D-π-A structured trisubstituted olefins with high affinity for α-synuclein, preparation method thereof, and application thereof

Abstract

The present invention relates to the field of medicinal chemistry. Specifically, it relates to a series of trisubstituted olefin compounds with a D-π-A structure, their preparation methods, and the application of such compounds as fluorescent probes for detecting Lewy pathologies (including Lewy bodies and Lewy neurites). The trisubstituted olefin compounds provided by the present invention have a D-π-A structure, and their structural general formula is shown as formula (I). The compound of formula (I) or its pharmaceutically acceptable salt, ester, amide or prodrug has a high affinity and high discrimination for α-synuclein, and thus can be used for fluorescent detection of α-synuclein aggregates in tissue samples and as a functional imaging agent for α-synuclein in the brain. When used for nuclear medicine imaging, it needs to be labeled with a suitable radioisotope.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular, to a series of trisubstituted olefin compounds having a D-π-A structure, a preparation method thereof, and the use of such compounds as fluorescent probes for detecting Lewy pathology (including Lewy bodies and Lewy neurites). Background Art

[0002] α-Synuclein is a small molecule protein composed of 140 amino acids. Under normal physiological conditions, it is widely distributed in the brain and peripheral nervous system in a random helix form.

[0003] At present, the physiological function of α-synuclein has not been fully clarified. A more widely recognized view is that it plays an important role in regulating and maintaining the normal function and plasticity of synapses. In some special diseases, soluble α-synuclein will abnormally fold, form a highly ordered β-sheet structure, and further induce aggregation to form oligomers and fibers, and finally form insoluble cell inclusions. Such diseases include Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and some rare diseases, which are collectively referred to as α-synucleinopathies.

[0004] Parkinson's disease is the most common α-synucleinopathy and the second most common neurodegenerative disease in the world after Alzheimer's disease (AD). At present, the diagnosis of Parkinson's disease mainly relies on family history and clinical symptoms, without the assistance of effective imaging techniques. However, due to the long time interval between the onset of the pathological process of Parkinson's disease and the manifestation of clinical symptoms, it is very difficult to achieve early detection and diagnosis of Parkinson's disease.

[0005] In the dopaminergic neurons of PD patients, α-synuclein will pathologically aggregate into a fibrous form and deposit as Lewy bodies (LBs) and Lewy neurites (LNs), collectively referred to as Lewy pathology, which makes PD different from other diseases with similar clinical characteristics. At the same time, according to the Braak analysis, the pathological process of PD can be divided into six stages. The early degeneration starts from the olfactory bulb and olfactory nucleus, and then gradually progresses to the substantia nigra, midbrain, and basal ganglia. Its disease progression is closely consistent with the appearance and spread of Lewy pathology. Therefore, α-synuclein has become an important biomarker for the preclinical diagnosis of PD, providing a window for further understanding and accurately monitoring the disease state.

[0006] In recent years, with the development of imaging technologies, optical imaging methods have been widely used in in vitro tissue section staining of pathological proteins and in vivo imaging of experimental animals due to their advantages such as low cost and intuitive results, which helps in the monitoring of disease progression and the establishment of animal models.

[0007] Currently, fluorescent probes targeting α-synuclein can be roughly classified into two categories according to their uses, namely for in vitro fiber monitoring and for staining and imaging in tissues or cells. Early probes include naphthalene sulfonate derivatives (ANS and TNS series), cyanine compounds (T-284, SH-516, SL-631, JC-1, etc.), 3,5-diphenylpyrazole derivatives (Anle138b, etc.), and aggregation-induced emission compounds TPE-TPP. Due to reasons such as their own charge, activity, or fluorescence properties, they are mainly used for in vitro monitoring of the aggregation process of α-synuclein. Later, with the discovery of benzofuranone derivative probes Tg-52 and benzothiazole derivative probes PP-BTA-4 and RB1, α-synuclein staining at the tissue section level and cell level was initially achieved.

[0008] However, generally speaking, the number of fluorescent probes for α-synuclein that can be used for tissue staining and in vivo imaging of experimental animals is very limited. In addition, due to the frequent co-occurrence of abnormal aggregation of amyloid-β (Aβ) during the pathological process of synucleinopathies, the development of specific fluorescent probes for α-synuclein is particularly challenging, and highly affinity and discriminatory specific α-synuclein probes still need to be continuously developed. Summary of the Invention

[0009] The object of the present invention is to provide a trisubstituted olefin compound with a D-π-A structure having high affinity and discrimination for α-synuclein, its preparation method, and its use for fluorescent detection of Lewy pathology in the brains of Parkinson's patients.

[0010] In a first aspect, the trisubstituted olefin compound provided by the present invention has a D-π-A structure, and its structural general formula is shown as formula (I):

[0011]

[0012] Among them, W is a six-membered aromatic ring or a benzo six-membered aromatic ring;

[0013] R1 is N,N-dimethylamino, methoxy, or hydrogen;

[0014] R2 is

[0015] n is 0 or 1, and the carbon-carbon double bonds are all trans double bonds.

[0016] Preferably, when W is a six-membered aromatic ring, R1 is N,N-dimethylamino at the para position, and the general formula of the corresponding compound is as shown in formula (II);

[0017]

[0018] Wherein, X is CH or N, that is, W is a benzene ring or a pyridine ring.

[0019] Preferably, when W is a benzo six-membered aromatic ring, the general formula corresponding to W is The general formula of the corresponding compound is as shown in formula (IV);

[0020]

[0021] Wherein, X, Y and Z each independently represent N or CH;

[0022] R1 is N,N-dimethylamino, methoxy or hydrogen at the 6th or 7th position.

[0023] In a second aspect, the present invention also provides a method for preparing the trisubstituted olefin compound represented by the above general formula (I). Specifically as follows:

[0024] When in the compound represented by the general formula (I), R2 is The preparation method of the corresponding compound is:

[0025] React with Dissolve in anhydrous methanol, add a methanol solution of saturated potassium carbonate, and let it stand at room temperature to precipitate a solid; obtain the compound represented by the above corresponding general formula (I). The obtained solid product is further post-treated, such as washed with absolute ethanol and petroleum ether.

[0026] The React with The molar ratio is 1:(0.66 - 3.11).

[0027] When in the compound represented by the general formula (I), R2 is The preparation method of the corresponding compound is:

[0028] React with Dissolve in anhydrous methanol, add piperidine, reflux, and let it stand at room temperature until a solid precipitates to obtain the compound represented by the corresponding general formula (I). The obtained solid product is further post-treated, such as washed with absolute ethanol and petroleum ether.

[0029] React with The molar ratio is 1:(1.02 - 1.04).

[0030] When in the compound represented by the general formula (I), R2 is When, the preparation method of the corresponding compound is as follows:

[0031] React with Dissolve in anhydrous methanol, then add a methanol solution of sodium methoxide, reflux overnight, and let stand at room temperature until a solid precipitates to obtain a compound represented by the corresponding general formula (I). The obtained solid product is further post-treated, such as washed with absolute ethanol and petroleum ether, or separated by column chromatography after removing the solvent.

[0032] React with The molar ratio of is 1:(1.2 - 1.4).

[0033] In a third aspect, the present invention also provides derivatives of the compound represented by the above general formula (I), and the derivatives include pharmaceutically acceptable salts, esters, amides or prodrugs of the compound represented by the general formula (I).

[0034] In a fourth aspect, the present invention also provides a radiopharmaceutical agent, the active ingredient of which is the compound represented by the above general formula (I) or its derivative. This radiopharmaceutical agent can be used to diagnose α-synuclein deposition diseases. The α-synuclein deposition diseases include but are not limited to Parkinson's disease, Lewy body dementia, and multiple system atrophy.

[0035] In a fifth aspect, the present invention also provides a fluorescent probe, the active ingredient of which is the compound represented by the above general formula (I) or its derivative. The fluorescent probe is used to detect α-synuclein aggregates in tissue samples.

[0036] The beneficial effects of the present invention are as follows:

[0037] The compound of the general formula (I) of the present invention or its pharmaceutically acceptable salt, ester, amide or prodrug has a high affinity and high discrimination for α-synuclein, and thus can be used for fluorescent detection of α-synuclein aggregates in tissue samples and as a radiopharmaceutical agent for the function imaging of α-synuclein in the brain. When used for nuclear medicine imaging, it needs to be labeled with a suitable radioactive isotope. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of the synthesis process of Compounds 1 - 20 in Examples 1 - 20 of the present invention; the reaction reagents and conditions designed therein are: (a) potassium carbonate, methanol, heated for 5 minutes or piperidine or sodium methoxide, methanol, refluxed for 1 hour to overnight; (b) 1-bromo-2-fluoroethane, potassium carbonate or cesium carbonate, acetone, refluxed overnight.

[0039] Figure 2 It is the competitive binding curve of Compounds 1 - 20 in the present invention to α-synuclein aggregates.

[0040] Figure 3 For the compound 1-20 in the present invention against Aβ 1-42 Aggregate competition binding curve.

[0041] Figure 4 They are respectively the fluorescence emission spectra of the compounds 1-9, 12, 13, 15-20 in the present invention in different solvents.

[0042] Figure 5 They are respectively the fluorescence excitation and emission spectra after the compounds 1-9, 13, 17-20 in the present invention bind to α-synuclein aggregates.

[0043] Figure 6 They are respectively the fluorescence excitation and emission spectra after the compounds 1-9, 13, 17-20 in the present invention bind to Aβ 1-42 Aggregate.

[0044] Figure 7 (A) to (I) are respectively the staining diagrams of the amyloid plaques on the brain sections of AD patients by the compounds 4, 6, 8, 9, 13, 17, 18, 19 and 20; (J), (M), (P) are respectively the staining diagrams of the Lewy pathology on the brain sections of PD patients by the compounds 20, 9 and 8; (K), (N), (Q) are the images of immunofluorescence (GFP) at the same positions; (L), (O), (R) are the superimposed images.

[0045] Figure 8 It is the fluorescence imaging diagram of the in vitro biodistribution experiment of the compound 9 in the present invention. Detailed implementation mode

[0046] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0047] Example 1:

[0048] Synthesis of (E)-2-(benzo[d]thiazol-2-yl)-3-(4-(dimethylamino)phenyl)acrylonitrile (Compound 1)

[0049] Dissolve 2-acetonitrile benzothiazole (28.4 mg, 0.16 mmol) by heating in 5 mL of anhydrous methanol, add p-dimethylaminobenzaldehyde (35.2 mg, 0.24 mmol), and then add two drops of saturated methanol solution of K2CO3 as a catalyst. Let it stand. After the reaction system cools to room temperature, a solid precipitates. Filter by suction and wash with a small amount of mixed solution of ethanol and petroleum ether, and dry to obtain 9.4 mg of orange-yellow solid. The yield is 18.9%.

[0050] The structure is as follows: 11H NMR (400 MHz, d6-DMSO) δ 8.11 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 9.2 Hz, 2H), 7.95 (d, J = 8.1 Hz, 1H), 7.52–7.47 (m, 1H), 7.43–7.38 (m, 1H), 6.83 (d, J = 9.2 Hz, 2H), 3.05 (s, 6H). 13 13C NMR (151 MHz, CDCl3) δ 164.88, 153.64, 152.98, 147.21, 134.57, 133.26, 126.70, 125.21, 122.84, 121.56, 120.41, 118.25, 111.84, 97.62, 40.14. HRMS: m / z calcd for C 18 H 16 N3S 306.10594; found 306.10565, M+H + 。

[0051] Example 2:

[0052] (E)-2-(benzo[d]thiazol-2-yl)-3-(6-(dimethylamino)pyridin-3-yl)acrylonitrile (Compound 2) synthesis

[0053] According to the method for synthesizing Compound 1, 2-acetonitrile benzothiazole (35.0 mg, 0.20 mmol) was reacted with p-dimethylaminonicotinaldehyde (32.4 mg, 0.22 mmol) to obtain orange solid 2 (48.9 mg, 79.4%). The structure is as follows: 1 1H NMR (600 MHz, d6-DMSO) δ 8.63 (s, 1H), 8.35 (d, J = 9.3 Hz, 1H), 8.14 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 9.3 Hz, 1H), 3.13 (s, 6H). 13 13C NMR (151 MHz, CF3COOD) δ 153.90, 141.82, 139.21, 135.09, 132.19, 131.80, 125.14, 124.12, 123.63, 118.31, 114.21, 113.58, 110.76, 109.73, 98.50, 51.75, 50.59. HRMS: m / z calcd for C17 H 15 N4S 307.10119; found 307.10098, M+H + 。

[0054] Example 3:

[0055] Synthesis of (E)-2-(1H-benzo[d]imidazol-2-yl)-3-(4-(dimethylamino)phenyl)acrylonitrile (Compound 3)

[0056] Dissolve 2-cyanobenzimidazole (407.6 mg, 2.59 mmol) and p-dimethylaminobenzaldehyde (381.4 mg, 2.56 mmol) in 20 mL of anhydrous methanol. Add 100 μL of piperidine as a catalyst. After refluxing for 1 hour, stop heating. Let it stand to room temperature and a solid precipitates. Filter by suction and wash with a small amount of petroleum ether to obtain light brown solid 3 (413.2 mg, 56.0%). The structure is as follows: 1 H NMR (400 MHz, d6-DMSO) δ 8.12 (s, 1H), 7.91 (d, J = 9.0 Hz, 2H), 7.59–7.54 (m, 2H), 7.22 (d, J = 3.2 Hz, 1H), 7.21 (d, J = 3.2 Hz, 1H), 6.87 (d, J = 9.1 Hz, 2H), 3.07 (s, 6H). 13 C NMR (151 MHz, d6-DMSO) δ 152.94, 149.43, 146.00, 144.04, 135.28, 132.39, 123.21, 122.46, 120.31, 119.12, 118.29, 112.31, 111.72, 94.25, 40.64. HRMS: m / z calcd for C 18 H 17 N4 289.14477; found 289.14456, M+H + 。

[0057] Example 4:

[0058] Synthesis of (2E,4E)-2-(benzo[d]thiazol-2-yl)-5-(4-(dimethylamino)phenyl)penta-2,4-dienenitrile (Compound 4)

[0059] According to the method for synthesizing Compound 1, a red solid 4 (6.4 mg, 20.5%) was prepared by reacting 2-cyanobenzothiazole (16.4 mg, 0.09 mmol) with p-dimethylaminocinnamaldehyde (16.6 mg, 0.09 mmol). The structure is as follows: 1 H NMR (600 MHz, CF3COOD) δ 8.46 (d, J = 11.2 Hz, 1H), 8.12 (d, J = 3.6 Hz, 1H), 8.11 (d, J = 3.5 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.88 (t, J = 7.9 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.74 (d, J = 15.2 Hz, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.62 (dd, J = 15.1, 11.2 Hz, 1H), 3.43 (s, 6H). 13 C NMR (151 MHz, CDCl3) δ 163.03, 159.71, 153.84, 151.73, 148.62, 147.15, 134.66, 130.40, 126.79, 125.50, 123.15, 121.59, 119.71, 116.31, 112.58, 99.99, 40.55. HRMS: m / z calcd for C 20 H 18 N3S3 32.12159; found 332.12131, M+H.

[0060] Example 5:

[0061] (2E,4E)-2-(benzo[d]thiazol-2-yl)-5-(6-(dimethylamino)pyridin-3-yl)penta-2,4-dienenitrile (Compound 5) Synthesis

[0062] According to the method for synthesizing Compound 1, a red metallic solid 5 (52.3 mg, 56.4%) was prepared by reacting 2-cyanobenzothiazole (48.6 mg, 0.28 mmol) with (E)-3-(6-dimethylaminopyridinyl)acrolein (55.8 mg, 0.32 mmol). The structure is as follows: 11H NMR (400 MHz, CF3COOD) δ 8.55–8.50 (m, 2H), 8.29 (s, 1H), 8.21 (d, J = 8.3 Hz, 2H), 7.98 (t, J = 7.9 Hz, 1H), 7.91 (t, J = 7.8 Hz, 1H), 7.71 (d, J = 15.2 Hz, 1H), 7.61 (dd, J = 15.0, 10.9 Hz, 1H), 7.32 (d, J = 9.8 Hz, 1H), 3.50 (s, 6H). 13 13C NMR (151 MHz, CF3COOD) δ 167.02, 156.06, 152.07, 147.26, 139.95, 138.69, 131.26, 129.88, 129.09, 124.02, 122.82, 120.45, 117.40, 112.80, 99.07, 38.94, 38.02. HRMS: m / z calcd for C 19 H 17 N4S3 333.11684; found 333.11642, M+H + 。

[0063] Example 6:

[0064] (E)-2-(benzo[d]thiazol-2-yl)-3-(6-(dimethylamino)naphthalen-2-yl)acrylonitrile (Compound 6) synthesis

[0065] According to the method for synthesizing Compound 1, a red solid 6 (37.2 mg, 99.0%) was prepared by reacting 2-acetonitrile benzothiazole (18.4 mg, 0.11 mmol) with 6-dimethylamino naphthalene-2-carboxaldehyde (25.0 mg, 0.13 mmol). The structure is as follows: 1 1H NMR (600 MHz, CDCl3) δ 8.28 (s, 1H), 8.26 (s, 1H), 8.14 (dd, J = 8.8, 1.6 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.53–7.50 (m, 1H), 7.41–7.39 (m, 1H), 7.16 (dd, J = 9.1, 2.4 Hz, 1H), 6.88 (s, 1H), 3.13 (s, 6H). 13CNMR(151MHz,CDCl3)δ164.02,153.86,150.42,147.46,137.28,134.92,133.61,130.71,127.03,126.78,126.04,125.80,125.57,123.33,121.62,117.57,116.43,105.74,101.75,40.53.HRMS:m / z calcd for C 22 H 18 N3S 356.12159;found 356.12109,M+H + 。

[0066] Example 7:

[0067] (E)-2-(benzo[d]thiazol-2-yl)-3-(2-(dimethylamino)quinolin-6-yl)acrylonitrile (Compound 7) Synthesis

[0068] According to the method for synthesizing Compound 1, orange solid 7 (23.5 mg, 46.3%) was prepared by reacting 2-cyanobenzothiazole (24.8 mg, 0.14 mmol) with 2-dimethylaminoquinoline-6-carbaldehyde (31.0 mg, 0.15 mmol). The structure is as follows: 1 H NMR(600MHz,CF3COOD)δ8.78(s,1H),8.73(s,1H),8.60(d,J = 8.9Hz,1H),8.49(d,J = 9.8Hz,1H),8.27(t,J = 8.7Hz,2H),8.11(d,J = 8.9Hz,1H),8.02(t,J = 7.8Hz,1H),7.95(t,J = 7.8Hz,1H),7.43(d,J = 9.8Hz,1H),3.64(s,3H),3.62(s,3H). 13 C NMR(101MHz,CF3COOD)δ167.99,154.39,152.80,143.21,140.07,140.02,139.83,134.58,133.70,131.34,130.07,129.34,128.29,122.85,121.03,118.98,117.64,112.37,98.18,39.73,38.39.HRMS:m / zcalcd for C 21 H 17 N4S 357.11684;found 357.11646,M+H +。

[0069] Example 8:

[0070] Synthesis of (E)-2-(benzo[d]thiazol-2-yl)-3-(6-(dimethylamino)quinoxalin-2-yl)acrylonitrile (Compound 8)

[0071] Following the method for synthesizing Compound 1, a purple-red solid 8 (19.4 mg, 54.3%) was prepared by reacting 2-cyanobenzothiazole (17.4 mg, 0.1 mmol) with 6-(dimethylamino)quinoxaline-2-carbaldehyde (21.9 mg, 0.11 mmol). The structure is as follows: 1 HNMR (600 MHz, CDCl3) δ 8.97 (s, 1H), 8.39 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 8.03 (d, J = 9.3 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.55–7.51 (m, 1H), 7.46–7.42 (m, 2H), 7.04 (d, J = 2.7 Hz, 1H), 3.23 (s, 6H). 13 C NMR (151 MHz, CF3COOD) δ 168.01, 158.02, 150.26, 149.94, 147.73, 144.44, 140.11, 137.73, 134.27, 133.76, 133.51, 131.44, 130.19, 129.44, 125.55, 122.85, 117.79, 98.02, 40.60. HRMS: m / z calcd for C 20 H 16 N5S 358.11209; found 358.11154, M+H + 。

[0072] Example 9:

[0073] Synthesis of (2E,4E)-2-(benzo[d]thiazol-2-yl)-5-(6-(dimethylamino)quinoxalin-2-yl)penta-2,4-dienenitrile (Compound 9)

[0074] Following the method for synthesizing Compound 1, a purple solid 9 (3.3 mg, 5.9%) was prepared by reacting 2-cyanobenzothiazole (25.3 mg, 0.15 mmol) with (E)-3-(6-(dimethylamino)quinoxalin)-2-carbaldehyde (34.5 mg, 0.15 mmol). The structure is as follows:1 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.11–8.05 (m, 2H), 8.02 (d, J = 14.4 Hz, 1H), 7.96–7.89 (m, 2H), 7.57–7.51 (m, 1H), 7.47–7.41 (m, 2H), 7.38 (d, J = 14.3 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 3.21 (s, 6H). 13 13C NMR (151 MHz, CF3COOD) δ 167.10, 157.63, 156.47, 155.33, 150.41, 146.87, 144.07, 140.08, 139.23, 134.17, 133.53, 132.94, 131.41, 129.27, 126.65, 125.09, 122.89, 117.52, 100.28, 40.09. HRMS: m / z calcd for C 22 H 18 N5S 384.12774; found 384.12735, M + H + 。

[0075] Example 10:

[0076] (E)-2-(benzo[d]thiazol-2-yl)-3-(quinoxalin-2-yl)acrylonitrile (Compound 10) Synthesis

[0077] According to the method for synthesizing Compound 1, yellow solid 10 (53.4 mg, 84.5%) was prepared by reacting 2-acetonitrile benzothiazole (35.0 mg, 0.2 mmol) with quinoxaline-2-carboxaldehyde (38.0 mg, 0.24 mmol). The structure is as follows: 1 1H NMR (600 MHz, CDCl3) δ 9.21 (s, 1H), 8.52 (s, 1H), 8.28–8.25 (m, 1H), 8.16–8.12 (m, 2H), 7.96 (d, J = 8.0 Hz, 1H), 7.87–7.85 (m, 2H), 7.57 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H). 1313C NMR(151MHz,CDCl3)δ161.83,153.77,146.00,145.86,142.74,142.38,140.25,135.81,132.08,131.22,130.25,129.37,127.33,126.64,124.13,121.93,115.74,111.55.HRMS:m / z calcd for C 18 H 11 N4S3 15.06989; found 315.06982, M+H + 。

[0078] Example 11:

[0079] (E)-2-(benzo[d]thiazol-2-yl)-3-(6-methoxyquinolin-2-yl)acrylonitrile (Compound 11) Synthesis

[0080] According to the method for synthesizing Compound 1, a light yellow solid 11 (39.6 mg, 77.3%) was prepared by reacting 2-acetonitrile benzothiazole (26.0 mg, 0.15 mmol) with 6-methoxyquinoline-2-carbaldehyde (35.7 mg, 0.19 mmol). The structure is as follows: 1 1H NMR(600MHz,d6-DMSO)δ8.55(s,1H),8.42(d,J = 8.2Hz,1H),8.22(d,J = 7.9Hz,1H),8.13(d,J = 8.0Hz,1H),8.03(d,J = 8.4Hz,1H),7.99(d,J = 9.1Hz,1H),7.62(t,J = 7.4Hz,1H),7.58–7.50(m,2H),7.45(s,1H),3.95(s,3H). 13 13C NMR(151MHz,d6-DMSO)δ163.68,159.51,153.59,148.34,146.19,143.95,136.30,135.19,131.29,130.33,127.80,127.03,124.79,124.24,123.85,123.12,116.46,108.27,106.17,56.33.HRMS:m / z calcd for C 20 H 14 ON3S3 344.08521; found 344.08502, M+H + 。

[0081] Example 12:

[0082] Synthesis of (E)-2-(benzo[d]thiazol-2-yl)-3-(7-(dimethylamino)quinoxalin-2-yl)acrylonitrile (Compound 12)

[0083] According to the method for synthesizing Compound 1, purple solid 12 (63.4 mg, 79.5%) was prepared by reacting 2-cyanobenzothiazole (40.0 mg, 0.23 mmol) with 7-(dimethylamino)quinoxaline-2-carbaldehyde (44.9 mg, 0.22 mmol). The structure is as follows: 1 H NMR (600 MHz, CDCl3) δ 8.86 (s, 1H), 8.44 (s, 1H), 8.14 (dd, J = 8.2, 0.6 Hz, 1H), 7.96 (dd, J = 7.9, 0.6 Hz, 1H), 7.94 (d, J = 9.3 Hz, 1H), 7.57 (ddd, J = 8.3, 7.2, 1.2 Hz, 1H), 7.51–7.49 (m, 1H), 7.49–7.46 (m, 1H), 7.13 (d, J = 2.8 Hz, 1H), 3.20 (s, 6H). 13 C NMR (151 MHz, CDCl3) δ 162.32, 153.81, 151.79, 145.62, 144.41, 141.53, 137.32, 135.68, 129.62, 127.20, 126.45, 124.04, 122.03, 121.87, 115.96, 110.45, 105.07, 90.77, 40.60. HRMS: m / z calcd for C 20 H 16 N5S358.11209; found 358.11172, M+H + 。

[0084] Example 13:

[0085] Synthesis of (E)-2-(1H-benzo[d]imidazol-2-yl)-3-(6-(dimethylamino)quinoxalin-2-yl)acrylonitrile (Compound 13)

[0086] According to the method for synthesizing Compound 3, magenta solid 12 (46.9 mg, 55.2%) was prepared by reacting 2-cyanobenzimidazole (40.8 mg, 0.26 mmol) with 6-(dimethylamino)quinoxaline-2-carbaldehyde (50.2 mg, 0.25 mmol). The structure is as follows: 11H NMR (600 MHz, d6-DMSO) δ 13.13 (s, 1H), 9.02 (s, 1H), 8.46 (s, 1H), 7.89 (d, J = 9.4 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.65 (dd, J = 9.4, 2.8 Hz, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.30 (t, J = 7.1 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 2.8 Hz, 1H), 3.19 (s, 6H). 13 13C NMR (151 MHz, d6-DMSO) δ 152.65, 148.12, 147.73, 144.67, 144.06, 141.30, 141.07, 135.91, 135.51, 130.63, 124.30, 122.96, 121.60, 119.77, 116.40, 112.29, 104.51, 103.58, 40.54. HRMS: m / z calcd for C 20 H 17 N6S 341.15092; found 341.15067, M+H + 。

[0087] Example 14:

[0088] (Z)-3-(6-(dimethylamino)naphthalen-2-yl)-2-(4-nitrophenyl)acrylonitrile (Compound 14) Synthesis

[0089] According to the method for synthesizing Compound 1, a deep red solid 13 (1.8 mg, 5.9%) was prepared by reacting 4-nitrobenzonitrile (45.3 mg, 0.28 mmol) with 6-dimethylaminonaphthalene-2-carbaldehyde (17.8 mg, 0.09 mmol). The structure is as follows: 1 1H NMR (600 MHz, CF3COOD) δ 8.56 (s, 1H), 8.45 (d, J = 8.8 Hz, 2H), 8.31 (d, J = 9.0 Hz, 1H), 8.27 (d, J = 8.6 Hz, 1H), 8.21 (d, J = 1.5 Hz, 1H), 8.19 (s, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 8.7 Hz, 2H), 7.77 (dd, J = 8.9, 2.1 Hz, 1H). 1313C NMR (151 MHz, CF3COOD) δ 147.80, 146.97, 140.30, 134.22, 133.52, 133.09, 132.88, 130.48, 129.28, 127.47, 126.75, 124.42, 119.10, 116.92, 115.58, 110.45, 47.15. HRMS: m / z calcd for C 21 H 18 O2N3 344.13935; found 344.13922, M+H + 。

[0090] Example 15:

[0091] (Z)-3-(6-(dimethylamino)quinolin-2-yl)-2-(4-nitrophenyl)acrylonitrile (Compound 15) Synthesis

[0092] According to the method for synthesizing Compound 1, a dark red solid 15 (27 mg, 62.8%) was prepared by reacting 4-nitrobenzyl cyanide (22.0 mg, 0.14 mmol) with 6-dimethylaminoquinoline-2-carbaldehyde (25.0 mg, 0.12 mmol). The structure is as follows: 1 1H NMR (600 MHz, CF3COOD) δ 9.50 (d, J = 8.9 Hz, 1H), 9.17 (d, J = 8.9 Hz, 1H), 8.87 (s, 1H), 8.77 (d, J = 9.3 Hz, 1H), 8.62–8.59 (m, 1H), 8.55 (d, J = 8.8 Hz, 2H), 8.46 (s, 1H), 8.19 (d, J = 8.8 Hz, 2H), 3.71 (s, 6H). 13 13C NMR (151 MHz, CF3COOD) δ 149.80, 149.72, 149.26, 143.25, 138.14, 137.00, 131.95, 129.26, 129.19, 128.28, 127.96, 124.74, 124.51, 122.59, 122.01, 100.00, 47.17. HRMS: m / z calcd for C 20 H 17 O2N4 345.13460; found 345.13499, M+H + 。

[0093] Example 16:

[0094] (Z)-3-(6-(dimethylamino)quinoxalin-2-yl)-2-(4-nitrophenyl)acrylonitrile (Compound 16) Synthesis

[0095] According to the method for synthesizing Compound 1, 4-nitrobenzyl cyanide (13.5 mg, 0.08 mmol) was reacted with 6-dimethylaminoquinoxaline-2-carbaldehyde (12.0 mg, 0.06 mmol) to obtain dark red solid 16 (5.8 mg, 28.2%). The structure is as follows: 1 H NMR (600 MHz, CDCl3) δ 9.04 (s, 1H), 8.34 (d, J = 7.2 Hz, 2H), 8.01 (d, J = 10.3 Hz, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.81 (s, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.08 (s, 1H), 3.23 (s, 6H). 13 C NMR (151 MHz, CF3COOD) δ 158.64, 150.54, 145.76, 142.04, 140.75, 135.79, 135.63, 134.91, 129.46, 127.31, 126.80, 116.85, 114.54, 102.35, 96.67, 42.32. HRMS: m / z calcd for C 19 H 16 O2N 5346.12985; found 346.12939, M+H + .

[0096] Example 17:

[0097] (Z)-3-(6-(dimethylamino)quinolin-2-yl)-2-(4-hydroxyphenyl)acrylonitrile (Compound 17) Synthesis

[0098] 4-Hydroxybenzyl cyanide (122.0 mg, 0.92 mmol) and 6-dimethylaminoquinoline-2-carbaldehyde (132.5 mg, 0.66 mmol) were dissolved in 20 mL of anhydrous methanol. 100 μL of a methanol solution of sodium methoxide (1 M) was added as a catalyst, and the mixture was refluxed overnight. After standing to room temperature, a solid precipitated. It was filtered by suction and washed with a small amount of petroleum ether to obtain light orange solid 17 (37.0 mg, 17.6%). The structure is as follows: 11H NMR (600 MHz, d6-DMSO) δ 8.14 (d, J = 8.6 Hz, 1H), 7.85 (s, 1H), 7.84 (d, J = 9.4 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.51 (dd, J = 9.4, 2.8 Hz, 1H), 6.93 (d, J = 2.8 Hz, 1H), 6.89 (d, J = 8.7 Hz, 2H), 3.32 (br, 1H), 3.09 (s, 6H). 13 13C NMR (151 MHz, d6-DMSO) δ 159.59, 149.52, 147.91, 141.65, 138.45, 134.48, 130.17, 129.86, 128.08, 125.30, 123.10, 120.55, 118.52, 116.55, 112.10, 104.67, 40.64. HRMS: m / z calcd for C 20 H 18 ON 4316.14444; found 316.14429, M+H + .

[0099] Example 18:

[0100] (Z)-3-(6-(dimethylamino)quinoxalin-2-yl)-2-(4-hydroxyphenyl)acrylonitrile (Compound 18) Synthesis

[0101] 4-Hydroxybenzonitrile (114.0 mg, 0.86 mmol) and 6-(dimethylamino)quinoxaline-2-carbaldehyde (142.9 mg, 0.71 mmol) were dissolved in 20 mL of anhydrous methanol. 100 μL of a methanol solution of sodium methoxide (1 M) was added as a catalyst, and the mixture was refluxed overnight. The solvent was removed under reduced pressure, and the residue was separated by column chromatography (dichloromethane:ethyl acetate = 2:1, v:v) to obtain 179.1 mg of an orange solid with a yield of 79.7%. The structure is as follows: 1 1H NMR (400 MHz, d6-DMSO) δ 10.00 (s, 2H), 8.88 (s, 2H), 7.92 (s, 2H), 7.80 (d, J = 9.4 Hz, 1H), 7.65 (d, J = 8.8 Hz, 4H), 7.55 (dd, J = 9.5, 2.9 Hz, 2H), 6.93 (d, J = 2.8 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 3.11 (s, 6H). 1313C NMR(151MHz, d6-DMSO) δ 160.63, 156.70, 153.62, 146.87, 144.75, 144.16, 137.52, 135.41, 131.14, 130.21, 128.86, 121.89, 118.83, 117.03, 104.74, 40.51. HRMS: m / z calcd for C 19 H 17 O2N 4317.13969; found 317.13947, M+H + .

[0102] Example 19:

[0103] Synthesis of (Z)-3-(6-(dimethylamino)quinoxalin-2-yl)-2-(4-hydroxyphenyl)acrylonitrile (Compound 19)

[0104] Compound 17 (34.0 mg, 0.11 mmol), 1-bromo-2-fluoroethane (200.8 mg, 1.58 mmol) and potassium carbonate (45.0 mg, 0.33 mmol) were dissolved in 10 mL of acetone and refluxed overnight. The solvent was removed under reduced pressure. The residue was separated by column chromatography (petroleum ether: ethyl acetate = 1:1, v:v) to give 16.5 mg of an orange solid with a yield of 42.3%. The structure is as follows: 1 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 8.7 Hz, 1H), 8.03–7.07 (m, 2H), 7.75–7.71 (m, 3H), 7.39 (dd, J = 9.4, 2.9 Hz, 1H), 7.02–6.99 (m, 2H), 6.79 (d, J = 2.8 Hz, 1H), 4.86–4.83 (m, 1H), 4.74–4.71 (m, 1H), 4.32–4.29 (m, 1H), 4.25–4.22 (m, 1H), 3.13 (s, 6H). 13 13C NMR (151 MHz, CDCl3) δ 159.55, 149.43, 147.69, 139.83, 134.67, 130.05, 129.83, 127.79, 127.57, 120.91, 120.06, 118.08, 115.17, 104.26, 81.83 (d, J = 171.4 Hz), 67.34 (d, J = 20.5 Hz), 40.62. HRMS: m / z calcd for C 22 H 21ON3F 362.16632; found 362.16608, M+H + 。

[0105] Example 20:

[0106] Synthesis of (Z)-3-(6-(dimethylamino)quinoxalin-2-yl)-2-(4-(2-fluoroethoxy)phenyl)acrylonitrile (20)

[0107] Compound 18 (35.0 mg, 0.11 mmol), 1-bromo-2-fluoroethane (71.5 mg, 0.56 mmol) and cesium carbonate (76.2 mg, 0.23 mmol) were dissolved in 10 mL of acetone and refluxed overnight. The solvent was removed under reduced pressure. The residue was separated by column chromatography (petroleum ether: ethyl acetate = 1:1, v:v) to give 9.6 mg of an orange solid with a yield of 23.9%. The structure is as follows: 1 H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 7.98 (d, J = 9.4 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.59 (s, 1H), 7.41 (dd, J = 9.4, 2.7 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 7.02 (d, J = 8.9 Hz, 2H), 4.87–4.71 (m, 2H), 4.34–4.23 (m, 2H), 3.20 (s, 6H). 13 C NMR (151 MHz, d6-DMSO) δ 159.83, 152.14, 147.58, 144.23, 142.70, 136.12, 135.55, 130.23, 128.13, 127.31, 121.22, 118.08, 115.76, 112.21, 104.79, 82.60 (d, J = 166.5 Hz), 67.89 (d, J = 18.9 Hz), 40.64. HRMS: m / z calcd for C 21 H 20 ON4F 363.16157; found 363.16144, M+H + 。

[0108] Example 21: In vitro competitive binding assay

[0109] I. Experimental procedure

[0110] (1) Prepare a dimethyl sulfoxide (DMSO) stock solution of the test compound at a concentration of 10 -3M, and serially dilute with absolute ethanol to prepare solutions of the test compound with concentrations of 10 -4 M, 10 -5 M, 10 -5.5 M, 10 -6 M, 10 -6.5 M, 10 -7 M, 10 -8 M, 10 -9 M;

[0111] (2) Prepare a DMSO stock solution of ThT with a concentration of 10 -3 M, and dilute it with absolute ethanol to 1.11×10 -5 M. Then further dilute it with PBS buffer (1×) to a concentration of 1.11×10 -6 M for use;

[0112] (3) Prepare the solutions for the blank group and the protein group in a black 96-well plate as follows:

[0113] a. Blank group: Use a pipette to sequentially transfer 20 μL of the gradient concentration solutions of the test compound and 180 μL of the ThT solution for use;

[0114] b. Protein group: Use a pipette to sequentially transfer 20 μL of the gradient concentration solutions of the test compound and 180 μL of the protein diluent.

[0115] When measuring the activity against α-synuclein, the protein diluent is prepared by uniformly mixing one tube (50 μL) of α-synuclein aggregates (the preparation of α-synuclein is carried out according to the existing method) with 7680 μL of the ThT solution for use, with a final concentration of 6.47 μg / mL;

[0116] When measuring the activity against Aβ 1-42 protein, the protein diluent is prepared by uniformly mixing two tubes (80 μL) of Aβ 1-42 protein aggregates (the preparation of Aβ 1-42 protein aggregates is carried out according to the existing method) with 7680 μL of the ThT solution for use, with a final concentration of 2.06 μg / mL;

[0117] Prepare 3 parallel groups for each gradient concentration group for testing;

[0118] (4) Place the 96-well plate in a shaker, incubate at 37 °C with a rotation speed of 100 rpm for 30 min. After the incubation, use a Tecan Infinite M200 PRO microplate reader to detect the fluorescence intensity, and then use GraphPad Prism 8 to analyze and process the data to obtain the IC 50 value, and use the formula K i = IC50 / (1 + [ligand] / K d ) to calculate the competitive binding constant values of the compound to be tested for the two proteins.

[0119] Among them, K d is the saturation binding constant of ThT to the aggregates of the two proteins, both measured according to the previous experimental methods. For α-synuclein and Aβ 1-42 they are 253.87 nM and 117.14 nM respectively; [ligand] is the concentration of the competitive ligand used, which is the final concentration of ThT, 1000 nM, in this method.

[0120] II. Experimental Results

[0121] The experimental results are shown in Table 1. The results show that except for Compound 11, the remaining compounds all have medium to high activity against α-synuclein aggregates, while they have relatively stronger affinity for Aβ 1-42 protein aggregates.

[0122] Among them, Compounds 5, 6, 9, 14, 16, and 20 have relatively high affinity for α-synuclein aggregates (K i < 10 nM), but lack selectivity;

[0123] Compounds 3, 17, and 19 have certain selectivity for α-synuclein aggregates, but the affinity of all three for the two proteins is only at a medium level.

[0124] Table 1 Activity of Compounds against α-Synuclein and Aβ 1-42 Protein Aggregates

[0125] Compound <![CDATA[K i-α-syn (nM)]]> <![CDATA[K i-Aβ (nM)]]> 1 22.51±3.52 4.08±0.23 2 51.78±13.79 25.97±13.73 3 29.04±11.32 45.99±17.56 4 13.68±2.66 1.14±0.08 5 8.32±1.34 2.26±0.29 6 4.68±1.59 0.84±0.04 7 28.64±8.89 11.04±1.26 8 11.62±2.30 1.89±0.94 9 8.2±1.85 0.94±0.03 10 60.13±18.06 25.47±8.26 11 Not detected 120.36±21.36 12 48.40±15.30 11.34±1.05 13 89.27±15.20 4.84±0.38 14 2.69±0.62 0.44±0.02 15 10.77±2.97 1.15±0.06 16 4.82±1.07 2.01±0.12 17 17.86±4.27 78.77±6.22 18 39.76±3.79 15.45±1.25 19 26.8±4.47 34.91±6.32 20 6.32±1.05 1.99±0.27

[0126] Example 22: Determination of Optical Properties of Fluorescent Molecules

[0127] I. Experimental Procedures

[0128] (1) Ultraviolet Absorption Spectrum: Accurately prepare DMSO solutions of the compound to be tested with 5 gradient concentrations, with concentrations of 6 - 50 μM. Use a UV-3600 ultraviolet-visible spectrometer (Shimadzu Corporation, Japan) to measure the ultraviolet absorption spectrum of the compound, and record the maximum absorption wavelength (λ abs , nm) and the absorbance values of the gradient concentration solutions at this wavelength. Calculate the molar extinction coefficient (ε, M -1 ·cm -1 ) of the compound according to the Lambert-Beer law.

[0129] (2) Fluorescence Spectrum: Prepare a DMSO stock solution of the compound to be tested, with a concentration of 10 -3Compound M was diluted with dichloromethane (DCM), tetrahydrofuran (THF), anhydrous methanol (MeOH), and DMSO to a solution with a concentration of 10 μM. The fluorescence excitation and emission spectra of the compound were measured and recorded using an RF-5310PC spectrofluorometer (Shimadzu Corporation, Japan).

[0130] (3) Fluorescence quantum yield: Prepare a DMSO stock solution of the compound to be tested with a concentration of 10 -3 μM, and dilute it to 5 μM with DCM. Use a Quantaurus-QY absolute quantum yield spectrofluorometer C11347 (Hamamatsu Corporation, Japan) to measure the fluorescence quantum yield of the compound to be tested.

[0131] II. Experimental Results

[0132] The experimental results are shown in Table 2. With the changes in the electron donor and electron acceptor in the system, the fluorescence properties of the compound showed obvious differences. Due to the solvation effect, the maximum emission wavelength of the vast majority of the probes increased with the increase in solvent polarity, showing a high sensitivity to solvent polarity.

[0133] Compounds with a quinoxaline structure (including 8, 9, 13, 16, 18, and 20) all had relatively high quantum yields (10.4% - 74.9%), which was beneficial for the highly sensitive detection of amyloid proteins in in vitro staining. The maximum emission wavelength of Compound 9 in DMSO reached 716 nm, indicating the potential of Compound 9 for near-infrared fluorescence imaging in experimental animals.

[0134] Table 2 Optical Properties of Compounds

[0135]

[0136]

[0137]

[0138] Example 23: In Vitro Binding Assays of Fluorescent Molecules to α-Synuclein Aggregates and Aβ 1-42 Protein Aggregates

[0139] I. Experimental Procedures

[0140] (1) Prepare a DMSO stock solution of the compound to be tested with a concentration of 10 -3 μM, and dilute it with absolute ethanol to a solution with a concentration of 5 μM for use;

[0141] (2) Prepare a PBS buffer (1×) solution of bovine serum albumin (BSA) with a concentration of 10 mg / mL;

[0142] (3) Prepare four test solutions, and the preparation methods are as follows:

[0143] a. Sequentially add 30 μL of the ethanol solution of the test compound to be used, 2930 μL of PBS buffer (1×), and 1 tube of α-synuclein aggregates (40 μL) into a borosilicate glass tube, and mix evenly;

[0144] b. Sequentially add 30 μL of the ethanol solution of the test compound to be used, 2890 μL of PBS buffer (1×), and 2 tubes of Aβ 1-42 protein aggregates (80 μL) into a borosilicate glass tube, and mix evenly;

[0145] c. Sequentially add 30 μL of the ethanol solution of the test compound to be used and 2970 μL of PBS buffer (1×) into a borosilicate glass tube, and mix evenly;

[0146] d. Add 3 mL of PBS buffer (1×) into a borosilicate glass tube, and this group serves as a blank control;

[0147] (4) Incubate the four solutions in a shaker at 37 °C with a rotation speed of 120 rpm for 30 min. After shaking well, use an RF-5310PC spectrofluorometer (Shimadzu Corporation, Japan) to measure and record the fluorescence excitation and emission spectra of the four solutions, and calculate the fluorescence enhancement factor after the compound binds to the protein at the maximum emission wavelength of the compound, which is expressed as: Fluorescence enhancement factor = FI probe+protein / FI probe . Among them, FI probe+protein is the fluorescence intensity after the compound binds to the corresponding protein, and FI Probe is the fluorescence intensity of the PBS solution of the compound.

[0148] II. Experimental Results

[0149] The experimental results are shown in Table 3. It can be seen from the experimental results that except for 3, 12, 14, 15, and 16, the remaining test compounds all showed a relatively obvious increase in fluorescence intensity after binding to α-synuclein aggregates and Aβ 1-42 protein aggregates.

[0150] Among them, the fluorescence enhancement factors of 4, 6, and 8 for both protein aggregates are greater than 50 times, with excellent optical properties and suitable for fluorescence imaging.

[0151] Except for compound 8, the remaining compounds did not show obvious fluorescence enhancement after binding to BSA, indicating that the non-specific binding level of other compounds except 8 is low.

[0152] The maximum emission wavelength of the compound after binding to α-synuclein aggregates is longer than that after binding to Aβ 1-42The maximum emission wavelength after protein aggregates bind is red-shifted, and the red-shift value is usually between 10nm and 20nm, indicating that such compounds have the potential to distinguish between two proteins at the spectral level.

[0153] Table 3 Fluorescence properties of compounds after binding to proteins

[0154]

[0155]

[0156] Example 24: In vitro fluorescence staining experiment

[0157] 1. Experimental steps

[0158] (1) Human brain paraffin sections from AD patients (88 years old, female) and PD patients (73 years old, female) were dewaxed by immersion in xylene for 5 min, then rinsed in anhydrous ethanol and pure water for 1 min respectively, and set aside;

[0159] (2) preparing compounds 4, 6, 8, 9, 13, 17, 18, 19 and 20 in 20% ethanol-80% aqueous solution at a concentration of 5 μM;

[0160] (3) The dewaxed human brain slices were completely covered with the test compound solution, left to stand at room temperature for 5 min, then rinsed with anhydrous ethanol for 30 s, then rinsed with pure water for 30 s, and observed under a fluorescence microscope.

[0161] 2. Experimental Results

[0162] Staining images are shown in Figure 7 Under the conditions used in Example 24, all the selected compounds could clearly stain Aβ plaques in brain sections of AD patients; however, only 8, 9 and 20 could stain Lewy pathology in brain sections of PD patients.

[0163] In particular, the staining results of compound 9 for Lewy pathology and amyloid plaques showed obvious spectral separation characteristics: under the same conditions equipped with Cy5 filters, amyloid plaques were difficult to observe while Lewy pathology was clearly visible.

[0164] Example 25: ICR mouse biodistribution experiment

[0165] 1. Experimental steps

[0166] 200 μL of the solution of Compound 9 (0.3 mg / kg, 30% DMSO, 70% 1,2-propanediol) was injected into ICR mice (4 - 6 weeks old, male) via the tail vein. The organs were dissected at 2 min, 10 min, 30 min, and 60 min after administration, respectively. The fluorescence signals were collected using an IVIS Lumina III in vivo imaging system to obtain fluorescence imaging maps (excitation wavelength was set at 520 nm, and emission wavelength was set at 640 nm). The data were analyzed using Living Image 4.2.1 software.

[0167] II. Experimental Results

[0168] The experimental results are shown in Figure 8 . The imaging results showed that Compound 9 could cross the blood-brain barrier, and the amount entering the brain reached the maximum at 10 min, which was suitable for in vivo near-infrared fluorescence imaging. The clearance rate was general, and there was still some residue at 30 min and it was basically completely cleared at 60 min.

[0169] Among each time point, the uptake value of the compound in the kidneys was not high. At 60 min, the fluorescence signal in the intestine increased significantly, indicating that Compound 9 might be metabolized through the hepatobiliary pathway.

[0170] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A trisubstituted olefin compound, characterized in that, is one of the following structural formulas: 、 、 。 2. The preparation method of the trisubstituted olefin compound according to claim 1, characterized in that, The structural formula of the trisubstituted olefin compound is as follows: or ; The preparation method of the trisubstituted olefin compound is: and dissolved in anhydrous methanol, a methanol solution of saturated potassium carbonate was added, and a solid was precipitated after standing at room temperature; the corresponding compound shown above was obtained; Among them, W is a benzene-based six-membered aromatic ring, and the corresponding structure is , where X, Y, and Z each independently represent CH, and n is 0; or X represents N, Y represents N, Z represents CH, and n is 1; R1 is at the 6-position N, N -dimethylamino; The 7-position is hydrogen; The carbon-carbon double bonds are all trans double bonds; R2 is .

3. The preparation method of the trisubstituted olefin compound according to claim 1, characterized in that, The structural formula of the trisubstituted olefin compound is as follows: or ; The preparation method of the trisubstituted olefin compound is: With dissolved in anhydrous methanol, and then a methanol solution of sodium methoxide was added, and the mixture was refluxed overnight and allowed to stand at room temperature until a solid precipitated to obtain the corresponding shown compound; Among them, W is a benzo six-membered aromatic ring, and the corresponding structure is , where X represents N, Y represents CH, Z represents CH, or X represents N, Y represents N, Z represents CH; R1 is at the 6-position N, N -dimethylamino; The 7-position is hydrogen; n is 0, and the carbon-carbon double bonds are all trans double bonds; R2 is .

4. A developer, characterized in that, Its active ingredient is the trisubstituted olefin compound described in claim 1.

5. A fluorescent probe, characterized in that, Its active ingredient is the trisubstituted olefin compound described in claim 1.

Images