Pyridinopyrrole amino acid derivative and application thereof in preparation of leucine-rich repeat kinase 2 inhibitor

Abstract

The invention discloses a pyridinopyrrole amino acid derivative and application thereof in preparation of a leucine-rich repeat kinase 2 inhibitor, the pyridinopyrrole amino acid derivative not only has a good inhibition effect on LRRK2 activity, but also has the structural characteristic of active transport by amino acid transporter on BBB, the ability of a drug to enter a center is increased, and the drug activity is improved. The compound is a potential therapeutic drug for treating central nervous degenerative diseases (such as Parkinson's disease).

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drugs and provides a pyridopyrrole amino acid derivative and an application thereof in preparing a leucine-rich repeat kinase 2 inhibitor. Background Art

[0002] Parkinson's disease (PD) is a chronic neurodegenerative disease that mainly affects the elderly. Its incidence rate ranks second only to Alzheimer's disease. Since it was first discovered and named in 1817, humans have been fighting against it for more than two centuries. According to my country's "Guidelines for the Treatment of Parkinson's Disease" (4th edition), the prevalence of Parkinson's disease in people over 65 years old is about 1.7%, while that in people over 80 years old is more than 4%. It is estimated that by 2030, the number of Parkinson's disease patients in my country will reach 5 million. At present, the awareness, consultation rate and diagnosis rate of Parkinson's disease are low, and there is no cure. Patients suffer from motor nervous system disorders such as tremor, limb stiffness, motor dysfunction and gait abnormalities throughout their lives, as well as non-motor symptoms such as hyposmia, sleep disorders and constipation. Existing drug treatments can only relieve symptoms to a certain extent, but cannot prevent the progression of the disease. Commonly used drugs in clinical practice are far from meeting the needs of patients with mid- to late-stage Parkinson's disease. There is an urgent need to develop a class of drugs that can prevent Parkinson's disease pathology and biochemical degeneration. Disease-modifying therapy is the current mainstream direction for the development of drugs for Parkinson's disease. It can affect the initial triggering factors of neuronal degeneration and promote neuronal compensatory responses or reduce pathological propagation and progression. Current mainstream research believes that the aggregation of α-Syn in Lewy bodies (LB) is an important cause of Parkinson's disease, and reducing the aggregation of α-Syn is a potential method for treating Parkinson's disease.

[0003] Leucine-rich repeat kinase 2 (LRRK2) blocks chaperone-mediated autophagy, resulting in the inability to degrade α-syn and causing toxicity. LRRK2 is involved in α-syn-mediated neurotoxicity. LRRK2 induces mitochondrial damage and endolysosomal dysfunction through oxidative mechanisms, inducing the progression of Parkinson's disease. LRRK2 kinase inhibitors can reduce pathological damage in Parkinson's disease models and improve patients' motor dysfunction. The safety and tolerability of two new LRRK2 kinase inhibitors, DNL201 and DNL151, were successfully tested in Phase IB clinical trials, and DNL151 has been launched into Phase IIb / III registration clinical studies. Therefore, the development of LRRK2 small molecule inhibitors is one of the most promising research directions for the development of Parkinson's treatment drugs.

[0004] Therefore, developing effective inhibitors of LRRK2 kinase and mutant LRRK2 kinase has become an important approach to treat neurodegenerative diseases. The present invention aims to invent a compound that can highly inhibit LRRK2 kinase, thereby further inventing a drug that can effectively treat neurodegenerative diseases.

[0005] Patent US8791112B2 discloses that Arrien's pyrido-pyrrole compounds are a class of LRRK2 inhibitors, and the general chemical structure formula is shown below.

[0006]

[0007] Although 148 in the patent has good LRRK2 inhibitory activity, with an IC50 activity of <0.5μM, experiments have found that this type of compound has low blood-brain barrier permeability and poor pharmacokinetic properties, so it has stopped at the preclinical research stage. This indicates that it is necessary to find better blood-brain barrier permeable compounds for this target in order to ensure effective drug administration to the brain while reducing drug concentration and toxic side effects in peripheral tissues. Summary of the invention

[0008] In view of this, the object of the present invention is to provide a pyridopyrrole amino acid derivative and its use in the preparation of leucine-rich repeat kinase 2 inhibitors.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] 1. A pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof, wherein the structure of the derivative is shown in the general formula (1):

[0011]

[0012] In the general formula (1),

[0013] R 1 Select from H or C 1-5 alkyl;

[0014] R 2 Select from H or C 1-5 Alkoxy.

[0015] Preferably, in the general formula (1),

[0016] R 1 Selected from H or methyl;

[0017] R 2 is selected from H or methoxy.

[0018] More preferably, in the general formula (1),

[0019] R1 is H or methyl;

[0020] R 2 For H.

[0021] Preferably, the derivative is (S)-2-amino-3-(3-(3-(cyclopropanecarbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)propanoic acid ethyl ester, and its chemical structure is as follows:

[0022]

[0023] Preferably, the derivative is (S)-2-amino-3-(3-(3-(cyclopropanecarbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)propanoic acid, and its chemical structure is as follows:

[0024]

[0025] Preferably, the derivative is (S)-2-amino-3-(4-(3-(cyclopropanecarbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)propanoic acid ethyl ester, and its chemical structure is as follows:

[0026]

[0027] Preferably, the derivative is (S)-2-amino-3-(4-(3-(cyclopropanecarbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)propanoic acid, and its chemical structure is as follows:

[0028]

[0029] Preferably, the derivative is (S)-2-amino-3-(3-(3-(cyclopropanecarbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-4-methoxyphenyl)propanoic acid, and its chemical structure is as follows:

[0030]

[0031] 2. Use of the aforementioned pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof in the preparation of a leucine-rich repeat kinase 2 inhibitor.

[0032] 3. Use of the aforementioned pyridopyrrole amino acid derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for inhibiting the activity of leucine-rich repeat kinase 2 to prevent and / or treat diseases.

[0033] 4. Use of the aforementioned pyridopyrrole amino acid derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for treating or preventing chronic neurodegenerative diseases.

[0034] 5. Use of the aforementioned pyridopyrrole amino acid derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for treating or preventing Parkinson's disease.

[0035] 6. A pharmaceutical composition or preparation comprising the aforementioned pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof.

[0036] Preferably, it further comprises pharmaceutically acceptable excipients, adjuvants or carriers.

[0037] The beneficial effects of the present invention are:

[0038] The applicant introduced amino acid fragments into the structure of existing lead compounds, aiming to maintain the inhibitory activity on LRRK2 while increasing the property of active transport of the compounds into the central nervous system through amino acid transporters on the BBB, in order to find potential drugs for the treatment of Parkinson's disease.

[0039] The present invention provides a class of amino acid derivatives with pyridopyrrole structures, which not only have a good inhibitory effect on LRRK2 activity, but also have the structural characteristics of being actively transported by amino acid transporters on the BBB, thereby increasing the ability of drugs to enter the central nervous system. The derivatives are potential therapeutic drugs for treating central nervous system degenerative diseases (such as Parkinson's disease).

[0040] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0042] Figure 1 This is the synthetic route of the target compound F1 of Example 1;

[0043] Figure 2 This is the synthetic route of the target compound F2 of Example 2;

[0044] Figure 3 This is the synthetic route of the target compound F3 of Example 3;

[0045] Figure 4 This is the synthetic route of the target compound F4 of Example 4;

[0046] Figure 5 This is the synthetic route of the target compound F5 in Example 5. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific implementation modes.

[0048] Definition and Description

[0049] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0050] The term "pharmaceutically acceptable" as used herein refers 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 humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0051] The term "pharmaceutically acceptable salt" refers to salts of compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or 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, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds of the present invention contain basic and acidic functional groups and can be converted into either base or acid addition salts.

[0052] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0053] The term "therapeutically effective dose" refers to an amount of a compound of the formula sufficient to be effective for treatment when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending on the specific activity of the therapeutic agent used, the age, physiological condition, presence of other disease states, and nutritional status of the patient. In addition, other drug treatments that the patient may be receiving will affect the determination of the therapeutically effective amount of the therapeutic agent to be administered.

[0054] The term "treat" means any treatment of a disease in a mammal, including: (i) preventing the disease, i.e., causing clinical symptoms of the disease not to develop; (ii) inhibiting the disease, i.e., arresting the development of clinical symptoms; and / or (iii) palliating the disease, i.e., causing regression of clinical symptoms.

[0055] The term "pharmaceutically acceptable excipient, adjuvant or carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0056] As used herein, the symbols and conventions used in these processes, schemes and examples are consistent with those used in contemporary scientific literature, such as the Journal of the American Chemical Society or the Journal of Biological Chemistry. Unless otherwise indicated, all starting materials were obtained from commercial suppliers and used without further purification. For example, the following abbreviations may be used in the examples and throughout the specification: 1,4-Dioxane (1,4-dioxane), 4.0M HCl in Dioxane (4.0M hydrochloric acid dioxane), 4.0M HCl in EA (4.0M hydrochloric acid ethyl ester), AcCl (acetyl chloride), ACN (acetonitrile), AcOK (potassium acetate, anhydrous), Ag 2 SO 4 (Silver sulfate, anhydrous), AlCl 3 (Aluminum chloride), aq.(aqueous solution), (Boc) 2 O(di-tert-butyl dicarbonate), BPD(biboronic acid pinacol ester), DCM(dichloromethane), EA(ethyl acetate), FA(formic acid), g(gram), h(hour), HCl(hydrogen chloride), Hz(hertz), I 2 (iodine), L (liter), L (microliter), LiOH (lithium hydroxide, anhydrous), m (milliliter), M (mole), mg (milligram), MHz (megahertz), min (minute), mM (millimole), mmol (millimole), mol (mole), Na 2 S 2 O 3 (Sodium thiosulfate, anhydrous), NaHCO 3 (Sodium bicarbonate, anhydrous), Pd(dppf)Cl 2([1,1-bis(diphenylphosphino)ferrocene]palladium dichloride), Pd(PPh 3 ) 4 (Tetrakis(triphenylphosphine)palladium);Na 2 CO 3 (sodium carbonate, anhydrous), rt (room temperature), TLC (thin layer chromatography).

[0057] The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art, and preferred embodiments include but are not limited to the embodiments of the present invention. Any intermediate or compound in the synthetic route that can be obtained by other reaction conditions is considered an alternative to the invention.

[0058] Example 1: Synthesis of target compound F1

[0059] Synthesis route such as Figure 1 shown.

[0060] The specific steps are as follows:

[0061] Step 1: Preparation of intermediate 3

[0062] First, AlCl 3 (54.1g, 405.6mmol) was dissolved in DCM (950ml), and then 5-bromo-7-azaindole (CAS: 183208-35-7, source: Shanghai Titan Technology Co., Ltd., 10g, 50.7mmol) was added. After the addition, it was stirred at room temperature of 25°C for 1h. Finally, cyclopropanecarbonyl chloride (CAS No.: 4023-34-1, source: Shanghai Titan Technology Co., Ltd., 7.43g, 71.0mmol) was added. After the addition, it was moved to an oil bath at 45°C to react overnight. The reaction process was monitored by TLC plate. After the reaction was completed, the unreacted AlCl 3 , rinse the filter cake with DCM. Pour the filtrate into water and extract with ethyl acetate. The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product is purified by silica gel column chromatography (elution gradient is ethyl acetate / petroleum ether volume fraction = 0-30%) to obtain intermediate 3 (8.44 g, 62%) as a white solid. [M+H] + =265; [M+H] + =267;

[0063] Step 2: Preparation of intermediate 4

[0064] Intermediate 3 (6 g, 22.63 mmol), BPD (5.75 g, 22.63 mmol), Pd(dppf)Cl 2 (828mg, 1.13mmol), AcOK (4.44g, 45.26mmol) were added to a 150ml sealed tube, and then 1,4-Dioxane (60ml) was added, and nitrogen was protected, and the reaction was carried out in an oil bath at 110℃ for 2h. The reaction process was monitored by TLC plate. After the reaction was completed, the reaction solution 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 obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient was methanol / dichloromethane volume fraction = 0-10%) to obtain intermediate 4 (2.61g, 36%) as a white solid. [M+H] + =313 (boronic acid); [M+H] + =231 (boric acid).

[0065] Step 3: Preparation of intermediate 6

[0066] (S)-3-(3-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid ethyl ester (CAS No.: 1159502-91-6, source: Shanghai Titan Technology Co., Ltd., 230 mg, 0.61 mmol), intermediate 4 (231.45 mg, 0.74 mmol), Pd(PPh 3 ) 4 (70.49 mg, 0.061 mmol), Na 2 CO 3 (129.31mg, 1.22mmol) was added to a 15ml sealed tube, and then 1,4-Dioxane (2ml) / water (400μl) was added, and nitrogen was protected, and the reaction was carried out in an oil bath at 100℃ for 2h. The reaction process was monitored by TLC plate. After the reaction was completed, the reaction solution 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 obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient was methanol / dichloromethane volume fraction = 0-10%) to obtain intermediate 6 (167mg, 56.61%) as a white solid. [M+H] + =478; [M-56] + =422; [M-Boc] + =378.

[0067] Step 4: Preparation of compound S9

[0068] Intermediate 6 (160 mg, 0.49 mmol) was added to a reaction tube, and then 4.0 M HCl in EA solution (2 ml) was added. The reaction was carried out at room temperature of 25°C for 2 h, and the reaction process was monitored by TLC plate. After the reaction was completed, the crude product was concentrated under reduced pressure. The crude product F1 was purified again by a preparative chromatography column (elution gradient was ACN / 0.1% FA volume fraction = 30% to 70%), and freeze-dried by a freeze dryer to obtain compound F1 (51.1 mg, 36.85%) as a white solid. [M+H] + =378; HPLC = 95.01%; 1 H NMR (600 MHz, DMSO-d 6 )δ12.60(s,1H),8.71(s,1H),8.66(d,J=2.3Hz,1H),8.59(d,J=2.2Hz,1H),8.16(s,1H),7. 54(dt,J=7.9,1.3Hz,1H),7.51(d,J=1.9Hz,1H),7.39(t,J=7.6Hz,1H),7.20(dt,J=7.6,1.4 Hz,1H),4.11–3.99(m,2H),3.75(t,J=6.8Hz,1H),2.95(qd,J=13.6,6.8Hz,2H),2.79(tt,J =7.9, 4.6Hz, 1H), 1.09 (t, J = 7.1Hz, 3H), 0.99 (p, J = 3.3Hz, 2H), 0.91 (dt, J = 8.0, 3.4Hz, 2H).

[0069] Example 2: Synthesis of target compound F2

[0070] Synthesis route such as Figure 2 shown.

[0071] The specific steps are as follows:

[0072] Step 1: Preparation of compound F2

[0073] Compound F1 (184 mg, 0.44 mmol) was added to a round-bottom flask, followed by ethanol (4 ml) / water (1 ml), and finally lithium hydroxide (85.15 mg, 3.55 mmol). The reaction was carried out at room temperature of 25°C for 2 h, and the reaction was monitored by TLC plate. After the reaction was completed, the pH of the reaction solution was adjusted to a weak acidic state using an aqueous formic acid solution (the volume ratio of formic acid to water was 1:10); ethyl acetate was used for extraction, and the extracted organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was filtered through a filter membrane to remove the residual impurities, and the filtrate was evaporated to dryness to obtain a crude product. The crude product F2 was purified again using a preparative chromatographic separation column (the elution gradient was ACN / 0.1% FA volume fraction = 30% to 70%), and the compound F2 (71.6 mg, 46%) was obtained as a white solid by freeze drying. [M+H] + =350; HPLC = 98.85%; 1 H NMR (600 MHz, DMSO-d 6 )δ8.72–8.67(m,2H),8.64–8.59(m,1H),7.59(d,J=1.9Hz,1H),7.54(dd,J=8 .4,7.0Hz,1H),7.39(t,J=7.6Hz,1H),7.27(d,J=7.5Hz,1H),3.44(dd,J=8.4, 4.3Hz,1H),3.22(dd,J=14.3,4.4Hz,1H),2.93(dd,J=14.3,8.2Hz,1H),2.79( ddd,J=12.3,7.9,4.6Hz,1H),1.05–0.96(m,2H),0.90(dq,J=7.1,3.3Hz,2H).

[0074] Example 3: Synthesis of F3

[0075] Synthesis route such as Figure 3 shown.

[0076] The specific steps are as follows:

[0077] Step 1: Preparation of intermediate 8

[0078] (S)-3-(4-bromophenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid ethyl ester (CAS: 591249-54-6, source: Shanghai Haohong Biopharmaceutical Technology Co., Ltd., 300 mg, 0.80 mmol), intermediate 4 (301.90 mg, 0.97 mmol), Pd(PPh 3 ) 4 (93.12 mg, 0.08 mmol), Na 2 CO 3(170.83mg, 1.61mmol) was added to a 15ml sealed tube, and then 1,4-Dioxane (5ml) / water (1ml) was added, and nitrogen was protected, and the reaction was carried out in an oil bath at 100℃ for 2h. The reaction process was monitored by TLC plate. After the reaction was completed, the reaction solution 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 obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient was methanol / dichloromethane volume fraction = 0-10%) to obtain intermediate 8 (106.7mg, 27.72%) as a white solid. [M+H] + =478; [M-56] + =422; [M-Boc] + =378.

[0079] Step 2: Preparation of Compound F3

[0080] Intermediate 8 (100 mg, 0.21 mmol) was added to a reaction tube, and then 4.0 M HCl in EA solution (2 ml) was added. The reaction was carried out at room temperature of 25°C for 2 h, and the reaction process was monitored by TLC plate. After the reaction was completed, the crude product was concentrated under reduced pressure. The crude product F3 was purified again by a preparative chromatography column (elution gradient was ACN / 0.1% FA volume fraction = 30% to 70%), and the compound F3 (18 mg, 20.93%) was obtained as a white solid by freeze drying. [M+H] + =378; HPLC = 96.27%; 1 HNMR (600 MHz, DMSO-d 6 )δ12.59(s,1H),8.71(s,1H),8.65(d,J=2.3Hz,1H),8.59(d,J=2.2Hz,1 H),8.14(s,1H),7.64–7.59(m,2H),7.34–7.24(m,2H),4.12–4.00(m,2H ),3.82(t,J=6.8Hz,1H),3.00–2.91(m,2H),2.79(tt,J=7.9,4.6Hz,1H) ,1.11(t,J=7.1Hz,3H),1.01–0.94(m,2H),0.90(dq,J=10.2,3.3Hz,2H).

[0081] Example 4: Synthesis of target compound F4

[0082] Synthesis Figure 4 shown.

[0083] The specific steps are as follows:

[0084] Step 1: Preparation of compound F4

[0085] Compound F3 (150 mg, 0.36 mmol) was added to a round-bottom flask, followed by ethanol (4 ml) / water (1 ml), and finally lithium hydroxide (69.44 mg, 2.89 mmol). The reaction was carried out at room temperature of 25°C for 2 h, and the reaction was monitored by TLC plate. After the reaction was completed, the pH of the reaction solution was adjusted to a weak acid using an aqueous formic acid solution (the volume ratio of formic acid to water was 1:10); ethyl acetate was used for extraction, and the extracted organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was filtered through a filter membrane to remove the residual impurities, and the filtrate was evaporated to dryness to obtain a crude product. The crude product F4 was purified again using a preparative chromatographic separation column (the elution gradient was ACN / 0.1% FA volume fraction = 30% to 70%), and the compound F4 (43.1 mg, 34%) was obtained as a white solid by freeze drying. [M+H] + =350; HPLC = 98.08%; 1 H NMR (600 MHz, DMSO-d 6 )δ8.70(s,1H),8.65(d,J=2.3Hz,1H),8.58(d,J=2.3Hz,1H),7.64–7.58(m,2H),7.41–7.35(m,2H),3.44(dd,J=8.2,4.5Hz,1H),3.18(dd, J=14.4,4.6Hz,1H),2.90(dd,J=14.3,8.2Hz,1H),2.78(ddd,J=12.5,7.9,4.6Hz,1H),0.98(p,J=3.5Hz,2H),0.90(dt,J=8.2,3.5Hz,2H).

[0086] Example 5: Synthesis of F5

[0087] Synthesis route such as Figure 5 shown.

[0088] The specific steps are as follows:

[0089] Step 1: Preparation of intermediate 10

[0090] 4-Methoxy-L-phenylalanine (CAS No.: 6230-11-1, source: Shanghai Titan Technology Co., Ltd., 500 mg, 2.56 mmol) was dissolved in methanol (7 mL), and AcCl (1 g, 12.8 mmol) was added dropwise to the reaction system at 0°C. After the addition was completed, stirring was continued for 30 minutes, and then the mixture was moved to an oil bath and heated under reflux for 4 hours. After the reaction was completed, the solvent was dried, and ethanol (6 mL) and NaHCO were added. 3(430 mg, 5.12 mmol), and (Boc) 2 O (559 mg, 2.56 mmol), reacted at room temperature at 25 ° C for 5 hours, and the reaction process was monitored by TLC plate. After the reaction was completed, the solvent was spin-dried, poured into water with a coarse filter and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient: ethyl acetate / petroleum ether volume fraction = 0-10%) to obtain intermediate 10 (783 mg, 99%) as a colorless transparent liquid. [M+H] + =310.

[0091] Step 2: Preparation of intermediate 11

[0092] Intermediate 10 (310 mg, 1.0 mmol) was dissolved in methanol (15 mL) and Ag was added at 0 °C. 2 SO4 (342 mg, 1.1 mmol), 1 2 (280 mg, 1.1 mmol) was added to the reaction system and stirred for 30 minutes. The reaction was monitored by TLC. 2 S 2 O 3 The solution was quenched and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient: ethyl acetate / petroleum ether volume fraction = 0-10%) to obtain intermediate 11 (431 mg, 90%) as a colorless transparent liquid. [M+H] + =436.

[0093] Step 3: Preparation of intermediate 12

[0094] Intermediate 11 (340 mg, 0.78 mmol), Intermediate 4 (390 mg, 1.25 mmol), Pd(dppf)Cl 2 (57 mg, 0.078 mmol), Na 2 CO 3 (207mg, 1.95mmol) was added to a 15ml sealed tube, and then 1,4-Dioxane (4ml) / water (800μl) was added, and nitrogen was protected, and the reaction was carried out in an oil bath at 100℃ for 2h. The reaction process was monitored by TLC plate. After the reaction was completed, the reaction solution 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 obtain a crude product. The crude product was purified by silica gel column chromatography (elution gradient was ethyl acetate / petroleum ether volume fraction = 0-20%) to obtain intermediate 12 as a white solid. [M+H] + =494.

[0095] Step 4: Preparation of Compound F5

[0096] After intermediate 12 (230 mg, 0.466 mmol) was dissolved in methanol / water (4 / 1 mL), LiOH (22 mg, 0.932 mmol) was added to the reaction system and stirred at room temperature at 25°C for 2 hours. The reaction process was monitored by TLC plate. After the reaction was completed, the pH was adjusted to about 4-5 with 10% acetic acid aqueous solution and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product without purification and directly carried out to the next step.

[0097] The obtained crude product was added to a 10 mL single-mouth bottle, and 4.0 M HCl in Dioxane solution (4 mL) was added, and stirred at room temperature 25°C for 2 hours. The reaction process was monitored by LC-MS. After the reaction was completed, the solvent was dried by spin drying and separated and purified by preparative chromatography (elution gradient was ACN / 0.1% FA volume fraction = 30% to 70%), and freeze-dried by a freeze dryer to obtain compound F5 (22 mg, 12.46%) as a white solid. [M+H] + =380; HPLC = 95.01%; 1 H NMR (400 MHz, DMSO-d 6 )δ12.68(s,1H),8.68(s,1H),8.57(s,1H),8.40(s,1H),7.25(s,2H),7.02(d,J=8.6Hz,1H),3.72(s,3H),3.48(dd,J=7.8,4.4Hz,1H ), 3.14(dd,J=14.4,4.3Hz,1H),2.92(dd,J=14.3,7.8Hz,1H),2.82–2.72(m,1H),0.98(t,J=3.9Hz,2H),0.89(dt,J=7.1,3.9Hz,2H).

[0098] Biological activity test

[0099] Protein binding assay:

[0100] Reagents and consumables:

[0101] LRRK2 G2019S enzyme (Thermo Fisher), substrate (LRRKtide) (Thermo Fisher), ATP (Thermo Fisher), TR-FRET diluent (Thermo Fisher), pLRRKtide antibody (Thermo Fisher), 384-well plate (PE) DMSO (Solarbio)

[0102] Experimental process:

[0103] All test compounds (including positive controls and test samples) were diluted to 1 mM with DMSO to obtain the corresponding test compound solutions. 35 μL of positive compound (structural formula is shown in Table 1, similar structural compounds disclosed by Arrien in patent US8791112B2, and synthesized with reference to the document) solution, 35 μL of test compound solution, and 35 μL of blank solution were added to a 384-well plate, and the plate was centrifuged at 2500 rpm for 1 minute. With 1 mM as the initial concentration, 3-fold gradient dilution was performed for 10 points, and 100 nL of positive compound, test compound, and blank well solution were added to another 384-well assay plate, 3 replicates, and the plate was centrifuged at 2500 rpm for 1 minute and sealed in foil for standby use.

[0104] Enzyme reaction: Dilute the LRRKtide substrate and LRRK2G2019S kinase mixed working solution (final concentration of LRRKtide substrate: 400nM and LRRK2 G2019S kinase: 580ng / mL) with assay buffer (Thermo Fisher TR-FRET Dilution buffer) and add it to all sample wells of the above 384 assay plate, 5μL per well, and incubate the 384 assay plate at 23°C for 20 minutes. After incubation, dilute the 2×ATP working solution (134μM) with assay buffer and add it to each well, 5mL per well, and incubate the 384 assay plate at 23°C for 60 minutes.

[0105] Detection: Dilute EDTA and pLRRKtide antibody with assay buffer (TR-FRET Dilution buffer) to obtain a mixed working solution (final concentration of EDTA: 10mM, pLRRKtide antibody: 2nM). Then add 10μL of the antibody mixed working solution to each well of the above 384 assay plate and incubate at 23°C for 60 minutes. Read the plate in the TE-FRET mode with 340nm excitation light, 520nm fluorescence emission light and 490nm terbium emission light.

[0106] Method reference: J. DeBicente Fidalgo et al. Compounds, compositions and methods: CN113939294A[P].2022-01-14.

[0107] The activity data of each compound are shown in Table 1.

[0108] Table 1. Compound activity data

[0109]

[0110]

[0111] As shown in Table 1, the series of new compounds provided in the embodiments of the present invention have a strong inhibitory effect on the kinase LRRK2 G2019S. Compared with the inhibitory activity of the positive structure on LRRK2 G2019S, especially the compound F2 provided in Example 2 of the present invention and the compound F4 provided in Example 4 have a better inhibitory effect on the mutant kinase LRRK2 G2019S, the compound F1 provided in Example 1 and the compound F3 provided in Example 3 also have a better inhibitory effect on the mutant kinase LRRK2G2019S, and the compound F5 provided in Example 5 has an inhibitory effect on the mutant kinase LRRK2 G2019S comparable to the positive structure. It has potential application value in the preparation of drugs for preventing and / or treating diseases related to increased gene LRRK2 activity in vivo.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the derivative is shown in the general formula (1): In the general formula (1), R1 is selected from H or C 1-5 alkyl; R2 is selected from H or C 1-5 Alkoxy.

2. The pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the general formula (1), R1 is selected from H or methyl; R2 is selected from H or methoxy.

3. The pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: In the general formula (1), R1 is H or methyl; R2 is H.

4. Use of the pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a leucine-rich repeat kinase 2 inhibitor.

5. Use of the pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug for preventing and / or treating diseases by inhibiting the activity of leucine-rich repeat kinase 2.

6. Use of the pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing chronic neurodegenerative diseases.

7. Use of the pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug for treating or preventing Parkinson's disease.

8. A pharmaceutical composition or preparation, characterized in that The invention comprises the pyridopyrrole amino acid derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

9. A pharmaceutical composition or preparation according to claim 8, characterized in that: It also contains pharmaceutically acceptable excipients, adjuvants or carriers.

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