Small molecule compound as well as preparation and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a small molecule compound as well as preparation and application thereof. The small molecule compound capable of identifying the alpha-synuclein fiber, provided by the invention, can be used as an independent reagent or a reagent composition and is used for detecting the alpha-synuclein fiber in vivo and in vitro; the method is further applied to diagnosis of alpha-synuclein fibers or other error folded protein related neurodegenerative diseases, and has a wide application prospect in the fields of treatment of the diseases and research and development of medicines.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a small molecule compound and its preparation and application. Background Art

[0002] Neurodegenerative disease (ND) is a chronic disease, and the incidence is closely related to age. The main NDs include Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), etc. The common feature of patients with these diseases is the abnormal aggregation of proteins, which leads to progressive neuronal loss and degeneration. Currently, there are more than 57 million ND patients in the world. Age is the most important cause of ND. With the increase of age, the incidence of ND increases significantly. As the number of elderly people in the world continues to increase, the number of ND patients will also continue to rise.

[0003] ND has a long disease cycle. Before obvious clinical symptoms appear, patients have pathological changes for decades. This stage is called the prodromal stage of ND. When patients show obvious ND symptoms, a large number of neurons in the patient's brain have been lost. Neurons in the human brain are non-regenerative. Diagnosing and treating early ND is an effective way to improve the efficacy of ND. Traditional ND diagnosis mainly relies on the patient's clinical symptoms. For example, AD patients will have obvious memory decline, and PD patients will have symptoms such as tremors and rigidity. However, these diagnostic methods cannot identify ND patients in the prodromal period, and miss the best time for therapeutic intervention. Therefore, developing early diagnostic methods for ND is an effective way to improve the efficacy of ND.

[0004] Abnormal aggregation of proteins is the most important pathological change of ND and the most important early diagnostic biomarker of ND. Under physiological conditions, these proteins exist in a disordered monomeric state, while under pathological conditions, these proteins form aggregated proteins rich in β sheets, and gradually aggregate to form oligomers and fibers, and finally form pathological markers unique to ND. There are two aggregating proteins in AD. Amyloid β (Aβ) forms amyloid plaques (AP) outside the cell and is deposited in the cortex, subcortical area and brainstem; at the same time, the pathological protein Tau is mainly deposited in the cell, and its abnormal aggregation forms neurofibrillary tangles (NFTs) and is deposited in the entorhinal cortex, hippocampus and basal cortex. α-Synuclein (α-syn) appears in the brains of patients with synuclein diseases, including PD, MSA and DLB. In the brains of PD and DLB patients, α-syn lesions mainly occur in neurons, forming Lewy bodies and Lewy neurites, which appear in tissues and organs such as the substantia nigra, striatum, brainstem, and olfactory bulb; while in MSA, α-syn lesions mainly occur in oligodendrocytes and form glial cytoplasmic inclusions (GCIs).

[0005] Positron emission tomography (PET) and single photon emission tomography (SPECT) are non-invasive imaging methods used to detect the structure and function of the body and to study the pathological progression and pathogenesis of the disease. This technology introduces radionuclides into small molecule tracers. After the tracer is injected into the body, the positrons or gamma photons emitted by the radionuclides can be detected and located by the imager's probe, and the tracer uptake and spatial distribution in the body are calculated by the processor and image reconstruction software, and changes in brain function and structure can be inferred from this. PET and SPECT are both important means of nuclear medicine imaging and are used for the diagnosis of clinical diseases. The working principle of PET is to inject a molecule containing radionuclides (such as 18 F. 15 O, etc.) are injected into the body. When these nuclides undergo positron emission decay, the positrons emitted react with electrons in the body to produce two beams of gamma rays with equal energy, which are emitted in opposite directions at 180 degrees. By using a surround detector to detect and calculate gamma rays, the emission position of the positrons can be determined, thereby obtaining information on the body's cross-sectional structure and metabolic function. SPECT is similar to PET in principle, except that the nuclides used, such as 99mTc, emit single gamma photons and do not require electrons. These technologies have high spatial resolution, and the visualization and quantification resolution of PET can reach 2 mm. Both PET and SPECT are non-invasive examinations that can be used for functional localization diagnosis of a variety of clinical diseases. They are particularly valuable in the detection, staging and evaluation of treatment effects of tumors, and are currently widely used in clinical practice. The development and exploration of new probes is an important future development direction for these two technologies.

[0006] Detecting pathological protein aggregation in the brain through PET and SPECT technology is an effective means to achieve early diagnosis of ND. In 2012, the FDA approved the first Aβ amyloid protein PET tracer AV-45 for AD diagnosis; in 2013, Bayer's amyloid protein tracer AV-1 was also approved by the FDA for marketing. Both AV-1 and AV-45 were developed by the University of Pennsylvania. They have good imaging effects on Aβ plaques in the brains of AD patients and can assist doctors in diagnosing AD. In 2014, GE's GE-067 became the third amyloid protein PET tracer approved for marketing. In 2020, Eli Lilly's Tau tracer AV-1451 was approved by the FDA for the diagnosis of AD. Unlike amyloid protein, the abnormal aggregation degree of Tau is closely related to the pathological development of AD, so AV-1451 can help doctors stage AD patients. However, AV-1451 has off-target imaging of MAO-B in the brain, which has an adverse effect on clinical diagnosis. Therefore, the development of Tau tracers without MAO-B off-target effects has become a research hotspot. Among them, Merck's MK-6240 and Xinxu's APN-1607 overcome the non-specific binding of MAO-B and have good imaging effects on Tau. Both molecules are in phase III clinical trials.

[0007] The successful application of PET and SPECT brain imaging in the early diagnosis of AD has made the development of α-syn fiber tracers a research hotspot. α-syn fibers are of great significance for the early diagnosis of synuclein diseases. Synuclein diseases include PD, PDD, DLB, and MSA, and patients with these diseases have α-syn fiber deposition in the brain. These pathological changes occur in the early stages of synuclein diseases and are closely related to the course of the disease. Therefore, imaging these misfolded α-syn through brain imaging technology can achieve early diagnosis of the disease, which is of great significance for the treatment of the disease and the development of new drugs. However, at this stage, there are still no α-syn fiber tracers on the market, so the development of this tracer is of great scientific significance. Summary of the invention

[0008] In order to solve the above problems, the purpose of the present invention is to provide a small molecule compound and its preparation and application.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] The first object of the present invention is to provide a small molecule compound, the chemical structure of which is shown below:

[0011]

[0012] In formula (I), A and B are independently selected from a substituted or unsubstituted 4- to 7-membered saturated cycloalkane, a substituted or unsubstituted 4- to 7-membered aromatic ring, or a substituted or unsubstituted 4- to 7-membered heterocycle;

[0013] The substitution means that one or more hydrogen atoms on the ring are replaced by the following substituents:

[0014] Amide, methylsulfone, ester, hydroxy, nitro, cyano, C1-6 alkoxy, C1-6 halohydroxyalkoxy, C1-6 alkyl, C1-6 alkylamino, halogen, C1-6 haloalkyl, C1-6 haloalkoxy, C2-6 alkenyl, C2-6 alkynyl, acyl, C1-6 hydroxyalkyl, carboxyl, amino or sulfonamide.

[0015] In one embodiment of the present invention, the halogen is selected from one of fluorine, chlorine, bromine or iodine.

[0016] In one embodiment of the present invention, the chemical structural formula of the small molecule compound is selected from one of the following structural formulas:

[0017]

[0018]

[0019] The second object of the present invention is to provide a method for preparing a small molecule compound, comprising the following steps:

[0020] (S1) dissolving 4-bromobenzyl bromide and mixing it with triethyl phosphite, and reacting under nitrogen protection to obtain a first compound;

[0021] (S2) dissolving the first compound prepared in step (S1) and adding sodium hydroxide, adding the second compound after pre-reaction under nitrogen protection, and performing post-treatment after the reaction to obtain a third compound;

[0022] (S3) dissolving the third compound prepared in step (S2) and sequentially adding the fourth compound, potassium tert-butoxide, Pd 2 (dba) 3 and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, react under nitrogen protection and then post-treat to obtain a small molecule compound;

[0023] Among them, the chemical structural formula of the first compound is shown in formula (II), the chemical structural formula of the second compound is shown in formula (III), the chemical structural formula of the third compound is shown in formula (IV), and the chemical structural formula of the fourth compound is shown in formula (V):

[0024]

[0025] In formula (III) and formula (IV), A is the same and is selected from a substituted or unsubstituted 4-7 membered saturated cycloalkane, a substituted or unsubstituted 4-7 membered aromatic ring, or a substituted or unsubstituted 4-7 membered heterocycle;

[0026] In formula (V), B is selected from a substituted or unsubstituted 4- to 7-membered saturated cycloalkane, a substituted or unsubstituted 4- to 7-membered aromatic ring, and a substituted or unsubstituted 4- to 7-membered heterocycle;

[0027] The substitution means that one or more hydrogen atoms on the ring are replaced by the following substituents:

[0028] Amide, methylsulfone, ester, hydroxy, nitro, cyano, C1-6 alkoxy, C1-6 halohydroxyalkoxy, C1-6 alkyl, C1-6 alkylamino, halogen, C1-6 haloalkyl, C1-6 haloalkoxy, C2-6 alkenyl, C2-6 alkynyl, acyl, C1-6 hydroxyalkyl, carboxyl, amino or sulfonamide.

[0029] In one embodiment of the present invention, in step (S1), the molar ratio of 4-bromobenzyl bromide to triethyl phosphite is 1:2-5; during the reaction, the temperature is 90-120°C and the reaction time is 6-8 hours.

[0030] Preferably, the molar ratio of 4-bromobenzyl bromide to triethyl phosphite is 1:3; during the reaction, the temperature is 110° C. and the reaction time is 8 hours.

[0031] In one embodiment of the present invention, in step (S2), the molar ratio of the first compound, sodium hydroxide and the second compound is 0.9-1.1:1.5-1.8:1; during the pre-reaction, the temperature is 0°C and the time is 0.3-1 hour; during the reaction, the temperature is 20-30°C and the time is 6-8 hours.

[0032] Preferably, the molar ratio of the first compound, sodium hydroxide and the second compound is 1.05:1.67:1; during the pre-reaction, the temperature is 0°C and the time is 0.5 hour; during the reaction, the temperature is 25°C and the time is 8 hours.

[0033] In one embodiment of the present invention, in step (S3), the third compound, the fourth compound, potassium tert-butoxide, Pd 2 (dba) 3 The molar ratio of 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl is 1:0.9-1.5:2-3:0.05-0.2:0.1-0.3; during the reaction, the temperature is 100-120°C and the time is 6-8 hours.

[0034] Preferably, the third compound, the fourth compound, potassium tert-butoxide, Pd 2(dba) 3 The molar ratio of 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl is 1:1.2:2.5:0.1:0.2; during the reaction, the temperature is 110°C and the time is 8 hours.

[0035] The third object of the present invention is to provide a use of the above-mentioned small molecule compound in the preparation of a reagent for diagnosing a neurological disorder associated with α-synuclein fibers, and / or a drug for treating and / or preventing α-synuclein fiber-related diseases.

[0036] In one embodiment of the present invention, the neurological disorder is selected from one of Parkinson's disease, multiple system atrophy or dementia with Lewy bodies;

[0037] The reagent is an imaging diagnostic reagent.

[0038] In one embodiment of the present invention, the imaging technique includes but is not limited to positron emission tomography, single photon emission computed tomography, near-infrared brain function imaging or optical imaging.

[0039] The fourth object of the present invention is to provide a reagent for diagnosing a neurological disorder associated with α-synuclein fibers, wherein the reagent contains the above-mentioned small molecule compound.

[0040] In one embodiment of the present invention, the diagnostic agent is the above-mentioned small molecule compound, or a composition comprising the above-mentioned small molecule compound and a medically acceptable salt or fusing agent.

[0041] The fifth object of the present invention is to provide a drug for treating and / or preventing α-synuclein fiber-related diseases, wherein the drug contains the above-mentioned small molecule compound.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention provides a small molecule compound that can identify α-synuclein fibers. The small molecule compound can be used as an independent reagent or a reagent combination for the detection of α-synuclein fibers in vivo and in vitro, and is further applied to the diagnosis of neurodegenerative diseases associated with α-synuclein fibers or other misfolded proteins. It has broad application prospects in the treatment of the above diseases and the research and development of drugs. DETAILED DESCRIPTION

[0044] The present invention is described in detail below with reference to specific embodiments.

[0045] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0046] In the following examples, the nucleotide sequence of α-synuclein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; specifically, as follows:

[0047] SEQ ID NO.1: ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAG GGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTGTGACAGCAGTA GCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTATGAACCTGAAGCCTAA

[0048] SEQ ID NO.2: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLY VGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAA ATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA

[0049] Example 1

[0050] This embodiment provides a small molecule compound, the chemical structure of which is shown below:

[0051]

[0052] The small molecule compound (I-1) is prepared by the following method (the synthesis process is described in the following formula):

[0053]

[0054] (S1) Dissolve 10.0 mmol of 4-bromobenzyl bromide 1 in 10 mL of toluene, add 30.0 mmol of triethyl phosphite to obtain a mixed solution; 2 The reaction was carried out at 110°C for 12 hours under protection, and then the solvent (toluene) was evaporated to dryness, and separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain a white solid: the first intermediate (2), with a yield of 97.6%, ESI-MS (positive): 307.0 (M+1) + .

[0055] (S2) 9.5 mmol of the first intermediate (2) prepared in step (S1) was added to 10 mL of dimethylformamide, and then 15.0 mmol of sodium hydroxide, N 2 The mixture was reacted at 0°C for 30 min under protection, and then 9.0 mmol 4-methoxybenzaldehyde (3-1) was added and reacted at room temperature for 8 hours; 50 mL ice water was then added to the reaction solution, filtered to obtain a yellow solid, and then recrystallized from ethyl acetate to obtain a yellow solid: the second intermediate (4-1), with a yield of 93.2%, ESI-MS (positive): 289.1 (M+1) + .

[0056] (S3) 1 mmol of the second intermediate (4-1) prepared in step (S2) was dissolved in 3 mL of toluene, and 1.2 mmol of 2-aminopyridine (5-1), 2.5 mmol of potassium tert-butoxide, 0.1 mmol of Pd 2 (dba) 3 , 0.2 mmol 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos) was transferred to the reaction flask, and the reaction flask was filled with N 2 Protection, 110 ° C reaction for 8h; then add 10mL ethyl acetate to the reaction solution, wash three times with 10mL saturated brine, dry over anhydrous sodium sulfate and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain a yellow solid: small molecule compound (I-1), yield 35.9%, ESI-MS (positive): 302.1 (M+1) + , 1 H NMR(600MHz,Chloroform-d)δ8.75-8.61(m,1H),7.81(t,J=8.1Hz,1H),7.53(d,J=8.0 Hz, 2H), 7.35-7.19 (m, 4H), 6.90 (d, J = 7.9Hz, 4H), 6.66 (d, J = 8.5Hz, 2H), 3.79 (s, 3H).

[0057] Example 2

[0058] This embodiment provides a small molecule compound (I-2), whose chemical structure is shown below:

[0059]

[0060] The preparation method of the small molecule compound (I-2) is the same as that of Example 1 except for the following contents:

[0061] In step (S2), 4-methoxybenzaldehyde is replaced with 4-fluorobenzaldehyde;

[0062] The small molecule compound (I-2) prepared in this example has a yield of 28.4%, ESI-MS (positive): 290.1 ​​(M+1) + , 1 H NMR (600MHz, Chloroform-d) δ 8.53-8.41 (m, 1H), 7.64 (t, J = 8.5Hz, 2H), 7.52 (d, J = 8.2Hz, 2H), 7.30-7.15 (m, 4H), 7.02-6.78 (m, 4H), 6.66 (d, J = 8.4Hz, 2H).

[0063] Example 3

[0064] This embodiment provides a small molecule compound (I-3), whose chemical structure is shown below:

[0065]

[0066] The preparation method of the small molecule compound (I-3) is the same as that of Example 1 except for the following contents:

[0067] In step (S2), 4-methoxybenzaldehyde is replaced with 2-methoxypyridine;

[0068] The small molecule compound (I-3) prepared in this example has a yield of 19%, ESI-MS (positive): 303.2 (M+1) + , 1 H NMR(600MHz,Chloroform-d)δ8.21(d,J=2.6Hz,1H),7.75(dd,J=8.6,2.5Hz,1H),7.43-7 .30(m,6H),7.28(d,J=6.2Hz,2H),6.95-6.70(m,3H),6.66(d,J=8.0Hz,2H),3.87(s,3H).

[0069] Example 4

[0070] This embodiment provides a small molecule compound (I-4), whose chemical structure is shown below:

[0071]

[0072] The preparation method of the small molecule compound (I-4) is the same as that of Example 1 except for the following contents:

[0073] In step (S2), 4-methoxybenzaldehyde is replaced with 2-dimethylamino-5-carboxythiazole;

[0074] In step (S3), 2-aminopyridine is replaced with 2-aminofuran;

[0075] The small molecule compound (I-4) prepared in this example is a brown solid; the yield is 7%, ESI-MS (positive): 312.1 (M+1) + , 1 H NMR (600MHz, Chloroform-d) δ8.41 (d, J = 2.0 Hz, 1H), 8.09 (d, J = 5.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 2H), 7.55-7.32 (m, 4H), 7.08-6.93 (m, 3H), 3.05 (s, 6H).

[0076] Example 5

[0077] This embodiment provides a small molecule compound (I-5), whose chemical structure is shown below:

[0078]

[0079] The preparation method of the small molecule compound (I-5) is the same as that of Example 1 except for the following contents:

[0080] In step (S2), 4-methoxybenzaldehyde is replaced with 4-dimethylaminobenzaldehyde;

[0081] In step (S3), 2-aminopyridine is replaced with aniline;

[0082] The small molecule compound (I-5) prepared in this example is a light yellow solid with a yield of 40%. ESI-MS (positive): 315.2 (M+1) + , 1H NMR(600MHz,Chloroform-d)δ8.20(d,J=2.2Hz,1H),7.62(dd,J=7.8,6.5Hz,2H) ,7.28(dd,J=8.2,8.0Hz,4H),7.01-6.90(m,5H),6.81-6.52(m,4H),2.94(s,6H).

[0083] Example 6

[0084] This embodiment provides a small molecule compound (I-6), whose chemical structure is shown below:

[0085]

[0086] The small molecule compound (I-6) is prepared by the following method (the synthesis process is described in the following formula):

[0087]

[0088] (S1) Dissolve 10.0 mmol of 4-bromobenzyl bromide 1 in 10 mL of toluene, add 30.0 mmol of triethyl phosphite to obtain a mixed solution; 2 The mixture was reacted at 110°C for 12 hours under protection, and then the solvent (toluene) was evaporated to dryness. The mixture was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain a white solid: the first intermediate (2);

[0089] (S2) 9.5 mmol of the first intermediate (2) prepared in step (S1) was added to 10 mL of dimethylformamide, and then 15.0 mmol of sodium hydroxide, N 2 The mixture was reacted at 0°C for 30 min under protection, and then 9.0 mmol 4-dimethylaminobenzaldehyde (3-6) was added and reacted at room temperature for 8 hours; 50 mL ice water was then added to the reaction solution, and a yellow solid was obtained by filtration, and then the yellow solid was recrystallized from ethyl acetate to obtain the second intermediate (4-6);

[0090] (S3) 1 mmol of the second intermediate (4-6) prepared in step (S2) was dissolved in 3 mL of toluene, and 1.2 mmol of 2-amino-5-methoxypyridine (5-6), 2.5 mmol of potassium tert-butoxide, 0.1 mmol of Pd 2 (dba) 3 , 0.2 mmol 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos) was transferred to the reaction flask, and the reaction flask was filled with N 2Protection, 110 ° C reaction for 8h; then add 10mL ethyl acetate to the reaction solution, wash three times with 10mL saturated brine, dry over anhydrous sodium sulfate and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain a yellow solid: the third intermediate (6-6); yield 26%, ESI-MS (positive): 345.2 (M+1) + ;

[0091] (S4) 0.2 mmol of the third intermediate (6-6) prepared in step (S3) was dissolved in 1 mL of dichloromethane, 2 mmol of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 2 h; then the solvent (dichloromethane) was evaporated to obtain a brown oil: the fourth intermediate (7-6), ESI-MS (positive): 332.2 (M+1) + .

[0092] (S5) The fourth intermediate (7-6) obtained in step (S4) was dissolved in 1 mL of DMF, 1 mmol of sodium hydroxide and 0.5 mmol of 1-bromo-2-fluoroethane were added, and the mixture was reacted at room temperature for 6 h. After the reaction, 5 mL of water was added, and the mixture was extracted with 10 mL of ethyl acetate. After drying over anhydrous sodium sulfate, the mixture was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2.5: 1) to obtain a yellow solid: a small molecule compound (I-6) with a yield of 53%. ESI-MS (positive): 378.2 (M+1) + , 1 H NMR(600MHz,Chloroform-d)δ8.24(d,J=2.4Hz,1H),7.60(dd,J=8.0,5.7Hz,1H),7.30(dd,J=8.1,7.9Hz ,4H),7.13-7.02(m,5H),7.00-6.74(m,3H),4.71(d,J=30.2Hz,2H),4.20(d,J=28.8Hz,2H),2.94(s,6H).

[0093] Example 7

[0094] This embodiment provides a small molecule compound (I-7), whose chemical structure is shown below:

[0095]

[0096] The small molecule compound (I-7) is prepared by the following method (the synthesis process is described in the following formula):

[0097]

[0098] (S1) Dissolve 10.0 mmol of 4-bromobenzyl bromide 1 in 10 mL of toluene, add 30.0 mmol of triethyl phosphite to obtain a mixed solution; 2 The mixture was reacted at 110° C. for 12 hours under protection, and then the solvent (toluene) was evaporated to dryness, and separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain a white solid: the first intermediate;

[0099] (S2) 9.5 mmol of the first intermediate prepared in step (S1) was added to 10 mL of dimethylformamide, and then 15.0 mmol of sodium hydroxide was added. 2 The mixture was reacted at 0°C for 30 min under protection, and then 9.0 mmol 4-nitrobenzaldehyde was added and reacted at room temperature for 8 hours. Subsequently, 50 mL ice water was added to the reaction solution, and a yellow solid was obtained by filtration. The yellow solid was then recrystallized from ethyl acetate to obtain the second intermediate.

[0100] (S3) 1 mmol of the second intermediate prepared in step (S2) was dissolved in 3 mL of toluene, and 1.2 mmol of 2-aminopyridine, 2.5 mmol of potassium tert-butoxide, 0.1 mmol of Pd 2 (dba) 3 , 0.2 mmol 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos) was transferred to the reaction flask, and the reaction flask was filled with N 2 Protection, 110 ° C reaction for 8h; then add 10mL of ethyl acetate to the reaction solution, wash three times with 10mL of saturated brine, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain a yellow solid: the third intermediate (I-7Pre), the yield is 19%, ESI-MS (positive): 318.1 (M+1) + .

[0101] (S4) 200mCi 18 F was enriched on a QMA column and eluted with 0.6 mL of eluent (150 mg Kryptofix 222, 40 mg K 2 CO 3 Dissolve in 9 mL acetonitrile and 1 mL water) 18F was eluted into a reaction bottle; the solvent was dried by heating at 110°C for 20 min under high-purity nitrogen; 0.5 mg of the third intermediate (I-7Pre) was added to the reaction bottle and reacted at 150°C for 15 min; the reaction solution was separated and purified by HPLC (HPLC conditions: CAPCELL PAK C18 UG80 10mm×250mm, acetonitrile: water=35:65, flow rate: 3mL / min), the product was collected and solid phase extracted with a C18 column, and the product was eluted with ethanol to obtain a small molecule compound (I-7), and the labeling efficiency was 10%.

[0102] Example 8

[0103] This embodiment provides a small molecule compound (I-8), whose chemical structure is shown below:

[0104]

[0105] The small molecule compound (I-7) is prepared by the following method (the synthesis process is described in the following formula):

[0106]

[0107] (S1) Dissolve 10.0 mmol of 4-bromobenzyl bromide 1 in 10 mL of toluene, add 30.0 mmol of triethyl phosphite to obtain a mixed solution; 2 The mixture was reacted at 110°C for 12 hours under protection, and then the solvent (toluene) was evaporated to dryness. The mixture was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 8:1) to obtain a white solid: the first intermediate (2);

[0108] (S2) 9.5 mmol of the first intermediate (2) prepared in step (S1) was added to 10 mL of dimethylformamide, and then 15.0 mmol of sodium hydroxide, N 2 The mixture was reacted at 0°C for 30 min under protection, and then 9.0 mmol 4-dimethylaminobenzaldehyde (3-6) was added and reacted at room temperature for 8 hours; 50 mL ice water was then added to the reaction solution, and a yellow solid was obtained by filtration, and then the yellow solid was recrystallized from ethyl acetate to obtain the second intermediate (4-6);

[0109] (S3) 1 mmol of the second intermediate (4-6) prepared in step (S2) was dissolved in 3 mL of toluene, and 1.2 mmol of 2-amino-5-methoxypyridine (5-6), 2.5 mmol of potassium tert-butoxide, 0.1 mmol of Pd 2 (dba) 3 , 0.2 mmol 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos) was transferred to the reaction flask, and the reaction flask was filled with N 2Protection, 110 ° C reaction for 8h; then add 10mL ethyl acetate to the reaction solution, wash three times with 10mL saturated brine, dry over anhydrous sodium sulfate and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain a yellow solid: the third intermediate (6-6); yield 26%, ESI-MS (positive): 345.2 (M+1) + ;

[0110] (S4) 0.2 mmol of the third intermediate (6-6) prepared in step (S3) was dissolved in 1 mL of dichloromethane, 2 mmol of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 2 h; then the solvent (dichloromethane) was evaporated to obtain a brown oil: the fourth intermediate (7-6), ESI-MS (positive): 332.2 (M+1) + .

[0111] (S5) The fourth intermediate (7-6) obtained in step (S4) was dissolved in 1 mL of DMF, 1 mmol of sodium hydroxide and 0.5 mmol of 1,2-dimethylbenzyloxyethane were added, and the mixture was reacted at room temperature for 6 h. After the reaction, 5 mL of water was added, and the mixture was extracted with 10 mL of ethyl acetate. After drying over anhydrous sodium sulfate, the mixture was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2.5: 1) to obtain a yellow solid: the fifth intermediate (I-8pre), ESI-MS (positive): 529.2 (M+1) + .

[0112] (S6) 200mCi 18 F was enriched on a QMA column and eluted with 0.6 mL of eluent (150 mg Kryptofix 222, 15 mg K 2 CO 3 Dissolve in 9 mL acetonitrile and 1 mL water) 18 F was eluted into a reaction bottle; the solvent was dried by heating at 110°C for 20 min under high-purity nitrogen; 0.5 mg of the fifth intermediate (I-8pre) was added to the reaction bottle and reacted at 140°C for 10 min; the reaction solution was separated and purified by HPLC (HPLC conditions: CAPCELL PAK C18 UG80 10mm×250mm, acetonitrile: water=37:63, flow rate: 3mL / min), the product was collected and solid phase extracted with a C18 column, and the product was eluted with ethanol to obtain a small molecule compound (I-8), and the labeling efficiency was 16%.

[0113] Example 9

[0114] This embodiment provides a study on the affinity of small molecule compounds (prepared in Examples 1 to 6) to α-syn fibers at the protein level (surface plasmon resonance and fluorescence methods).

[0115] (A1) Preparation of α-syn fibers:

[0116] Add 100 μL of α-synuclein monomer (synthesized and purified by a commissioned company, 500 μM) to 400 μL of 1*PBS Buffer, mix by pipetting with a pipette tip to obtain a mother solution with a final protein concentration of 100 μM (about 5 mg / mL), and then incubate the mother solution in a shaker at 37°C and 1000 rpm for 7 days to obtain an α-syn fiber solution.

[0117] (A2) SPR protein level binding test

[0118] The purchased CMD 500 chip (GE) was washed with double distilled water, air-dried, and then run the "dock" program to enter the biacore T200 instrument, run the "desorb" program to remove impurities in the instrument, run the "Prime" program to remove bubbles, and then proceed to the next step of the experiment. The α-syn fiber solution (5 mg / mL) prepared in step (A1) was diluted to 20 μg / mL of sample with sodium acetate buffer at pH 4.0, 4.5, 5.0, and 5.5, respectively, and injected for 60 seconds in sequence. After injection, NaOH was used for elution, and the best coupling buffer was selected according to the coupling amount (the best coupling buffer was sodium acetate buffer at pH 4.0, and the buffer was used for subsequent experiments).

[0119] 200 μL chip activation solution (provided by GE) was injected for 900 s to activate the carboxyl groups on the chip. The α-syn fiber solution was diluted with sodium acetate buffer at pH 4.0 to a 50 μg / mL protein sample, and then the protein sample was injected for 110 s, repeated 10 times, so that the protein was fully coupled to the chip. The blocking solution (provided by GE) was injected for 900 s to block the unreacted carboxyl groups in the chip. The small molecule compounds (I-1, I-2, I-3, I-4, I-5, I-6) prepared in Examples 1 to 6 were diluted to different concentrations (10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, μM, 0.156 μM, 0.078 μM) using PBS, and the "Kinetic" program in Biacore was run to inject the samples in sequence. The binding force and kinetic spectrum of the small molecule compounds were derived using the system software, and the binding force of the small molecule compounds to α-syn fibers was calculated (as shown in Table 1).

[0120] (A3) Fluorescence protein level binding test

[0121] 30 μL of small molecule compounds (I-1, I-2, I-3, I-4, I-5, I-6) of different concentrations (10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, μM, 0.156 μM, 0.078 μM) were added to a 384-well plate, 30 μL of the α-syn fiber solution (1 μM) prepared in step (A2) was added to the experimental group, and 30 μL of PBS was added to the control group. The 384-well plate was shaken (50 rpm) and incubated for 1 hour at room temperature; the maximum absorption and emission wavelengths of the small molecules were detected by an ELISA reader, and the fluorescence value was detected at the wavelength. The binding force of the small molecule compound and the α-syn fiber was calculated using the Saturation binding module of GraphPad Prism (as shown in Table 1).

[0122] Table 1 Test results of the binding ability of small molecule compounds to α-syn fibers

[0123] Small molecule compounds Maximum absorption wavelength Maximum emission wavelength Fluorescence method SPR I-1 360nm 415nm B C I-2 355nm 410nm B A I-3 360nm 410nm B A I-4 360nm 415nm C D I-5 355nm 425nm A B I-6 360nm 430nm A A

[0124] In Table 1, A represents K d Value is less than 0.5 μM, B represents K d The value is between 0.5 and 2 μM, and C represents K d The value is between 2 and 10 μM, and D represents K d The value was greater than 10 μM.

[0125] It can be found from Table 1 that the small molecule compounds prepared in Examples 1 to 6 have good binding force with α-syn fibers; further, the small molecule compounds can be used as independent reagents or reagent compositions for the detection of α-synuclein fibers in vivo and in vitro, and further applied to the diagnosis of neurodegenerative diseases associated with α-synuclein fibers or other misfolded proteins.

[0126] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A small molecule compound, characterized in that: The chemical structural formula of the small molecule compound is shown below: In formula (I), A and B are independently selected from a substituted or unsubstituted 4- to 7-membered saturated cycloalkane, a substituted or unsubstituted 4- to 7-membered aromatic ring, or a substituted or unsubstituted 4- to 7-membered heterocycle; The substitution means that one or more hydrogen atoms on the ring are replaced by the following substituents: Amide, methylsulfone, ester, hydroxy, nitro, cyano, C1-6 alkoxy, C1-6 halohydroxyalkoxy, C1-6 alkyl, C1-6 alkylamino, halogen, C1-6 haloalkyl, C1-6 haloalkoxy, C2-6 alkenyl, C2-6 alkynyl, acyl, C1-6 hydroxyalkyl, carboxyl, amino or sulfonamide.

2. A small molecule compound according to claim 1, characterized in that: The chemical structure of the small molecule compound is selected from one of the following structural formulas:

3. A method for preparing a small molecule compound as claimed in any one of claims 1 to 2, characterized in that: The following steps are involved: (S1) dissolving 4-bromobenzyl bromide and mixing it with triethyl phosphite, and reacting under nitrogen protection to obtain a first compound; (S2) dissolving the first compound prepared in step (S1) and adding sodium hydroxide, adding the second compound after pre-reaction under nitrogen protection, and performing post-treatment after the reaction to obtain a third compound; (S3) dissolving the third compound prepared in step (S2), and sequentially adding the fourth compound, potassium tert-butoxide, Pd2(dba)3 and 2-dicyclohexylphospho-2',4',6'-triisopropylbiphenyl, reacting under nitrogen protection, and then post-treating to obtain a small molecule compound; Among them, the chemical structural formula of the first compound is shown in formula (II), the chemical structural formula of the second compound is shown in formula (III), the chemical structural formula of the third compound is shown in formula (IV), and the chemical structural formula of the fourth compound is shown in formula (V): In formula (III) and formula (IV), A is the same and is selected from a substituted or unsubstituted 4-7 membered saturated cycloalkane, a substituted or unsubstituted 4-7 membered aromatic ring, or a substituted or unsubstituted 4-7 membered heterocycle; In formula (V), B is selected from a substituted or unsubstituted 4- to 7-membered saturated cycloalkane, a substituted or unsubstituted 4- to 7-membered aromatic ring, and a substituted or unsubstituted 4- to 7-membered heterocycle; The substitution means that one or more hydrogen atoms on the ring are replaced by the following substituents: Amide, methylsulfone, ester, hydroxy, nitro, cyano, C1-6 alkoxy, C1-6 halohydroxyalkoxy, C1-6 alkyl, C1-6 alkylamino, halogen, C1-6 haloalkyl, C1-6 haloalkoxy, C2-6 alkenyl, C2-6 alkynyl, acyl, C1-6 hydroxyalkyl, carboxyl, amino or sulfonamide.

4. The method for preparing a small molecule compound according to claim 3, characterized in that: In step (S1), the molar ratio of 4-bromobenzyl bromide to triethyl phosphite is 1:2-5; during the reaction, the temperature is 90-120° C. and the reaction time is 6-8 hours.

5. The method for preparing a small molecule compound according to claim 3, characterized in that: In step (S2), the molar ratio of the first compound, sodium hydroxide and the second compound is 0.9-1.1:1.5-1.8:1; during the pre-reaction process, the temperature is 0°C and the time is 0.3-1 hour; during the reaction process, the temperature is 20-30°C and the time is 6-8 hours.

6. The method for preparing a small molecule compound according to claim 3, characterized in that: In step (S3), the molar ratio of the third compound, the fourth compound, potassium tert-butoxide, Pd2(dba)3 and 2-dicyclohexylphospho-2',4',6'-triisopropylbiphenyl is 1:0.9-1.5:2-3:0.05-0.2:0.1-0.3; during the reaction, the temperature is 100-120°C and the time is 6-8 hours.

7. Use of the small molecule compound according to any one of claims 1 to 2 in the preparation of a reagent for diagnosing a neurological disorder associated with α-synuclein fibers, and / or a drug for treating and / or preventing α-synuclein fiber-related diseases.

8. The use according to claim 7, characterized in that: The neurological disorder is selected from one of Parkinson's disease, multiple system atrophy or dementia with Lewy bodies; The reagent is an imaging diagnostic reagent.

9. A reagent for diagnosing a neurological disorder associated with α-synuclein fibers, characterized in that The reagent contains the small molecule compound according to any one of claims 1 to 2.

10. A drug for treating and / or preventing α-synuclein fiber-related diseases, characterized in that: The medicine contains the small molecule compound according to any one of claims 1 to 2.

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