Benzothiazole-carbazole hetero-conjoined derivative as well as preparation method and application thereof

Abstract

The invention discloses a benzothiazole-carbazole hetero-conjoined compound as well as a preparation method and application thereof. The general formula of the compound is # imgabs0. The benzothiazole-carbazole hetero-conjoined compound provided by the invention has multi-target anti-AD (Alzheimer's Disease) effects of nerve protection, cholinesterase activity inhibition, amyloid protein deposition inhibition, copper ion chelation and the like.

Description

Technical Field

[0001] The invention relates to the technical field of benzothiazole-carbazole heteromer compounds, in particular to a benzothiazole-carbazole heteromer derivative and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a common type of dementia, characterized by progressive, irreversible memory loss, cognitive and language dysfunction. It is also one of the most common and serious chronic diseases among the elderly. Since the current drugs for the treatment of this disease can only delay the progression of the disease and cannot fundamentally improve the patient's symptoms, drug research on AD is imminent. However, the complex pathogenesis of AD is an important reason that hinders drug development. It is currently believed that the occurrence and development of AD is related to a variety of pathological mechanisms, including amyloid protein deposition, tau protein phosphorylation, oxidative stress, acetylcholine deficiency, metal ion disorders, etc. Research evidence shows that single-target drug treatment is difficult to prevent the disease or improve the deterioration of AD. Therefore, the development of multi-target drugs is considered to be an effective way to treat the disease.

[0003] Benzothiazole heterocycle is a multifunctional scaffold in medicinal chemistry with a wide range of biological activities. Studies have found that unsubstituted 2-benzothiazole compounds and their derivatives can effectively inhibit the aggregation of Aβ and reduce the content of soluble amyloid oligomers in vivo and in vitro. Chinese patent application number 201110434465.4 discloses a benzothiazole derivative and its use, which has an inhibitory and imaging effect on the aggregation and deposition of β-amyloid protein. When used as an imaging agent, it needs to be labeled with a suitable radioactive isotope or a contrast agent suitable for magnetic resonance detection. The compound can be used in the preparation of drugs for the prevention and treatment of Alzheimer's disease. However, the benzothiazole derivative only inhibits the aggregation of Aβ, and the pharmacological effect is relatively single. The present invention aims to provide a new compound with multiple pharmacological activities, such as anti-oxidative damage, copper ion chelation, inhibition of Aβ aggregation, inhibition of cholinesterase activity, etc., so as to be used for the treatment of Alzheimer's disease. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention designs benzothiazole-carbazole heteromers and their derivatives, which can be used to prepare drugs for treating Alzheimer's disease through multiple targets and synergistic effects. Another object of the present invention is to provide a preparation method for this series of derivatives.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows: a benzothiazole-carbazole heteroconjugate compound and its derivatives, the general formula of the compound is:

[0006]

[0007] Wherein X is a benzene ring or -NC-C6H6 or -N=C-C6H6; R1 is OH or -O-CH3;

[0008] R2 is

[0009] Preferably, the compound is selected from one or more of 4a, 4b, 4c, 5a, 5b, 5c, 6a, 6b, 6c, 7a, 7b, 7c, 8a and 9a;

[0010] 4a: 4b:

[0011] 4c: 5a:

[0012] 5b: 5c:

[0013] 6a: 6b:

[0014] 6c: 7a:

[0015] 7b: 7c:

[0016] 8a: 9a:

[0017] Preferably, one or more isotopically enriched atoms are present in the compound, specifically as follows: deuterium or tritium is used to replace hydrogen, or 13 C or 14 C-rich carbon atoms to replace carbon atoms, or with 15 N-enriched nitrogen replaces the nitrogen atom.

[0018] The second aspect of the present invention provides the use of the benzothiazole-carbazole heteromer compound and its derivatives in the preparation of anti-AD drugs.

[0019] Preferably, the compound has antioxidant damage, copper ion chelation, Aβ aggregation inhibition and

[0020] Inhibits cholinesterase activity.

[0021] In a third aspect of the present invention, a method for preparing the benzothiazole-carbazole heteroconjugate compound is further provided, wherein the preparation methods of compounds 4a, 4b and 4c are as follows:

[0022] Compound 2 2-amino-5-methoxybenzenethiol and compound 3a-c Dissolve in DMSO, heat to react, cool the mixture to room temperature after the reaction, pour 3-5 times (volume) of ice water as the reaction solvent, extract three times with dichloromethane, wash the dichloromethane solution with water and dry over anhydrous Na2SO4; filter, evaporate the solvent under vacuum; chromatograph the obtained crude product on a silica gel column with a mixture of n-hexane / CH2Cl2 at a ratio of 2.5-5:1 v / v as the eluent to obtain 4a, 4b and 4c, respectively;

[0023] The molar ratio of compound 2 to compound 3a-c is 2-5:1; the ratio of compound 3a-c to DMSO is 1.5-2.0 mmol:15-25 mL; the reaction temperature is 160-180° C., and the reaction time is 10-15 h;

[0024] The preparation methods of compounds 5a, 5b and 5c are as follows:

[0025] Slowly add 0.3-0.8 mmol of compound 4a-c in argon at -80℃~-75℃ 25-35 ml of CH2Cl2 solution and 3-5 ml of anhydrous CH2Cl2 solution containing 3-5 mmol BBr3; the mixture was gradually heated to room temperature and stirred for 18-25 h; after neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2; the obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under vacuum, and the crude product was chromatographed on a silica gel column with CH2Cl2 as the eluent to obtain white powders 5a, 5b and 5c;

[0026] In compounds 3a-c and 4a-c, R is one of the structures:

[0027] Preferably, the preparation methods of compounds 6a, 6b and 6c are as follows:

[0028] Compound 1 6-methoxybenzothiazole-2-amine, compound 3a-c A mixture of the product and glacial acetic acid was refluxed in ethanol for 5-8 h, and then purified by recrystallization from ethoxyethanol and dichloromethane to obtain compounds 6a, 6b and 6c;

[0029] The molar ratio of compound 1 to compound 3a-c is 1.5-2.5:1; the ratio of compound 3a-c, glacial acetic acid and ethanol is 1.5-2.0 mmol: 8-15 mL: 15-25 mL;

[0030] The preparation methods of compounds 7a, 7b and 7c are as follows:

[0031] Slowly add compound 6a-c in argon at 75℃~85℃ THF solution and LiAlH4, stirred for 15-25h, extracted with CH2Cl2 three times, the obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under vacuum. The crude product was purified by recrystallization from ether and dichloromethane to obtain white powders 7a, 7b and 7c;

[0032] The ratio of the compound 6a-c, LiAlH4 and THF is 0.8-1.5mmol:3-4mmol:15-25mL.

[0033] Preferably, the preparation method of compound 8a is as follows: 25-35 ml of a CH2Cl2 solution containing 0.8-1.2 mmol 7a and 3-5 ml of anhydrous CH2Cl2 solution containing 3-5 mmol BBr3 are slowly added in argon at -80°C to -75°C, the mixture is gradually heated to room temperature, stirred for 15-25 h, neutralized with a saturated NaHCO3 solution, extracted three times with CH2Cl2, the obtained dichloromethane solution is washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated under vacuum. The crude product is eluted with CH2Cl2 and chromatographed on a silica gel column to obtain compound 8a.

[0034] Preferably, the preparation method of compound 9a is as follows: 25ml-35ml of a CH2Cl2 solution containing 0.4-0.7mmol 6a and 3-5ml of anhydrous CH2Cl2 solution containing 3-5mmol BBr3 are slowly added in argon at -80°C to -75°C, the mixture is gradually heated to room temperature, stirred for 15-25h, neutralized with a saturated NaHCO3 solution, extracted three times with CH2Cl2, the obtained dichloromethane solution is washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated under vacuum. The crude product is eluted with CH2Cl2 and chromatographed on a silica gel column to obtain compound 9a.

[0035] The compounds provided by the present invention have novel structures and have antioxidant damage protection, anti-amyloid protein aggregation (inhibition of Aβ aggregation) and inhibition of cholinesterase activity. In addition, compounds 5a and 8a also have copper ion chelation, which is also beneficial for the treatment of AD. Excessive copper ions are considered to be one of the causes of AD. Therefore, the compounds provided by the present application can be used to prepare drugs for the treatment of Alzheimer's disease through multiple targets and synergistic effects.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention connects benzothiazole and carbazole through different group chains, the preparation method is simple to operate, and the yield is high; the prepared benzothiazole-carbazole heteromer compound is a small molecule drug that exerts multi-target anti-AD effects, including antioxidant damage, copper ion chelation, inhibition of Aβ aggregation, inhibition of cholinesterase activity, etc., and can be used to prepare anti-AD drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A synthetic route for a benzothiazole-carbazole heteromer derivative proposed in the present invention;

[0038] Figure 2 This is a bar graph showing the cytotoxic effect of the present invention on HT22 cells;

[0039] Figure 3 This is a bar graph showing the protective effect of the present invention against L-glutamate-induced HT22 cell toxicity;

[0040] Figure 4 It is the UV-visible absorption spectrum diagram of the interaction between compounds 5a and 8a of the present invention and copper ions. DETAILED DESCRIPTION

[0041] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. The reagents 1-bromo-1,2,2-triphenylethylene, 4-formylphenylboric acid, tetrabutylammonium bromide (TBAB), tetrakis(triphenylphosphine)palladium [Pd(PPh3)4], potassium hydroxide (KOH) and boron tribromide (BBr3) used in the following examples were purchased from Alfa Aesar Reagent Company. Super water (18MΩcm) was used for the experiment. All other reagents and solvents were purchased from Guangzhou Dongzheng Company (China) of analytical grade without further purification.

[0042] The present invention provides a benzothiazole-carbazole heteromer derivative, and the compound of general formula I is prepared as follows:

[0043]

[0044] Wherein X is a benzene ring or -NC-C6H6 or -N=C-C6H6; R1 is OH or -O-CH3;

[0045] R2 is

[0046] Its synthetic route is Figure 1 The preparation process of each compound is described in the following examples.

[0047] The compounds of formula (I) described in the present invention also include pharmaceutically acceptable derivatives thereof, such as any pharmaceutically acceptable salts, prodrugs or salts of prodrugs of the compounds of the present invention, or any other compounds that can provide the compounds of the present invention after administration to a patient.

[0048] The pharmaceutically acceptable salt of the compound of formula (I) of the present invention refers to the salt derived from inorganic or organic acid and alkali. Such acid addition salts include the following salts: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, cyclopentane propionate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, hemisulfate, caproate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, mesylate, nicotinate, oxalate, persulfate, picrate, pivalate, propionate, succinate, tartrate, toluenesulfonate and undecanoate. Base addition salts include ammonium salts, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts formed with organic bases such as dicyclohexylamine salts, and salts formed with amino acids such as arginine, lysine, etc.

[0049] The compounds of formula (I) of the present invention can also be modified by adding suitable functional groups to improve the selective biological characteristics. Such modifications are known in the art and include modifications for improving the biopenetration into a given biological system (e.g., blood, lymphatic system, central nervous system), improving oral availability, improving solubility to allow injection, changing metabolism and changing excretion rate.

[0050] In one embodiment, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0051] Pharmaceutically acceptable carriers that can be used in these pharmaceutical compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

[0052] The pharmaceutical composition can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the composition can be administered orally, intraperitoneally or intravenously.

[0053] The amount of the compound that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will depend on the patient being treated and the particular mode of administration. Preferably, the composition should be formulated so that a dosage of 0.01-100 mg / kg body weight / day of the compound can be administered.

[0054] Example 1

[0055] This embodiment provides Figure 1 The preparation method of 4a in the synthetic route, the structural formula of 4a is as follows:

[0056]

[0057] The preparation method of compound 4a is:

[0058] 2-Amino-5-methoxythiophenol (2) (5.55mmol, 0.86g) and compound 3a (1.85mmol, 0.5g) were dissolved in 20ml DMSO and heated at 170℃ for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, poured into ice water 3-5 times (volume) as the reaction solvent, and extracted with dichloromethane three times. The dichloromethane solution was washed with water and dried over anhydrous Na2SO4. Filtered and the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with a mixture of n-hexane / CH2Cl2 (3:1, v / v) as the eluent to obtain a yellow-brown powder (0.344g, yield 40%). 1 H NMR (300MHz, CDCl3) δ (ppm): 7.71 (d, J = 7.2, 1.4Hz, 2H), 6.82 (d, 2H), 6.64 (dd, J = 8.9Hz, 2H), 7.71 (d, J = 7.2, 1.4Hz, 2H), 5.80 (m, 4H) ),7.53(dd,J=7.5,1.5Hz,2H),7.14(d,J=8.9,2.5,1.0Hz,1H),3.80(d,J=1.5Hz,3H),3.81(dd,J=7.0Hz,2H),2.33(dd,J=7.0,2H); 13C NMR (75MHz, CDCl3) δppm: 169.0, 156.7, 149.6, 146.7, 136.8, 136.1, 134.1, 129.7, 122. 6,122.5,122.4,120.5,114.6,112.7,104.9,78.9,55.8,34.3; EI-MS, m / z: 410.15([M] + ,calcd forC 26 H 22 N2OS,410.15).

[0059] Example 2

[0060] This embodiment provides Figure 1 The preparation method of 4b in the synthetic route, the structural formula of 4b is as follows:

[0061]

[0062] The preparation method of compound 4b is:

[0063] 2-Amino-5-methoxythiophenol (2) (5.55mmol, 0.86g) and compound 3b (1.85mmol, 0.5g) were dissolved in 20ml DMSO and heated at 170℃ for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, poured into ice water 3-5 times (volume) as the reaction solvent, and extracted with dichloromethane three times. The dichloromethane solution was washed with water and dried over anhydrous Na2SO4. Filtered and the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with a mixture of n-hexane / CH2Cl2 (3:1, v / v) as the eluent to obtain a yellow-brown powder (0.774g, yield 90%). 1 H NMR (300MHz, CDCl3) δ (ppm): 7.85 (dd, J=8.8, 1.5Hz, 2H), 7.27 (m, J=1.5, 7.8Hz, 2H) ,7.53(dd,J=8.8,2.6Hz,2H),7.35(d,J=7.8Hz,1H),7.24–7.0(m,10H),3.81(s,3H). 13 C NMR (75MHz, CDCl3) δppm: 169.0, 156.7, 146.7, 145.9, 129.7, 129.6, 126.8, 125.7, 123.2, 122.6, 114.6, 55.8; EI-MS, m / z: 408 (calcd. for C 26 H 20 N2OS,408).

[0064] Example 3

[0065] This embodiment provides Figure 1 The preparation method of 4c in the synthetic route, the structural formula of 4c is as follows:

[0066]

[0067] The preparation method of compound 4c is as follows:

[0068] 2-Amino-5-methoxythiophenol (2) (5.55mmol, 0.86g) and compound 3c (1.85mmol, 0.67g) were dissolved in 20ml DMSO and heated at 170℃ for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, poured into ice water 3-5 times (volume) as the reaction solvent, and extracted with dichloromethane three times. The dichloromethane solution was washed with water and dried over anhydrous Na2SO4. Filtered and the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with a mixture of n-hexane / CH2Cl2 (3:1, v / v) as the eluent to obtain a yellow-brown powder (0.4472g, yield 52%). 1 H NMR (300MHz, CDCl3) δppm: 3.89 (s, J = 0.40Hz, 3H), 6.96-7.20 (m, 18H), 7.32 (dd, J = 0.40Hz, 2.47Hz, 1H), 7.78 (dd, J = 0.40Hz, 8.24Hz, 2H), 7.91 (dd, J = 0.40 8.92Hz,1H); 13 C NMR (75MHz, CDCl3) δppm: 165.54, 157.85, 148.75, 146.45, 143.41, 141.93, 139.97, 136.30, 132. 13,131.48,127.99,126.99,126.80,123.75,115.81,104.39,56.12; MS (EI), m / z: 495 (calcd.for C 34 H 25 NOS495).

[0069] Example 4

[0070] This embodiment provides Figure 1 The preparation method of 5a in the synthetic route, the structural formula of 5a is as follows:

[0071]

[0072] The preparation method of compound 5a is:

[0073] A solution of 4a (0.5 mmol, 0.2 g) in CH2Cl2 (30 ml) and an anhydrous CH2Cl2 (4 ml) solution containing BBr3 (4 mmol, 1.00 g) were slowly added in -78°C argon. The mixture was gradually heated to room temperature and stirred for 20 h. After neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2. The obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with CH2Cl2 as eluent to obtain a white powder (0.1734 g, yield 86.7%). 1H NMR (300MHz, CDCl3) δ (ppm): 7.71 (d, J = 1.5 7.6Hz, 2H), 7.29-5.80 (m, 8H), 7.36 (ds, J = 7.5Hz, 1H), 7.10 (s, J = 1.5Hz, 1H), 6.82 (d, J = 1.57.5Hz 2H), 6.71 (d, J=1.5 7.5Hz, 1H), 6.0 (s, 1H), 3.81 (m, J=7.0Hz, 2H), 2.33 (m, J=7.0Hz, 2H). 13 C NMR (75MHz, CDCl3) δppm: 169.0,155.1,149.6,147.0,137.2,139.6,134.1,129.7,123 .0,122.5,122.4,120.5,114.9,112.7,106.5,78.9,34.3; EI-MS, m / z: 396([M]+,calcd forC 25 H 20 N2OS,396).

[0074] Example 5

[0075] This embodiment provides Figure 1 The preparation method of 5b in the synthetic route, the structural formula of 5b is as follows:

[0076]

[0077] The preparation method of compound 5b is:

[0078] A solution of 4b (0.5 mmol, 0.2 g) in CH2Cl2 (30 ml) and a solution of BBr3 (4 mmol, 1.00 g) in anhydrous CH2Cl2 (4 ml) were slowly added under argon at -78°C. The mixture was gradually heated to room temperature and stirred for 20 h. After neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2. The obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with CH2Cl2 as the eluent to obtain a white powder (0.1702 g, yield 85.1%). 1H NMR (300MHz, CDCl3) δ (ppm): 7.85 (dd, J = 7.2 1.4Hz, 2H), 7.54 -8.18(m,10H),7.27(m,2H),7.36(d,J=8.9Hz,1H),7.1(dd,J=7.2,2.4Hz,1H),6.71(dd,J=8.92.4Hz,1H),6.0(d,1H). 13 C NMR (75MHz, CDCl3) δppm: 169.0,155.1,147.0,145.9,137.2,129.7,129.6,126.8,125 .7,125.4,123.2,123.0,114.9,106.5.EI-MS, m / z: 394 (calcd.forC25H18N2OS,394).

[0079] Example 6

[0080] This embodiment provides Figure 1 The preparation method of 5c in the synthetic route, the structural formula of 5c is as follows:

[0081]

[0082] Preparation method of compound 5c:

[0083] A solution of 4c (0.5 mmol, 0.25 g) in CH2Cl2 (30 ml) and an anhydrous CH2Cl2 (4 ml) solution containing BBr3 (4 mmol, 1.00 g) were slowly added to -78°C argon. The mixture was gradually heated to room temperature and stirred for 20 h. After neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2. The obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with CH2Cl2 as eluent to obtain a white powder (0.184 g, yield 92%). 1H NMR (400MHz, DMSO-d6) δppm: 6.96-7.08(m,7H),6.96-7.08(m,7H),7.41(s,1H) ,7.78(d,2H,J=0.40Hz,7.89Hz),7.82(d,1H,J=0.40Hz,8.81Hz),9.92(s,1H); 13 C NMR (100MHz, DMSO-d6) δppm: 163.45, 156.26, 147.60, 146.16, 143.33, 142.03, 140.19, 136.38, 132 .02,131.64,131.16,128.46,127.23,126.67,123.89,116.62,107.23; MS (EI), m / z: 481 (calcd.for C 33 H 23 NOS 481).

[0084] Example 7

[0085] This embodiment provides Figure 1 The preparation method of 6a in the synthetic route, the structural formula of 6a is as follows:

[0086]

[0087] Preparation method of compound 6a:

[0088] A mixture of 6-methoxybenzothiazole-2-amine (1) (3.7 mmol, 0.67 g), compound 3a (1.85 mmol, 0.5 g) and glacial acetic acid (10 mL) was refluxed in ethanol (20 mL) for 6 h. After removing the solvent, the mixture was recrystallized and purified using ethoxyethanol and dichloromethane (volume ratio 1:1) to obtain a light yellow solid (0.576 g, yield 86%). 1 H NMR (300MHz, CDCl3) δ (ppm): 8.39 (s, J = 7.7, 1H), 7.53 (d, J = 8.6Hz, 2H), 7.29 (dd, J = 8.8 Hz,2H),7.5(dd,J=8.8Hz,2H),7.0(dd,J=8.8Hz,1H),6.78-6.64(m,4H),5.80(td,J=8.6 2.5Hz,4H),3.81(m,2H),3.83(s,3H),2.33(m,2H). 13C NMR (75MHz, CDCl3) δppm: 174.6,160.0,156.7,151.9,141.0,136.1,134.1,128.3,125.9 ,122.6,122.5,122.4,114.6,111.9,104.9,78.9,55.8,34.2; EI-MS, m / z: 437 (calcd.for C27H23N3OS,437).

[0089] Example 8

[0090] This embodiment provides Figure 1 The preparation method of 6b in the synthetic route, the structural formula of 6b is as follows:

[0091]

[0092] Preparation method of compound 6b:

[0093] A mixture of 6-methoxybenzothiazole-2-amine (1) (3.7 mmol, 0.67 g), compound 3b (1.85 mmol, 0.5 g) and glacial acetic acid (10 mL) was refluxed in ethanol (20 mL) for 6 h. After removing the solvent, the product was recrystallized and purified using ethoxyethanol and dichloromethane (volume ratio 1:1) to obtain a light yellow solid (0.609 g, yield 91%). 1 H NMR (300MHz, CDCl3) δ (ppm): 7.45 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 1.6 Hz, 1H), 7.23 (m, J = 5. 1, 2.1Hz, 4H), 7.15–6.97 (m, 9H), 6.90 (dd, J = 8.8, 2.6Hz, 1H), 4.56 (s, 2H), 3.83 (s, 3H). 13 C NMR (75MHz, CDCl3) δppm: 174.6,160.0,156.7,148.2,145.9,141.0 136.1,130.8,129.6,128.3,126.8,125.7,125.1,122.6,114.6,104.9,55.8; EI-MS, m / z: 435 (calcd.for C 27 H 21 N3OS,435).

[0094] Example 9

[0095] This embodiment provides Figure 1 The preparation method of 6c in the synthetic route, the structural formula of 6c is as follows:

[0096]

[0097] Preparation method of compound 6c:

[0098] A mixture of 6-methoxybenzothiazole-2-amine (1) (3.7 mmol, 0.67 g), compound 3c (1.85 mmol, 0.67 g) and glacial acetic acid (10 mL) was refluxed in ethanol (20 mL) for 6 h. After removing the solvent, the mixture was recrystallized and purified using ethoxyethanol and dichloromethane (volume ratio 1:1) to obtain a light yellow solid (0.629 g, yield 94%). 1 H NMR (300MHz, CDCl3) δppm: 3.83 (s, 3H, J = 0.40Hz), 7.53-7.0 (m, 18H), 8.39 (s, 1H), 7.75 (d, J = 0.40Hz, 8.24Hz, 2H), 7.31 (m, J = 0.40Hz, 8.92Hz, 2H); 13 C NMR (75MHz, CDCl3) δppm: 174.6, 160.0, 156.7, 142.3, 141.0, 136.1, 135.6, 129. 4,129.1,128.6,127.9,122.6,114.6,104.9,55.8; MS (EI), m / z: 522 (calcd.for C 35 H 26 NOS 522).

[0099] Example 10

[0100] This embodiment provides Figure 1 The preparation method of 7a in the synthetic route, the structural formula of 7a is as follows:

[0101]

[0102] Preparation method of compound 7a:

[0103] Slowly add THF (20 mL) solution containing 6a (1.1 mmol, 0.5 g) and LiAlH4 (3.4 mmol, 0.13 g) in argon at 80 ° C, stir for 20 h, extract with CH2Cl2 three times, wash the obtained dichloromethane solution with water, and dry over anhydrous sodium sulfate. After filtration, evaporate the solvent under vacuum. The crude product is purified by recrystallization from ether and dichloromethane (volume ratio 1:1) to obtain a white powder (0.47 g, yield 94%). 1H NMR (300MHz, CDCl3) δ (ppm): 8.39 (s, J = 7.7, 1H), 7.53 (d, J = 8.6Hz, 2H), 7.29 (dd, J = 8.8 Hz,2H),7.5(dd,J=8.8Hz,2H),7.0(dd,J=8.8Hz,1H),6.78-6.64(m,4H),5.80(td,J=8.6 2.5Hz,4H),4.49(s,2H),3.96(m,1H),3.83(s,3H),2.33(m,2H). 13 C NMR (75MHz, CDCl3) δppm: 174.4,156.7,147.6,144.5,136.1,134.1,131.8,129.4,127.8,12 2.5,122.4,118.2,112.9,114.7,104.9,79.9,55.8,49.1,34.4; EI-MS, m / z: 439 (calcd.for C 27 H 25 N3OS,439).

[0104] Embodiment 11

[0105] This embodiment provides Figure 1 The preparation method of 7b in the synthetic route, the structural formula of 7b is as follows

[0106]

[0107] Preparation method of compound 7b:

[0108] Slowly add THF (20 mL) solution containing 6b (1.1 mmol, 0.5 g) and LiAlH4 (3.4 mmol, 0.13 g) in argon at 80 ° C, stir for 20 h, extract with CH2Cl2 three times, wash the obtained dichloromethane solution with water, and dry over anhydrous sodium sulfate. After filtration, evaporate the solvent under vacuum. The crude product is purified by recrystallization from ether and dichloromethane (volume ratio 1:1) to obtain a white powder (0.455 g, yield 91%). 1 H NMR (300MHz, CDCl3) δ (ppm): 7.42 (d, J = 8.8 Hz, 1H), 7.24–7.21 (m, 4H), 7.13–6.98 (m, 10H), 6.90 (m, 2H), 4.54 (s, 2H), 3.96 (s, 1H), 3.82 (s, 3H). 13C NMR (75MHz, CDCl3) δppm: 174.5,156.7,145.9,145.5,143.9,134.3,131.9,129.6, 127.8,126.6,125.9,118.2,114.6,104.7,55.9,49.1; EI-MS, m / z: 437 (calcd.forC 27 H 23 N3OS,437).

[0109] Example 12

[0110] This embodiment provides Figure 1 The preparation method of 7c in the synthetic route, the structural formula of 7c is as follows:

[0111]

[0112] Preparation method of compound 7c:

[0113] Slowly add THF (20 mL) solution containing 6c (1.1 mmol, 0.57 g) and LiAlH4 (3.4 mmol, 0.13 g) in argon at 80 ° C, stir for 20 h, extract with CH2Cl2 three times, wash the obtained dichloromethane solution with water, and dry over anhydrous sodium sulfate. After filtration, evaporate the solvent under vacuum. The crude product is purified by recrystallization from ether and dichloromethane (volume ratio 1:1) to obtain a white powder (0.475 g, yield 95%). 1 H NMR (300MHz, CDCl3) δ (ppm): 7.20-7.53 (m, 21H), 6.93 (dd, J = 8.8, 1.3Hz, 1H), 4.60 (d, J = 27.6Hz, 2H), 3.97 (s, 1H), 3.81 (d, J = 0.8Hz, 3H). 13 C NMR (75MHz, CDCl3) δppm: 174.5,156.7,145.6,141.3,140.0,139.1,138.0,134.4,131.6, 128.4,128.3,128.0,127.6,118.2,124.7,104.9,55.7,49.3; EI-MS, m / z: 524 (calcd.for C 35 H 28 N2OS,524).

[0114] Example 13

[0115] This embodiment provides Figure 1 The preparation method of 8a in the synthetic route, the structural formula of 8a is as follows:

[0116]

[0117] Preparation method of compound 8a:

[0118] A solution of 7a (1 mmol, 0.44 g) in CH2Cl2 (30 ml) and a solution of BBr3 (4 mmol, 1.00 g) in anhydrous CH2Cl2 (4 ml) were slowly added under argon at -78°C. The mixture was gradually heated to room temperature and stirred for 20 h. After neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2. The obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with CH2Cl2 as the eluent to obtain a white powder (0.19 g, yield 43%). 1 H NMR (300MHz, CDCl3) δ (ppm): 7.25 (m, 3H), 7.1 (m, 3H), 6.71-6.64 (m, 5H), 6.0 (s, 1H), 5.80 (m, J = 8.6 2.5Hz,4H),4.50(s,2H),3.96(s,1H),3.81(m,2H),2.33(m,2H). 13 CNMR (75MHz, CDCl3) δppm: 174.6,155.7,147.6,145.5,136.3,134.1,132.8,129.4,127.6 ,122.5,122.4,117.9,114.9,112.8,106.6,78.9,49.3,34.1; EI-MS, m / z: 425(calcd.forC 26 H 23 N3OS,425).

[0119] Embodiment 14

[0120] This embodiment provides Figure 1 The preparation method of 9a in the synthetic route, the structural formula of 9a is as follows:

[0121]

[0122] Preparation method of compound 9a:

[0123] A solution of 6a (0.5 mmol, 0.22 g) in CH2Cl2 (30 ml) and an anhydrous CH2Cl2 (4 ml) solution containing BBr3 (4 mmol, 1.00 g) were slowly added to -78°C argon. The mixture was gradually heated to room temperature and stirred for 20 h. After neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2. The obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under vacuum. The crude product was chromatographed on a silica gel column with CH2Cl2 as eluent to obtain a white powder (0.11 g, yield 52%). 1 H NMR (300MHz, CDCl3) δ (ppm): 8.39 (s, J = 7.7, 1H), 7.36-7.29 (m, 3H), 7.50 (dd, J = 8.8Hz, 2H), 7.10 (s ,J=8.8Hz,1H),6.78-6.64(m,5H),6.0(s,1H),5.80(td,J=8.62.5Hz,4H),3.82(m,2H),2.33(m,2H). 13 C NMR (75MHz, CDCl3) δppm: 174.6,160.0,155.9,155.2,141.3,136.4,134.1,128.3,125 .6,123.0,122.5,122.4,114.9,111.9,106.4,78.9,34.1; EI-MS, m / z: 423 (calcd.for C 26 H 21 N3OS,423).

[0124] Example 15 Toxic effects of the compounds of the present invention on HT22 cells

[0125] Mouse hippocampal neuron cell line HT22 was routinely cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C in a carbon dioxide incubator containing 5% CO2 and 95% air. Cells in the logarithmic growth phase were digested with 0.25% trypsin, resuspended in complete medium, counted under a microscope, and the cell concentration was adjusted to 10×10 4 / ml, inoculate 96-well cell culture plates, 100μL / well, culture overnight to allow cells to adhere. Aspirate the culture medium in the 96-well plate, dissolve the test compound with DMSO, dilute with complete culture medium, and add to the 96-well plate, 100μL / well. After pre-incubation for 24h, add 10μL of 5mg / mL MTT to each well, incubate for 2h, discard the supernatant, add DMSO 100μL / well, shake to fully dissolve the product formazan, and measure the absorbance of each well on an enzyme reader, with a measurement wavelength of 570nm. The formula compound promotes cell survival rate (%) = 100% * (A test compound-A model group) / (A model group-A blank) is used to calculate the cell survival rate. From the results of the MTT experiment, it can be found that except for 9a, the other compounds did not show obvious cytotoxicity at a concentration of 30μM. The results are shown in Figure 2 .

[0126] Example 16 The compounds of the present invention inhibit L-glutamate-induced cytotoxicity

[0127] Mouse hippocampal neuron cell line HT22 was routinely cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C, saturated humidity, and a carbon dioxide incubator containing 5% CO2 and 95% air by volume. Cells in the logarithmic growth phase were digested with 0.25% trypsin, resuspended in complete medium, counted under a microscope with a cell counting plate and adjusted to a cell concentration of 10×104 / ml, inoculated into a 96-well cell culture plate, 100 μL / well, and cultured overnight to allow cells to adhere to the wall. The medium in the 96-well plate was aspirated, the test compound was dissolved with DMSO, diluted with complete medium, and added to the 96-well plate, 100 μL / well. After pre-incubation for 30 minutes, 2 μL 100mM L-glutamate was added. The model group did not add the test compound, and 2 μL 100mM L-glutamate was directly added. After incubation for 24 hours, add 10 μL 5mg / mL MTT to each well, incubate for 2 hours, discard the supernatant, add 100 μL / well DMSO, shake to fully dissolve the resulting formazan, and measure the absorbance of each well on an ELISA reader at a wavelength of 570 nm. The cell survival rate is calculated using the formula: Compound-promoted cell survival rate (%) = 100% * (A test compound-A model group) / (A model group-A blank). From the MTT results, we can find that compounds 4a, 5a, and 8a have significant anti-glutamate-induced cell damage effects at a concentration of 10 μM. Results are shown in Figure 3 .

[0128] Example 17 - Inhibitory effects of the compounds of the present invention on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities

[0129] The colorimetric method reported by Ellman (Ellman, GL; et al. Biochem. Pharmacol. 1961.) was used to evaluate the AChE / BuChE inhibitory activity at 37°C. The test solution consisted of the following: 0.1M phosphate buffer pH 8.0, 0.5mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent), 0.03 units of AChE / BuChE (Sigma, derived from electric eel), and 0.5mM acetylthiocholine iodide (or butyrylthiocholine iodide) as the substrate for the enzymatic reaction. The compound to be tested was added to the assay solution and pre-incubated with the enzyme at 37°C for 20 minutes before adding the substrate. The absorbance change at 412nm within 5 minutes was measured with a spectrophotometer, the reaction rates were compared, and the percentage of reaction rate inhibition due to the presence of the test compound was calculated. The reaction rate was calculated from at least triplicate measurements, and the percent inhibition due to the presence of the test compound relative to the control without the compound was calculated. The concentration of compound that reduced the rate of the AChE enzymatic reaction by 50% (IC50) was determined. The IC 50 It is defined as the concentration of the compound at which the enzyme activity is reduced by 50% after the addition of the compound relative to the absence of the inhibitor. From the experimental results, it can be seen that except for 5a and 6c, the other compounds have good anticholinesterase activity, and the activity of inhibiting butyrylcholinesterase activity is stronger. The results are shown in Table 1.

[0130] Table 1. Compounds inhibiting acetylcholinesterase and butyrylcholinesterase activities

[0131]

[0132] Example 18 - Inhibition of Aβ self-induced aggregation by compounds of the present invention

[0133] Aβ was monomerized and freeze-dried using hexafluoroisopropanol (HFIP). (1-42) The test compound was dissolved in DMSO and diluted with 0.215M PBS (pH8.0). The test solution consisted of the following: 10μL Aβ(1-42) solution and 10μL test compound (final concentration was 10μM) or 10μL 0.215M PBS (pH 8.0). After incubation at 37°C for 24h, 180μL 1.5μM Thioflavin T solution was added, mixed, and the fluorescence intensity was scanned for 300 seconds ((λexc=446nm; λem=490nm). The inhibition rate of the test compound on Aβ self-induced aggregation was calculated using the formula inhibition rate=100-(IFi / IF0*100]. Among them, IF0 and IFi are the measured values ​​of Aβ group and Aβ+test compound group, respectively. From the experimental results, it can be found that the compounds have different degrees of inhibition of Aβ. (1-42)Aggregation, among which compounds 5a and 8a had stronger inhibitory activity. The results are shown in Table 2.

[0134] Table 2. Th-T fluorescence assay to determine whether compounds inhibit the formation of amyloid fibrils Aβ

[0135]

[0136] Example 19 - Interaction of compounds 5a and 8a of the present invention with copper ions

[0137] The interaction between compounds 5a and 8a and copper ions was studied by UV-visible spectrophotometry. Experimental procedure: The mother solution of the compound to be tested was diluted with methanol and placed in a 1 cm cuvette for UV-visible absorption spectrum measurement. Then, copper ions and compounds were added to the cuvette solution in a ratio of 1:1, and the UV-visible absorption spectrum of copper ions / compounds was measured after appropriate stirring. The results are shown in Figure 4 .

[0138] Figure 4 Figure A (compound 5a), Figure 4 In Figure B (compound 8a), after adding copper ions, the ultraviolet absorption curve shifted. The shift indicates that the compound can chelate with copper ions, indicating that compounds 5a and 8a have a certain copper ion chelating effect.

[0139] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A benzothiazole-carbazole heteroconjugate compound and its derivatives, characterized in that: The general formula of the compound is: Wherein X is a benzene ring or -NC-C6H6 or -N=C-C6H6; R1 is OH or -O-CH3; 2. The benzothiazole-carbazole heteromeric compound and its derivative according to claim 1, characterized in that: The compound is selected from one or more of 4a, 4b, 4c, 5a, 5b, 5c, 6a, 6b, 6c, 7a, 7b, 7c, 8a and 9a; 3. The benzothiazole-carbazole heteromeric compound and its derivative according to claim 1, characterized in that: The compound contains one or more isotopically enriched atoms, specifically as follows: hydrogen is replaced by deuterium or tritium, or 13 C or 14 C-rich carbon atoms to replace carbon atoms, or with 15 N-enriched nitrogen replaces the nitrogen atom.

4. Use of the benzothiazole-carbazole heteromer compound and its derivatives according to any one of claims 1 to 3 in the preparation of anti-AD drugs.

5. The use of the benzothiazole-carbazole heteroconjugate compound and its derivatives in the preparation of anti-AD drugs according to claim 4, characterized in that: The compound has the effects of anti-oxidative damage, copper ion chelation, Aβ aggregation inhibition and cholinesterase activity inhibition.

6. The method for preparing the benzothiazole-carbazole heteromeric compound and its derivatives according to claim 2, characterized in that: The preparation methods of compounds 4a, 4b and 4c are as follows: Compound 2 2-amino-5-methoxybenzenethiol and compound 3a-c Dissolve in DMSO, heat to react, cool the mixture to room temperature after the reaction, pour into ice water 3-5 times the amount of the reaction solvent, extract with dichloromethane three times, wash the dichloromethane solution with water and dry over anhydrous Na2SO4; filter, evaporate the solvent under vacuum; chromatograph the obtained crude product on a silica gel column using a mixture of n-hexane / CH2Cl2 at 2.5-5:1 v / v as the eluent to obtain compounds 4a, 4b and 4c, respectively; The molar ratio of compound 2 to compound 3a-c is 2-5:1; the ratio of compound 3a-c to DMSO is 1.5-2.0 mmol:15-25 mL; the reaction temperature is 160-180° C., and the reaction time is 10-15 h; The preparation methods of compounds 5a, 5b and 5c are as follows: Slowly add 0.3-0.8 mmol of compound 4a-c in argon at -80℃~-75℃ 25-35 ml of CH2Cl2 solution and 3-5 ml of anhydrous CH2Cl2 solution containing 3-5 mmol BBr3; the mixture was gradually heated to room temperature and stirred for 18-25 h; after neutralization with saturated NaHCO3 solution, it was extracted three times with CH2Cl2; the obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under vacuum, and the crude product was chromatographed on a silica gel column with CH2Cl2 as the eluent to obtain white powders 5a, 5b and 5c; In compounds 3a-c and 4a-c, R is one of the structures: The preparation methods of compounds 6a, 6b and 6c are as follows: Compound 1 6-methoxybenzothiazole-2-amine, compound 3a-c A mixture of the product and glacial acetic acid was refluxed in ethanol for 5-8 h, and then purified by recrystallization from ethoxyethanol and dichloromethane to obtain compounds 6a, 6b and 6c; The molar ratio of compound 1 to compound 3a-c is 1.5-2.5:1; the ratio of compound 3a-c, glacial acetic acid and ethanol is 1.5-2.0 mmol: 8-15 mL: 15-25 mL; The preparation methods of compounds 7a, 7b and 7c are as follows: Slowly add compound 6a-c in argon at 75℃~85℃ THF solution and LiAlH4, stirred for 15-25h, extracted with CH2Cl2 three times, the obtained dichloromethane solution was washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under vacuum. The crude product was purified by recrystallization from ether and dichloromethane to obtain white powders 7a, 7b and 7c; The ratio of the compound 6a-c, LiAlH4 and THF is 0.8-1.5mmol:3-4mmol:15-25mL; The preparation method of the compound 8a is as follows: slowly add 25-35 ml of a CH2Cl2 solution containing 0.8-1.2 mmol 7a and 3-5 ml of anhydrous CH2Cl2 solution containing 3-5 mmol BBr3 in argon at -80°C to -75°C, gradually heat the mixture to room temperature, stir for 15-25 hours, neutralize with a saturated NaHCO3 solution, extract with CH2Cl2 three times, wash the obtained dichloromethane solution with water, dry over anhydrous sodium sulfate, filter, evaporate the solvent under vacuum, and chromatograph the crude product on a silica gel column using CH2Cl2 as an eluent to obtain compound 8a; The preparation method of compound 9a is as follows: 25ml-35ml of a CH2Cl2 solution containing 0.4-0.7mmol 6a and 3-5ml of an anhydrous CH2Cl2 solution containing 3-5mmol BBr3 are slowly added in argon at -80°C to -75°C, the mixture is gradually heated to room temperature, stirred for 15-25h, neutralized with a saturated NaHCO3 solution, extracted three times with CH2Cl2, the obtained dichloromethane solution is washed with water and dried over anhydrous sodium sulfate, filtered, and the solvent is evaporated under vacuum. The crude product is eluted with CH2Cl2 and chromatographed on a silica gel column to obtain compound 9a.

Images