

Example 20 2-Cyclopropyl-4- (l-cyclopropyl-lH-pyrazol-4ylamino) pyrido [4,3- d] pyrimidin-5 (6H)

The synthesis of this compound was synthesized using procedures analogous to those described in Preparation 9 and Example 1 using intermediate 4.

MS (ESI) m / z 309.4 [M + H] < ⁺ >.

Example 21.

In this example, the inhibitory effect of the in vitro LRRK2 (Leucine Rich Repeat Kinase 2) kinase on the compounds of the present invention was measured. In the results analysis in this example, IC50 values were calculated and used so as to enable quantitative comparison of in vitro activity.

Test Methods

Compounds of the present invention were reconstituted in 100 mM DMSO to 10 mM and serially diluted from 1 [mu] M to 10 [mu] M with LRRK2 kinase activity measuring solutions. Purified LRRK2 (CARNA BIOSCIENCES) was added to a black U-bottom 96-well microtiter plate containing 6 [mu] l of serially diluted compounds and incubated at room temperature for 30 minutes. For kinase reaction, ATP and substrate solution specific urolite-labeled polypeptides (Ulight ^™ -poly TK, PerkinElimer) were added. The reaction was carried out at room temperature for 1 hour, and the solution was assayed with LANCE (trademark) detection solution containing EDTA. The lanes detection solution containing the europium-labeled antibody (LRRK2 specific PT66) was added and the kinase experiment was terminated by incubation at room temperature for 50 minutes. The phosphorylated substrate was detected by 665 nm emission measurement. IC50 values were calculated by nonlinear regression analysis using Prism ^TM software based on the absence of kinase inhibitor.

result

The in vitro inhibitory effects of LRRK2 on the compounds of the present invention are described in Table 2 below.

In vitro LRRK2 activity inhibitory effect  Compound No. IC50 (uM) LRRK2-IN-1 0.0082 One 0.0087 2 0.0045 10 0.0083 13 0.0011 14 0.0024 18 0.0094

As shown in the results of this experiment (Table 2), compounds 2, 13 and 14 of the compounds of the present invention exhibited inhibitory effect on LRRK2 activity at the pM level at nM, LRRK2-specific activity Inhibitory properties of the present invention. On the other hand, a 6-tyrosine kinase panel test was further performed on Compounds 2, 13 and 14 in which high LRRK2 activity inhibitory effect was confirmed in this Example. As a result, all three compounds were found to have high LRRK2 specific activity inhibition selectivity Respectively.

Example 22.

In this example, in order to confirm whether the inhibitory effect of the compounds of the present invention in Example 42 is due to LRRK2 inhibitory effect at the cell level, in a dopamine secretory cell line (HMG9) overexpressing the LRRK2 G2019S mutant protein, And inhibition of cell death was carried out. The LRRK2G2091 mutant protein has been shown to cause neurotoxicity in the expression of Parkinson's disease by inducing dopamine neuronal cell death. Therefore, inhibition of phosphorylation of the LRRK2 G2019S mutation is known to be important for the treatment of Parkinson's disease.

Test Methods

The mouse dopamine producing cell line (HMG9) expressing the human G2019S mutant LRRK2 was obtained from the Wonkwang University Cancer Neuropsychiatric Research Center and was treated with 10% fetal bovine serum (Gibco), penicillin / streptomycin (Gibco) Cells were cultured in DMEM medium at 37 ° C in 5% CO ₂ . Cell growth by treatment of the compounds of the present invention under cell death conditions induced by oxidative stress was examined. Cells were seeded at a cell concentration of 0.5 x ¹⁰ 4/100 μl in a 96-well plate, and 6 μM of 6-hydroxydopamine (6-OHDA), which induces oxidative stress, Lt; / RTI > Cell numbers were determined by measuring the total ATP amount in each well using an ATPlite assay kit (PerkinElmer).

result

The inhibitory effect of the compounds of the present invention on neuronal cell death is shown in Table 3 below.

cell rescue underoxidativestress (% of Control) Compound No. DMSO
control 1 uM 3 uM 10 uM LRRK2-IN-1 100 55 38 24 One 100 107 115 118 2 100 90 74 31 10 100 105 83 60 13 100 111 107 85 14 100 99 70 37 18 100 106 110 108

Compounds of the present invention exhibited dose-dependent neuronal cell protection in this experiment (Table 3). Compounds 1, 13, and 18 showed relatively low LRRK2 inhibitor-dependent cell protection against LRRK2-IN- .

Example 23.

In this example, experiments were carried out to confirm that the compounds of the present invention protect dopamine cells damaged by LRRK2 G2019S overexpression and further that neuronal cells contribute to neurite growth.

Test method: Measurement of nerve pole germination

The effect of the compound treatment of the present invention on neurite outgrowth formation was investigated. Cells were seeded at a cell concentration of 0.5x10 ⁴ / well in a 6-well plate coated with collagen, followed by incubation for 3 days with 1 mM n-butyric acid and 3 μM of the inventive compound. Cells were fixed by treatment with 1% paraformaldehyde solution and stained with Alexa 488 fluorescence-beta-tubulin antibody (Santa Cruz Biotechnology) to observe the nerve pole under fluorescence microscope. We tried to analyze the length of the nerve pole using Image J (NIH) software.

result

The effect of the compounds of the present invention on neurite neurite growth is shown in Fig. As shown in FIG. 1, Compounds 1 and 13 of the present invention were found to be superior in neuronal cell protection and neurogenic germination growth as compared with the DMSO comparison group. Statistical significance was p <0.05.

Claims (6)

A prophylactic and therapeutic agent for degenerative brain diseases such as Parkinson's disease comprising a compound of the formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, prodrug or isomer thereof:
(I)

Wherein R ₁ is selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-7 cycloalkyl, aryl, benzyl, heteroaryl, hydroxy-C _1-6 alkyl, C _2-14 heterocycloalkyl, -CONR ₇ R _8, or is -COR _9, n is an integer from 0 to _2, R 2 is C _{1 -6} alkyl, C _{3 -7-cycloalkyl,} C _{2 -14} heterocycloalkyl, C _6-14 aryl or heteroaryl to be. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, sulfate, acetate, formate, succinate, malate, fumarate, toluenesulfonate and methanesulfonate. Prevention and treatment of degenerative brain diseases such as diseases. The method of claim 1, wherein, R ₁ is C _{2 -14} heterocycloalkyl alkyl. The compound of claim 1, wherein R ₂ is C _{2 -14} heterocycloalkyl, C _{6 -14} aryl, or heteroaryl. The compound of formula I according to claim 1, wherein the compound of formula I is selected from the group consisting of 4- (3-isopropylphenylamino) -2- (tetrahydro-2H-pyran-4-yl) pyrido [ (6H) -one, 4- (3-isopropylphenylamino) -2- (piperidin-4-yl) pyrido [ 4-ylamino) -2- (piperidin-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (1-cyclopropyl-1H 4-ylamino) -2- (piperidin-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) 4- (3-isopropylamino) -2- (1-isopropylpyridin-4-yl) Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (l-isopropyl- lH- pyrazol- Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (l-Isopropyl-lH- pyrazol- (1-iso Yl) pyrido [4,3-d] pyrimidin-5 (6H) -one and 4- (2- isopropylpyridin- 4- ylamino) -2- (tetrahydro- Pyrazol-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) Pyrido [4,3-d] pyrimidin-5 (6H) -one, 4- (l -cyclopropyl- lH- pyrazol- Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (3-t- butylphenylamino) -2- (tetrahydro- Yl) pyrido [4,3- d] pyrimidin-5 (6H) -one, 4- (1-t- butyl- lH-pyrazol-4-ylamino) -2 - (tetrahydro-2H-pyran-4-yl) pyrido [4,3-d] pyrimidin-5 (6H) [4,3-d] pyrimidin-5 (6H) -one, 2-isopropyl- (6H) -one, 4- (l-cyclopropyl-lH-pyrazol-4-ylamino) -2-isopropylpyrido [4,3-d] pyrimidin- , 2- (4-chloropyridin-4-yl) propyl] -4- (3-isopropylphenylamino) pyrido [ Pyrimido-5 (6H) -one and 2-cyclopropyl-4- (l-cyclopropyl-lH-pyrazol-4ylamino) pyrido [4,3 -d] pyrimidine-5 (6H) -one. &lt; / RTI &gt; The compound according to claim 1, which is useful as a preventive and therapeutic agent for degenerative brain diseases such as Parkinson's disease.

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