Nitrogen-containing heterocyclic compound, pharmaceutical composition and application thereof

Abstract

The present invention discloses a nitrogen-containing heterocyclic compound, a pharmaceutical composition and application thereof. The present invention provides a compound as shown in formula I or a pharmaceutically acceptable salt thereof. The compound of the present invention has a strong inhibitory effect on OGA and has a good application prospect in preventing and / or treating various diseases related to OGA.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to nitrogen-containing heterocyclic compounds, pharmaceutical compositions and applications thereof. Background Art

[0002] Many proteins in cells, including those in the nucleus and cytoplasm, can be post-translationally modified by adding the monosaccharide 2-acetylamino-2-deoxy-β-D-glucoside (N-acetylglucosamine). This modification is called O-linked N-acetylglucosamine (O-GlcNAc). The enzyme responsible for this modification is O-GlcNAc transferase (OGTase), and another enzyme O-GlcNAcase (OGA) can remove this modification, allowing the protein to perform its function.

[0003] O-GlcNAc-modified proteins regulate a variety of cellular functions, including transcription, protein degradation, and cell signaling. O-GlcNAc is also present on the cytoskeletal protein "tau," which is important for stabilizing the intracellular microtubule network.

[0004] Tau protein in the brain of patients with Alzheimer's disease (AD) is overphosphorylated, which leads to the destruction of its normal function and the formation of deformed neurofibrillary tangles (NFTs). Studies have found that hyperphosphorylation inside neurons is related to O-GlcNAc modification, and increased phosphorylation levels lead to decreased O-GlcNAc levels, and vice versa. This may be due to damage to glucose transport and metabolism, causing hyperphosphorylation of tau protein. Inhibiting O-GlcNAcase enzymes can prevent the hyperphosphorylation of tau, thereby compensating for the attenuation of brain glucose metabolism, which may become a potential target for the treatment of AD and other neurodegenerative diseases (progressive supranuclear palsy or Parkinson's). Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect that there are few types of O-GlcNAcase (OGA) inhibitors in the prior art, and to provide a class of nitrogen-containing heterocyclic compounds, pharmaceutical compositions and applications thereof. Such compounds have a strong inhibitory effect on OGA and have good application prospects in preventing and / or treating various diseases related to OGA.

[0006] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt thereof,

[0007]

[0008] Among them, the "The carbon atom is in R configuration, S configuration or a mixture of the two;

[0009] Each R 1are independently halogen;

[0010] Each R 2 are independently C1-C6 alkyl;

[0011] R 3 is a halogen;

[0012] R 4 and R 5 are each independently hydrogen or ;

[0013] R 4-1 is a C1-C6 alkyl group;

[0014] m and n are each independently 1, 2 or 3.

[0015] In certain preferred embodiments of the present invention, certain groups in the compound of Formula I or a pharmaceutically acceptable salt thereof are defined as follows, and the unmentioned groups are the same as those described in any embodiment of the present invention (referred to as "in a certain embodiment of the present invention").

[0016] In one embodiment of the present invention, each "C1-C6 alkyl" is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; for example, methyl.

[0017] In one embodiment of the invention, each "halogen" is independently fluorine, chlorine, bromine or iodine; for example, fluorine.

[0018] In one embodiment of the present invention, for ;For example, .

[0019] In one embodiment of the present invention, for ;For example, , where end a and connected;

[0020] Preferably, for ;For example, ; a end and connected.

[0021] In one embodiment of the present invention, R 4 For hydrogen.

[0022] In one embodiment of the present invention, R 5 for .

[0023] In one embodiment of the present invention, m and n are 1.

[0024] In one embodiment of the present invention, the compound as shown in formula I is .

[0025] The present invention also provides a pharmaceutical composition, comprising:

[0026] (1) the compound of formula I or a pharmaceutically acceptable salt thereof; and

[0027] (2) Pharmaceutically acceptable excipients.

[0028] The present invention also provides the use of the compound shown in Formula I, a pharmaceutically acceptable salt thereof or the pharmaceutical composition in the preparation of an O-acetylglucosaminidase (O-GlcNAcase, referred to as OGA) inhibitor.

[0029] The present invention also provides the use of the compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a method for preventing and / or treating a disease or condition associated with OGA; the disease or condition may be Alzheimer's disease.

[0030] The present invention also provides the use of the compound as shown in Formula I, its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of a drug for preventing and / or treating Alzheimer's disease, progressive supranuclear palsy or Parkinson's disease; for example, Alzheimer's disease.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those of the claimed subject matter.

[0032] The standard meaning in the art. If there are multiple definitions for a term, the definition in this document shall prevail.

[0033] " " means that the structural fragment is connected to other fragments in the molecule through this site. For example, It refers to acetylamino.

[0034] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0035] The term "alkyl" refers to a straight or branched chain alkyl group having a specified number of carbon atoms (e.g., C1-C6). Alkyl groups include, but are not limited to, methyl, ethyl, and the like.

[0036] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, 2002).

[0037] The term "pharmaceutical composition" refers to a mixture or solution containing a therapeutically effective amount of an active drug ingredient and a pharmaceutically acceptable excipient, ready for administration to a mammal, such as a human, in need thereof.

[0038] The term "pharmaceutically acceptable excipient" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient, and representative excipients include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc. Their preparations are well known to those skilled in the field of cosmetics or topical medicines.

[0039] The term "treat" refers to therapeutic treatment. With respect to a specific condition, treatment means: (1) ameliorating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0040] The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0041] The inhibitor drug can be used in mammals; it can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the art to provide rapid detection of the effect of inhibiting O-acetylglucosaminidase.

[0042] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0043] The reagents and raw materials used in the present invention are commercially available.

[0044] The positive progress of the present invention is that the present invention provides a class of nitrogen-containing heterocyclic compounds, pharmaceutical compositions and applications thereof. The compounds have a strong inhibitory effect on OGA and have good application prospects in preventing and / or treating various diseases related to OGA. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Two-dimensional NMR image of compound B-4.

[0046] Figure 2 : The structural formula of compound 1. DETAILED DESCRIPTION

[0047] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0048] Intermediate A

[0049] Synthesis route:

[0050]

[0051] first step

[0052] Compound A-1 (6.80 g, 41.8 mmol) and pyridine (13.2 g, 167 mmol) were dissolved in dichloromethane (80 mL), and acetic anhydride (12.8 g, 125 mmol) was slowly added dropwise under stirring at 0 °C, and the reaction solution was stirred at 25 °C for 20 hours. Water (100 mL) was added to the reaction solution, and ethyl acetate (100 mL x 3) was used for extraction. The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 2 / 1, v / v) to obtain compound A-2. 1 H NMR (400 MHz, DMSO-d6): δ 13.00 (s,1H), 9.90 (s, 1H), 2.22 (s, 3H). ESI-MS calculated value: [M+H] + = 204.98, measured value 204.9.

[0053] Step 2

[0054] Compound A-2 (3.00 g, 14.7 mmol) and cesium fluoride (22.3 g, 147 mmol) were dissolved in dimethyl sulfoxide (50 mL) and stirred at 140 °C for 20 hours in a nitrogen atmosphere. The reaction solution was cooled to room temperature, and water (200 mL) and ethyl acetate (200 mL x 3) were added for extraction. The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 2 / 1, v / v) to obtain a crude product. The crude product was purified by high performance liquid chromatography (Waters-spherical-C18-35µm-19*250mm, mobile phase: acetonitrile-0.1% formic acid aqueous solution, flow rate: 100 mL / min, gradient: 5-25%, retention time: 32-40 min) to obtain compound intermediate A. 1 H NMR (400 MHz, DMSO-d6) δ 13.00 (s, 1H), 9.87 (s, 1H), 2.21(s, 3H). ESI-MS calculated value: [M+H] + = 189.01, measured value 189.0.

[0055] Intermediate B

[0056] Synthesis route:

[0057]

[0058] first step

[0059] Dissolve the compound ethyl propiolate (16.1 g, 164 mmol) in tetrahydrofuran (125 mL). Under a nitrogen atmosphere, add a solution of n-butyl lithium in tetrahydrofuran (56.3 mL, 141 mmol, 2.5 mol / L) at -70 °C and stir at -70 °C for 0.5 hour. Slowly add a solution of compound B-1 (10 g, 46.9 mmol) in tetrahydrofuran (125 mL) and stir at -70 °C for 1 hour. After the reaction is completed, slowly add a solution of acetic acid (10.5 mL) in tetrahydrofuran (125 mL) and stir at 25 °C for 0.5 hour. Saturated aqueous sodium bicarbonate solution (300 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (200 mL x 3). The organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 6 / 1, v / v) to obtain compound B-2. 1H NMR (400 MHz,CDCl3): δ 4.40-4.33 (m, 1H), 4.28-4.20 (m, 2H), 3.93-3.89 (m, 1H), 3.26-3.16 (m, 1H), 2.28 (s, 1H), 2.07-2.01 (m, 1H), 1.92-1.87 (m, 3H), 1.46 (s, 9H),1.33-1.27 (m, 6H). ESI-MS calculated value: [M+Na] + = 334.17, measured value 333.9.

[0060] Step 2

[0061] Compound B-2 (14.0 g, 45.0 mmol) was dissolved in anhydrous ethanol (200 mL), and then 5% wet palladium / carbon (2.78 g) was added. The mixture was stirred at 25 °C for 16 hours under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with anhydrous ethanol (100 mL x 3), and the filtrate was concentrated under reduced pressure. The residue was compound B-3. ESI-MS theoretical calculated value: [M+Na] + =338.20, measured value 338.0.

[0062] Step 3

[0063] Compound B-3 (13.0 g, 41.2 mmol) was dissolved in toluene (200 mL) and stirred at 100 °C for 3 hours under nitrogen atmosphere. The reaction solution was cooled to room temperature, water (300 mL) was added to the reaction solution, and ethyl acetate (200 mL x 3) was used for extraction. The organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 3 / 1, v / v) to obtain compound B-4, whose two-dimensional NMR is shown in the attached Figure 1 ; H11 and H6 showed no NOE signal, confirming that the chirality of B-4 was correct. 1H NMR (400 MHz, CDCl3): δ 4.48-4.40 (m, 1H), 4.06-3.95 (m, 1H), 3.22-2.97 (m, 1H), 2.67-2.53 (m, 2H), 2.29-2.09 (m, 1H), 2.05-1.98 (m, 1H), 1.85-1.72 (m, 3H), 1.64-1.58 (m, 1H), 1.46 (s,9H), 1.31-1.19 (m, 3H). ESI-MS calculated value: [M+Na] + = 292.16, measured value 292.0.

[0064] Step 4

[0065] Compound B-4 (10.2 g, 38.0 mmol) was dissolved in dichloromethane (120 mL). Under nitrogen atmosphere, a hexane solution of diisobutylaluminum hydride (37.6 mL, 37.6 mmol, 1.0 mol / L) was added dropwise at -30 °C, stirred at -30 °C for 2 hours, and then stirred at 0 °C for 2 hours. Water (1.5 mL), 15% aqueous sodium hydroxide solution (1.5 mL) and water (3.0 mL) were added to the reaction solution, and stirred at 25 °C for 0.5 hours. Anhydrous magnesium sulfate (30.0 g) was added, filtered, and the filter cake was washed with dichloromethane (100 mL x 3). The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 4 / 1, v / v) to obtain compound B-5. 1 H NMR (400 MHz, CDCl3): δ 5.57-5.22 (m, 1H), 4.44-4.26 (m, 1H), 3.99-3.82 (m, 1H), 3.27-3.06 (m, 1H), 2.11-1.79 (m, 5H), 1.75-1.50 (m, 4H), 1.46 (s, 9H), 1.32-1.24 (m, 3H). ESI-MS calculated value: [M+Na] + =294.18, measured value 294.0.

[0066] Step 5

[0067] Compound B-5 (9.60 g, 35.4 mmol) was dissolved in dichloromethane (80 mL). Trimethylsilyl cyanide (5.27 g, 53.1 mmol) was added dropwise at -70 °C under nitrogen atmosphere. The mixture was stirred at -70 °C for 0.5 hours. Boron trifluoride ether (5.52 g, 38.92 mmol) was added dropwise and stirred at 0 °C for 1 hour. Saturated sodium bicarbonate aqueous solution (60 mL) was slowly added dropwise, di-tert-butyl dicarbonate (7.72 g, 35.4 mmol) was added, and the mixture was stirred at 25 °C for 1 hour. After the reaction was completed, dichloromethane (100 mLx 3) was used for extraction, and the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 3 / 1, v / v) to obtain compound B-6. 1 H NMR (400 MHz, DMSO-d6): δ 4.98-4.94 (m, 1H), 4.28-4.19 (m, 1H), 3.82-3.75 (m,1H), 3.03-2.91 (m, 1H), 2.35-2.27 (m, 1H), 2.23-2.16 (m, 1H), 1.85-1.74 (m,3H), 1.72-1.65 (m, 1H), 1.56-1.43 (m, 2H), 1.39 (s, 9H), 1.20-1.14 (m, 3H). ESI-MS calculated value: [M+H-56] + = 225.18, measured value 225.0.

[0068] Step 6

[0069] Compound B-6 (3.20 g, 11.4 mmol) was dissolved in methanol (30 mL), sodium methoxide (925 mg, 17.1 mmol) was added, and the mixture was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was completed, 1 molar hydrochloric acid aqueous solution (40 mL) was added, and ethyl acetate (40 mL x 3) was used for extraction. The organic phase was washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 5 / 1, v / v) to obtain compound B-7. 1H NMR (400 MHz, CDCl3): δ 4.55-4.50 (m, 1H), 4.37-4.31 (m, 1H), 3.91-3.83 (m, 1H), 3.71 (s, 3H), 3.28-3.10 (m, 1H), 2.32-2.18 (m, 1H), 2.09-2.00(m, 1H), 1.82-1.67 (m, 5H), 1.58-1.50 (m, 1H), 1.44 (s, 9H), 1.28-1.20 (m,3H). ESI-MS calculated value: [M+H] + = 314.19, measured value 314.1.

[0070] Step 7

[0071] Compound B-7 (3.00 g, 9.57 mmol) was dissolved in tetrahydrofuran (15 mL) and water (15 mL), and lithium hydroxide monohydrate (803 mg, 19.1 mmol) was added, and stirred at 25 °C for 16 hours. After the reaction was completed, 1 molar hydrochloric acid aqueous solution (40 mL) was added, and ethyl acetate (50 mL x 3) was used for extraction. The organic phase was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 3 / 1, v / v) to obtain compound intermediate B. 1 H NMR (400 MHz, DMSO-d6): δ 12.45 (s, 1H), 4.42-4.36 (m, 1H), 4.24-4.18 (m, 1H), 3.76-3.72 (m, 1H), 3.17-3.03 (m, 1H), 2.28-2.13 (m, 1H), 1.99-1.91 (m, 1H), 1.70-1.53 ​​(m, 5H), 1.42-1.33 (m, 10H), 1.21-1.16 (m, 3H). ESI-MS calculated value: [M+H] + = 300.17, measured value 300.1.

[0072] Example 1

[0073] Synthesis route:

[0074]

[0075] first step

[0076] In a glove box filled with nitrogen, compound nickel chloride dimethoxyethane (14.5 mg, 0.066 mmol) and 4,4'-di-tert-butyl-2,2'-bipyridine (13.3 mg, 0.050 mmol) were dissolved in N,N-dimethylformamide (2 mL). Compound intermediate B (100 mg, 0.33 mmol), compound 1-1 (116 mg, 0.66 mmol), anhydrous cesium carbonate (215 mg, 0.66 mmol) and bis[2-(2,4-difluorophenyl)-5-trifluoromethylpyridine][2-2'-bi(4-tert-butylpyridine)]iridium di(hexafluorophosphate) salt (3.70 mg, 0.0033 mmol) were added. The reaction solution was placed under 34 W blue LED (420 nm) and stirred at 25 °C for 16 hours. After the reaction was completed, the blue light was turned off, water (20 mL) was added to the reaction solution, and ethyl acetate (20 mL x 3) was used for extraction. The organic phases were combined and washed with saturated brine (20 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, 3 / 1, v / v) to obtain compound 1-2. ESI-MS theoretical calculated value: [M+H-56] + = 294.21, measured value 294.1.

[0077] Step 2

[0078] Dissolve compound 1-2 (58.0 mg, 0.17 mmol) in dichloromethane (5 mL), add trifluoroacetic acid (1.0 mL, 13.5 mmol), and stir at 25 °C for 1 hour under nitrogen atmosphere. After the reaction is completed, the reaction solution is concentrated under reduced pressure to obtain the trifluoroacetate salt of compound 1-3. ESI-MS theoretical calculated value: [M+H] + = 250.15, measured value 250.0.

[0079] Step 3

[0080] The trifluoroacetate of compound 1-3 (58.1 mg, 0.16 mmol) was dissolved in ethyl acetate (3 mL), and N,N-diisopropylethylamine (31.0 mg, 0.24 mmol), intermediate A (45.2 mg, 0.24 mmol) and sodium triacetoxyborohydride (102 mg, 0.48 mmol) were added, and stirred at 40 °C for 2 hours. Saturated sodium bicarbonate aqueous solution (10 mL) was added to the reaction solution, extracted with ethyl acetate (10 mL x 3), and washed with saturated brine (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (Waters-XBridge-C18-10µm-19*250mm, mobile phase: acetonitrile-0.1% aqueous ammonia solution, gradient: 42-55%, retention time: 9.3-10.4 min, running time: 16 min) to obtain compound 1, see attached Figure 2 . 1 H NMR (400 MHz, DMSO-d6): δ 12.22(s, 1H), 7.35-7.32 (m, 2H), 7.15-7.10 (m, 2H), 4.89-4.85 (m, 1H), 3.83 (d, J= 14.8 Hz, 1H), 3.61 (d, J = 14.8 Hz, 1H), 3.29 (s, 3H), 2.82-2.78 (m, 1H), 2.35-2.28 (m, 2H), 2.11 (s, 3H), 1.72-1.64 (m, 4H), 1.48-1.43 (m, 1H), 1.13-1.10 (m, 3H). ESI-MS theoretical calculated value: [M+H] + = 422.16, measured value 422.0.

[0081] Effect Example 1

[0082] Inhibitory effects of compounds on O-linked N-acetylglucosaminyl hydrolase (O-GlcNAcase, OGA)

[0083] 1. Experimental Materials

[0084]

[0085] 2. Experimental instruments and equipment

[0086]

[0087] 3. OGA enzyme reaction method

[0088] 3.1 Enzyme and reaction substrate configuration

[0089] The OGA enzyme buffer system contained 50 mM NaH2PO4, 100 mM NaCl, and 0.1% BSA;

[0090] The substrate 4-MUF-NAG was added to the buffer to prepare a 2× substrate solution of 280 μM;

[0091] Add OGA enzyme to the buffer to prepare a 2x enzyme solution with a concentration of 5 nM;

[0092] 3.2. Compound configuration

[0093] The test compound was prepared into a stock solution with DMSO at the desired concentration.

[0094] 3.3 Experimental Procedure

[0095] 1) 45 µL of the compound to be tested is used as the highest test concentration (10 μM) in a 384-well Echo plate. 15 µL of the compound is taken from the first concentration well and added to the second concentration well. The concentration is diluted 3-fold in sequence, for a total of 10 concentrations. 40 µL of 100% DMSO is transferred to two empty wells as controls without compound and enzyme.

[0096] 2) Use Echo 650 to transfer 100 nL of different concentrations of the test compound to a 384-well assay plate;

[0097] 3) Transfer 10 μL of 2 x OGA enzyme solution into the reaction wells of the 384-well assay plate containing the compound;

[0098] 4) Transfer 10 μL of 2 x substrate solution to the reaction wells of the 384 assay plate containing the compound to start the reaction, centrifuge at 1000 rpm for 1 minute, and incubate at room temperature for 15 minutes;

[0099] 5) Take out the test plate and put it into the Synergy multi-function microplate reader to read the value.

[0100] 4. Data Reading

[0101] Dynamically read RFU value on Synergy (Ex355 / Em460)

[0102] 5. Data Analysis

[0103] 1) Copy the RFU reading value

[0104] 2) Convert the above data into inhibition percentage using the formula

[0105] Percent inhibition = (max-sample RFU) / (max-min) 100

[0106] "min" is the reading of the control well without enzyme reaction; "max" is the reading of the control well with DMSO added

[0107] 3) Import data into GraphPad Prism for curve fitting

[0108] Fitting formula:

[0109] Experimental results:

[0110]

[0111] Experimental conclusion:

[0112] The experimental samples (compounds) were prepared from the corresponding examples, and the results are shown in the table above. The compounds of the present application have an inhibitory effect on O-linked N-acetylglucosaminyl hydrolase (O-GlcNAcase, OGA) in this test system.

[0113] Effect Example 2

[0114] Evaluation of the brain penetration properties of compounds

[0115] Purpose:

[0116] The brain tissue / plasma partition coefficient of the compound obtained in the example of the present invention in CD-1 mice was evaluated.

[0117] Experimental Materials:

[0118]

[0119] Experimental procedures:

[0120] The candidate compound was prepared into a clear solution of 0.50 mg / mL with a specified solvent and administered orally to two CD-1 mice. The solvent for oral administration was 2% dimethyl sulfoxide + 98% (5% sulfobutyl-β-cyclodextrin aqueous solution). Whole blood samples and brain tissue samples were collected 1 hour after administration into commercial EDTA2K anticoagulation tubes, centrifuged to obtain upper plasma and brain tissue homogenate samples, and acetonitrile solution containing internal standard was added to precipitate protein. The supernatant was centrifuged and added with an equal volume of water. After centrifugation, the supernatant was sampled and injected. The drug concentration in plasma samples and brain tissue samples was quantitatively analyzed by LCMS / MS analysis method.

[0121] Experimental methods:

[0122]

[0123] Experimental results:

[0124]

[0125] Experimental conclusion:

[0126] The test samples were prepared from the corresponding examples. The results showed that the compound of the present application had a larger brain-blood partition coefficient, indicating that it had good brain-penetrating properties.

[0127] Effect Example 3

[0128] Bidirectional permeability test in MDR1-MDCK II cells

[0129] Cell culture:

[0130] MDR1-MDCK II cells obtained from the Netherlands Cancer Institute will be seeded onto PET membranes in 96-well inserts and cultured for 4-7 days before use in experiments.

[0131] Verification of monolayer integrity:

[0132] Verify the integrity of the cell monolayer by performing a Lucifer Yellow exclusion assay.

[0133] Monolayer cell quality verification:

[0134] Validation was performed in duplicate wells by measuring the unidirectional (A→B) permeability of nadolol (low permeability marker), metoprolol (high permeability marker), and the bidirectional permeability of digoxigenin (P-glycoprotein substrate marker).

[0135] Standard test conditions:

[0136] Buffer: HBSS containing 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.40 ± 0.05;

[0137] The compound was first dissolved in DMSO solution to 1 mM, then diluted to the test concentration in buffer and added to the A side of the cells. Test compound concentration: 2.0 µM;

[0138] Number of repetitions: n=2;

[0139] Direction: including bidirectional transportation from A to B and from B to A;

[0140] Incubation time: single time point, 2.5 hours;

[0141] Incubation conditions: 37±1°C, 5% CO2, relative saturated humidity.

[0142] Dosage solution preparation:

[0143] The dose solution (2.0 μM solution of the test compound) will be mixed with buffer and stop solution (250 nM tolbutamide and 250 nM labetalol) containing appropriate internal standards (IS) as the T0 sample.

[0144] Sample processing:

[0145] After incubation, the sample solution was removed from the donor and acceptor wells and immediately mixed with the stop solution.

[0146] Sample Analysis:

[0147] All samples (including T0 samples, donor samples, and acceptor samples) will be analyzed using LC-MS / MS. The concentration of the test compound will be expressed as the peak area ratio of the analyte to the IS, without the need for a standard curve.

[0148] Experimental results:

[0149]

[0150] Experimental conclusion:

[0151] The test samples were prepared from the corresponding examples. The results showed that the compounds of the present application have the characteristics of high iso-permeability and low efflux.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof, characterized in that: ； Among them, the "The carbon atom is in R configuration, S configuration or a mixture of the two; Each R 1 are independently halogen; Each R 2 are independently C1-C6 alkyl; R 3 is a halogen; R 4 is hydrogen; R 5 for ; R 4-1 is a C1-C6 alkyl group; m and n are each independently 1, 2 or 3.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1) Each "C1-C6 alkyl" is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; (2) each "halogen" is independently fluorine, chlorine, bromine or iodine; (3) for ; (4) for ; (5) R 4 is hydrogen; and (6) R 5 for .

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: It meets one or more of the following conditions: (1) each "C1-C6 alkyl" is independently methyl; (2) each "halogen" is independently fluorine; (3) for ; and (4) for , where end a and connected.

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: for .

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound shown in formula I is .

6. A pharmaceutical composition, comprising: (1) The compound of formula I according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof; and (2) Pharmaceutically acceptable excipients.

7. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 6 in the preparation of an O-acetylglucosaminidase inhibitor; wherein, The inhibitor is used in vitro for experimental purposes.

Images