Amide-substituted tryptophanone derivatives, their preparation and use in medicine

Abstract

The application belongs to the technical field of pharmacotherapy, and particularly relates to an amide-substituted tryptophan ketone derivative, preparation and application thereof in medicine. The structural formula of the tryptophan ketone derivative is shown in formula (A) or formula (B). In the formula, n=1 or 2, and R is any one of pyridyl, trifluoropyridyl and methyl imidazolyl. The compound can selectively inhibit the activity of acetylcholinesterase, and has the abilities of nerve protection and the like, and has the potential to develop into a drug for treating Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical therapeutics, specifically relating to an amide-substituted tryptamine ketone derivative, its preparation, and its application in pharmaceuticals. Background Technology

[0002] Alzheimer's disease (AD) is one of the most common progressive neurodegenerative diseases, characterized by widespread brain atrophy, cognitive loss, sleep disturbances, mood swings, and behavioral changes. According to a 2023 report by Alzheimer's Disease International (ADI), more than 55 million people worldwide suffer from Alzheimer's disease, and its incidence is projected to increase dramatically, reaching 139 million by 2050. Therefore, AD poses a serious threat to global health and presents a significant challenge to society. To date, the pathogenesis of AD is complex and not fully understood. Fortunately, several hypotheses have been proposed and extensively studied, including low levels of acetylcholine (ACh), deposition of β-amyloid (Aβ) peptides, neuroprotection, neuroinflammation (NI), oxidative stress, and metal ion homeostasis dysregulation. Among these, the cholinergic hypothesis is currently the most classic hypothesis for AD.

[0003] According to the cholinergic hypothesis first described in 1976, the neurotransmitter acetylcholine (ACh) is responsible for nerve cells involved in learning and memory, and abnormally low ACh levels in the hippocampus and neocortex directly lead to cognitive decline. Accumulated evidence suggests that high levels of AChE contribute to the hydrolysis of ACh, resulting in memory deficits in the early stages of Alzheimer's disease (AD). Butyrylcholinesterase (BuChE), an enzyme closely associated with AChE, acts as a co-regulator of cholinergic neurotransmission by hydrolyzing ACh. Growing evidence supports that BuChE is a more beneficial target in patients with moderate to severe AD compared to the well-known AChE. Therefore, inhibiting ChE to restore ACh levels appears to be the most helpful approach for AD.

[0004] Tryptophan (TRYP) is an alkaloid widely found in the roots and leaves of Isatis indigotica. Recent studies have shown that tryptophan possesses a variety of pharmacological properties, including anti-inflammatory, antibacterial, antioxidant, antiviral, and antitumor activities. Seungjun Lee et al. pointed out that TRYP significantly inhibits the protein expression of iNOS and COX-2, reduces the levels of its products (NO and PGE2) in LPS-stimulated BV2 microglia, and downregulates the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. Many studies have confirmed that natural products with diverse pharmacological properties provide novel lead templates for drug discovery and development, playing a crucial role. Therefore, it is necessary to optimize and modify natural compounds. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention first provides an amide-substituted tryptamine ketone derivative, the structural formula of which is shown in formula (A) or formula (B):

[0006] (A)

[0007] (B)

[0008] In the formula, n = 1 or 2, and R is any one of pyridyl, trifluoropyridyl, or methylimidazolyl.

[0009] This invention provides a method for preparing amide-substituted tryptamine ketone derivatives as described above. The method for preparing tryptamine ketone derivatives with the structural formula shown in formula (A) includes the following steps:

[0010] S1. Under nitrogen protection, indigo anhydride reacts with indigo in a one-pot reaction with triethylamine and toluene to yield tryptamine ketone;

[0011] S2. Nitrate tryptophanone under HNO3 conditions to obtain 8-nitrotryptophanone;

[0012] S3. 8-Nitrotryptamine ketone is reduced by SnCl2 and HCl to give 8-aminotryptamine ketone;

[0013] S4. 8-Aminotryptamine ketone reacts with chloroacetyl chloride or chloropropionyl chloride to give the corresponding acylated products;

[0014] S5. The acylated products prepared in step S4 are reacted with amines having the corresponding R groups in acetonitrile solution. The purified products are amide-substituted tryptamine ketone derivatives having the structure shown in formula (A); the R group is any one of pyridyl, trifluoropyridyl, and methylimidazolyl.

[0015] Preferably, in step S5, the reaction temperature is 110 ℃ and the stirring time is 8-12 hours.

[0016] The above synthesis path is represented as follows:

[0017]

[0018] I. Indigo anhydride and indigo undergo a dehydration condensation reaction under the protection of triethylamine and nitrogen to give compound a; II. Compound a undergoes a nitration reaction in an ice-water bath of concentrated sulfuric acid and concentrated nitric acid to generate compound b; III. Compound b and stannous chloride dihydrate are added to an ethanol-hydrochloric acid solution and undergo a hydrogenation reduction reaction to give compound c; IV. Compound c is purified by column chromatography and then reacted with chloroacetyl chloride and chloropropionyl chloride, respectively, to give compounds d and e; V. Compounds d and e react with amines having various substituents R (110℃), and are purified by column chromatography to give compounds f1-f3, g1-g3; wherein the R group is any one of pyridinyl, trifluoropyridinyl, or methylimidazolyl.

[0019] The method for preparing tryptophan ketone derivatives with the structure shown in formula (B) includes the following steps:

[0020] S1. Indomethacin anhydride yields the precursor of the compound in concentrated H2SO4 in the presence of KNO3;

[0021] S2. The precursor compound reacts with indigo under the protection of triethylamine and nitrogen to undergo a dehydration condensation reaction, yielding 2-nitrotryptamine ketone;

[0022] S3.2-nitrotryptamine and stannous chloride dihydrate were added to an ethanol-hydrochloric acid solution and then hydrogenated to reduce the 2-aminotryptamine.

[0023] S4. 2-Aminotryptamine ketone reacts with chloroacetyl chloride or chloropropionyl chloride to give the corresponding acylated products;

[0024] S5. The acylated products prepared in step S4 are reacted with amines having the corresponding R groups in acetonitrile solution. The purified products are amide-substituted tryptamine ketone derivatives having the structure shown in formula (B); the R group is any one of pyridyl, trifluoropyridyl, and methylimidazolyl.

[0025] Preferably, in step S5, the reaction temperature is 110 ℃ and the stirring time is 8-12 hours.

[0026] The above synthesis path is represented as follows:

[0027]

[0028] VI. In the presence of KNO3, in concentrated H2SO4, indigo anhydride reacts to give compound h; I. Compound h reacts with indigo under the protection of triethylamine and nitrogen to give compound i; III. Compound i and stannous chloride dihydrate are added to ethanol-hydrochloric acid solution and hydrogenated to give compound j; IV. Compound j is purified by column chromatography and reacted with chloroacetyl chloride and chloropropionyl chloride to give compounds k and i, respectively; V. Compounds k and i react with amines having various substituents R (110℃) and purified by column chromatography to give compounds m1-m3, n1-n3; wherein the R group is any one of pyridinyl, trifluoropyridinyl, or methylimidazolyl.

[0029] The above-described preparation route is primarily for illustrative purposes and not for limiting the invention.

[0030] This invention provides the use of the amide-substituted tryptamine derivatives as described above in the preparation of acetylcholinesterase inhibitors.

[0031] This invention provides the use of the amide-substituted tryptamine ketone derivatives as described above in the preparation of medicaments for treating Alzheimer's disease.

[0032] The present invention also provides an acetylcholinesterase inhibitor or a medicament for treating Alzheimer's disease, said medicament containing a pharmaceutically effective dose of the tryptophan derivative as described above.

[0033] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0034] Preferably, the pharmaceutically acceptable carrier includes one or more excipients with functions such as excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents; such as starch, lipids, waxes, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.

[0035] Preferably, the drug is any one of injection, tablet, pill, capsule, suspension or emulsion.

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

[0037] 1. Structural modification of natural compounds can improve their potency and selectivity, enhance their pharmacological properties, and significantly reduce their harmful effects. Many studies have shown that introducing side chains with terminal amino groups into compounds can greatly improve the inhibitory activity of AChE and the selectivity of AChE / BuChE. Amide bonds are an important component of bioactive molecules and are widely present in many anti-AD drugs. Inspired by the above, we retained the tryptophan ketone structure and connected the parent nucleus and side chains with amide bridges of different lengths to obtain the expected cholinesterase inhibitory activity, ultimately screening and obtaining 12 tryptophan ketone derivatives provided in this invention.

[0038] 2. The amide-substituted tryptophanone derivatives involved in this invention have a selective inhibitory effect on AChE activity, indicating that these compounds have the potential to be developed into effective AChE inhibitors.

[0039] 3. The amide-substituted tryptophan derivatives involved in this invention have a neuroprotective effect on H2O2-induced PC12 cells, indicating that these compounds have the potential to be developed into an effective treatment for Alzheimer's disease.

[0040] 4. The amide-substituted tryptophan derivatives involved in this invention have the effect of inhibiting ROS accumulation in PC12 cells.

[0041] 5. The compound n2 involved in this invention significantly inhibits cholinesterase activity and has a better effect than donepezil, a first-line clinical drug, indicating that this type of compound has the potential to be developed into an effective treatment for Alzheimer's disease.

[0042] 5. The compounds involved in this invention have the potential to be developed into drugs for the treatment of Alzheimer's disease. Attached Figure Description

[0043] Figure 1 The interaction mode between compound n2 and acetylcholine (PDB: 4EY7): Figure 1 A is an n2 surface view of the AChE active site. Figure 1 In the middle, B represents the 2D mode of the interaction between n2 and AChE;

[0044] Figure 2 The cytotoxic effect of compound n2 on PC12;

[0045] Figure 3 The protective effect of compound n2 against H2O2-induced PC12 cell death;

[0046] Figure 4 and Figure 5 The effect of compound n2 on ROS accumulation in PC12 cells. Detailed Implementation

[0047] Unless otherwise stated, the terms used herein have the meanings commonly understood by those skilled in the art.

[0048] The technical solution of the present invention will be described in more detail below with reference to the embodiments.

[0049] Example 1

[0050] Synthesis of compound f1

[0051]

[0052] Compound d (339 mg, 1 mM) was dissolved in pyridine solution (5 mL) and refluxed at 110°C for 8–12 h. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solvent was removed. Compound f1 was purified by column chromatography with dichloromethane:methanol (20:1) to give a yellow solid in 75% yield. 1 H NMR (500 MHz, DMSO- d6 ) δ 11.61 (s, 1H), 9.21 –9.04 (m, 2H), 8.71 (tt, J = 7.8, 1.4 Hz, 1H), 8.44 (d, J = 8.6 Hz, 1H), 8.30(dt, J = 7.8, 1.0 Hz, 1H), 8.24 (dd, J = 7.8, 6.5 Hz, 2H), 8.17 (d, J = 2.2Hz, 1H), 8.03 – 7.89 (m, 3H), 7.77 – 7.69 (m, 1H), 5.77 (s, 2H). 13 C NMR (126MHz, DMSO- d6 ) δ 182.77, 164.35, 157.92, 152.30, 146.96, 146.85, 145.74,142.19, 137.46, 135.61, 128.12, 128.05, 127.36, 123.74, 123.23, 121.87,121.31, 118.25, 115.08, 62.59.

[0053] Example 2

[0054] Synthesis of compound f2

[0055]

[0056] The method is the same as in Example 1, except that the R group is replaced with a trifluoropyridyl group, yielding a yellow solid with a yield of 72%. 1 HNMR (500 MHz, DMSO-d6) δ 11.60 (s, 1H), 9.52 (t, J = 3.1 Hz, 1H), 9.06 (d, J= 5.8 Hz, 1H), 8.82 – 8.77 (m, 1H), 8.45 (d, J = 8.7 Hz, 1H), 8.40 – 8.34 (m,1H), 8.31 (d, J = 7.7 Hz, 1H), 8.16 (d, J = 2.2 Hz, 1H), 8.00 – 7.91 (m, 3H), 7.74 (dt, J = 8.1, 4.2 Hz, 1H), 5.79 (s, 2H). 13 C NMR (126 MHz, DMSO-d6) δ182.76, 163.79, 157.93, 146.85, 145.74, 144.49, 142.26, 137.33, 136.42,135.64, 134.63, 130.42, 129.47, 129.41, 128.16, 127.37, 126.45, 123.74,123.25, 118.30, 115.13, 62.92.

[0057] Example 3

[0058] Synthesis of compound f3

[0059]

[0060] The method is the same as in Example 1, except that the R group is replaced with a methylimidazolium group, yielding a yellow solid with a yield of 71%. 1 HNMR (600 MHz, DMSO-d6) δ 11.49 (s, 1H), 9.18 (s, 1H), 8.43 (d, J = 8.7 Hz,1H), 8.31 (s, 1H), 8.20 (s, 1H), 7.97 (d, J = 26.9 Hz, 3H), 7.78 (d, J = 31.0Hz, 3H), 5.34 (s, 2H), 3.94 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 182.79,164.89, 157.90, 146.86, 145.75, 142.10, 138.39, 137.61, 135.59, 130.39,128.04, 127.34, 125.44, 124.42, 123.75, 123.58, 123.20, 118.20, 115.01,51.68, 36.36.

[0061] Example 4

[0062] Synthesis of compound g1

[0063]

[0064] The method is the same as in Example 1, except that the compound is replaced with e, yielding a yellow solid with a yield of 76%. 1 H NMR(500 MHz, DMSO-d6) δ 10.84 (s, 1H), 9.21 – 9.15 (m, 2H), 8.63 – 8.58 (m, 1H), 8.38 (d, J = 8.7 Hz, 1H), 8.32 – 8.28 (m, 1H), 8.19 – 8.15 (m, 3H), 7.95 –7.92 (m, 2H), 7.85 (dd, J = 8.7, 2.3 Hz, 1H), 7.73 (dt, J = 8.2, 4.2 Hz, 1H), 4.94 (t, J = 6.4 Hz, 2H), 3.24 (t, J = 6.4 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6)δ 181.80, 167.78, 156.80, 145.79, 145.16, 144.81, 144.70, 140.83, 136.76,134.50, 129.32, 129.29, 127.20, 126.92, 126.26, 122.71, 122.00, 117.02,113.95, 56.13, 35.84.

[0065] Example 5

[0066] Synthesis of compound g2

[0067]

[0068] The method is the same as in Example 2, except that the compound is replaced with e, yielding a yellow solid with a yield of 61%. 1 H NMR(500 MHz, DMSO-d6) δ 10.28 (d, J = 4.6 Hz, 1H), 8.94 (d, J = 3.4 Hz, 1H), 8.48 (d, J = 6.1 Hz, 1H), 8.01 (td, J = 8.2, 2.6 Hz, 1H), 7.72 (d, J = 8.7Hz, 1H), 7.67 – 7.58 (m, 2H), 7.52 (d, J = 2.2 Hz, 1H), 7.27 (d, J = 4.1 Hz, 2H), 7.22 (dd, J = 8.7, 2.3 Hz, 1H), 7.07 (dt, J = 8.2, 4.2 Hz, 1H), 4.30 (t,J = 6.4 Hz, 2H), 2.50 (s, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 182.88, 168.79,157.86, 146.85, 145.76, 143.33, 141.86, 137.94, 135.57, 133.79, 133.65,130.39, 129.56, 129.50, 127.99, 127.33, 123.77, 123.04, 118.04, 115.04,57.79, 49.06, 36.74.

[0069] Example 6

[0070] Synthesis of compound g3

[0071]

[0072] The method is the same as in Example 3, except that the compound is replaced with e, yielding a yellow solid with a yield of 70%. 1H NMR(500 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.43 (d, J = 1.7 Hz, 1H), 7.48 (d, J =8.7 Hz, 1H), 7.45 – 7.37 (m, 2H), 7.11 (dd, J = 8.7, 2.3 Hz, 1H), 7.06 (d, J= 3.5 Hz, 2H), 7.00 (d, J = 1.9 Hz, 1H), 6.90 – 6.80 (m, 2H), 3.02 (s, 3H), 2.50 (s, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 182.86, 169.09, 157.80, 146.83,145.73, 141.71, 138.25, 137.56, 135.52, 130.36, 130.33, 127.90, 127.30,123.97, 123.76, 122.98, 122.94, 117.88, 115.01, 45.37, 36.46, 36.23.

[0073] Example 7

[0074] Synthesis of compound m1

[0075]

[0076] Compound k (354 mg, 1 mM) was dissolved in pyridine solution (5 mL) and refluxed at 110 °C for 8–12 h. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solvent was removed. Compound m1 was purified by column chromatography with dichloromethane:methanol (20:1) to give a yellow solid in 76% yield. 1 H NMR (500 MHz, DMSO-d6) δ 12.26 (s, 1H), 9.18(d, J = 6.1 Hz, 2H), 8.75 (d, J = 2.6 Hz, 1H), 8.60 (d, J = 4.9 Hz, 1H), 8.47(d, J = 8.0 Hz, 1H), 8.26 (t, J = 7.0 Hz, 2H), 7.95 (d, J = 8.7 Hz, 1H), 7.87(d, J = 7.5 Hz, 3H), 7.48 (t, J = 7.6 Hz, 1H), 5.90 (s, 2H). 13C NMR (126 MHz, DMSO-d6) δ181.69, 164.76, 156.82, 145.21, 144.37, 143.37, 142.63, 141.67,139.22, 137.07, 130.34, 127.28, 126.30, 125.40, 124.05, 123.43, 121.75,116.47, 115.13, 42.97.

[0077] Example 8

[0078] Synthesis of compound m2

[0079]

[0080] The method is the same as in Example 7, except that the R group is replaced with a trifluoropyridyl group, yielding a yellow solid with a yield of 73%. 1 HNMR (600 MHz, DMSO-d6) δ 12.42 (s, 1H), 9.66 (s, 1H), 9.17 (d, J = 6.1 Hz,1H), 8.86 – 8.80 (m, 1H), 8.77 (d, J = 2.5 Hz, 1H), 8.50 – 8.46 (m, 1H), 8.38 (dt, J = 8.9, 5.8 Hz, 1H), 8.19 (dd, J = 8.8, 2.5 Hz, 1H), 7.97 (d, J = 8.7Hz, 1H), 7.87 (t, J = 7.6 Hz, 2H), 7.48 (t, J = 7.5 Hz, 1H), 5.96 (s, 2H). 13 CNMR NMR (126 MHz, ) δ 182.76, 165.17, 157.87, 146.27, 144.45, 142.70, 140.29,138.44, 138.15, 134.92, 131.45, 127.39, 126.34, 125.14, 124.49 (d, J = 29.9Hz), 123.59, 122.82, 119.99, 117.52, 116.07, 51.87.

[0081] Example 9

[0082] Synthesis of compound m3

[0083]

[0084] The method was the same as in Example 7, except that the R group was replaced with a methylimidazolium group, yielding a yellow solid with a yield of 71%; ¹H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1H), 9.22 (d, J = 1.8 Hz, 1H), 8.71 (d, J = 2.5 Hz, 1H), 8.45 (dd, J = 7.9, 0.9 Hz, 1H), 8.11 (dd, J = 8.8, 2.5 Hz, 1H), 7.94 (s, 1H), 7.88 – 7.82 (m, 3H), 7.77 (t, J = 1.8 Hz, 1H), 7.47 (td, J = 7.5, 0.9 Hz, 1H), 5.40 (s, 2H), 3.95 (s, 3H).13C NMR (126 MHz, DMSO-d6) 13CNMR (126 MHz, DMSO-d6) δ 182.76, 165.16, 157.87, 146.26, 144.44, 142.70,140.29, 138.44, 138.15, 131.45, 127.39, 126.34, 125.14, 124.54, 124.44,123.59, 122.82, 117.52, 116.06, 51.86, 36.38.

[0085] Example 10

[0086] Synthesis of compound n1

[0087]

[0088] The method is the same as in Example 7, except that the compound is replaced with i, yielding a yellow solid with a yield of 70%. 1 H NMR(500 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.26 (d, J = 5.9 Hz, 2H), 8.69 – 8.61 (m,2H), 8.43 (d, J = 8.2 Hz, 1H), 8.20 (t, J = 7.1 Hz, 2H), 8.04 (dd, J = 8.8,2.5 Hz, 1H), 7.88 – 7.82 (m, 3H), 7.46 (t, J = 7.5 Hz, 1H), 5.00 (t, J = 6.5Hz, 2H), 3.34 (t, J = 6.5 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 182.72,169.13, 157.87, 146.24, 146.22, 145.91, 144.19, 142.36, 140.69, 138.12,131.21, 128.30, 127.35, 126.31, 125.10, 124.37, 122.79, 117.48, 115.87,57.23, 37.26.

[0089] Example 11

[0090] Synthesis of compound n2

[0091]

[0092] The method is the same as in Example 8, except that the compound is replaced with i, yielding a yellow solid with a yield of 68%. 1 H NMR(500 MHz, DMSO-d6) δ 11.38 (s, 1H), 9.70 (t, J = 3.4 Hz, 1H), 9.22 (d, J =6.1 Hz, 1H), 8.68 (dd, J = 12.3, 2.5 Hz, 2H), 8.42 (d, J = 8.3 Hz, 1H), 8.29(dt, J = 8.9, 5.9 Hz, 1H), 8.04 (dd, J = 8.8, 2.5 Hz, 1H), 7.87 – 7.80 (m,3H), 7.45 (t, J = 7.5 Hz, 1H), 5.01 (t, J = 6.5 Hz, 2H), 3.36 (d, J = 6.5 Hz, 2H). 13 C NMR (126MHz, DMSO-d6) δ 182.71, 169.06, 159.04, 157.87, 146.21,144.18, 143.37, 142.35, 140.73, 138.11, 133.79, 133.65, 131.20, 129.58,127.34, 126.32, 125.10, 124.36, 122.79, 117.47, 115.87, 57.78, 37.06.

[0093] Example 12

[0094] Synthesis of compound n3

[0095]

[0096] The method is the same as in Example 9, except that the compound is replaced with i, yielding a yellow solid with a yield of 73%. 1 H NMR(500 MHz, DMSO-d6) δ 11.32 (s, 1H), 9.29 (d, J = 1.7 Hz, 1H), 8.70 (d, J =2.4 Hz, 1H), 8.45 – 8.39 (m, 1H), 8.06 (dd, J = 8.8, 2.5 Hz, 1H), 7.88 – 7.79(m, 4H), 7.72 (t, J = 1.8 Hz, 1H), 7.45 (td, J = 7.5, 0.9 Hz, 1H), 4.53 (t, J= 6.4 Hz, 2H), 3.87 (s, 3H), 3.14 (t, J = 6.4 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 182.71, 169.36, 157.88, 146.21, 144.15, 142.31, 140.83, 138.11, 137.57,131.18, 127.34, 126.31, 125.09, 124.36, 123.98, 122.99, 122.78, 117.48,115.86, 45.30, 36.56, 36.23.

[0097] Example 13

[0098] The inhibitory effects of the compounds on acetylcholinesterase and butyrylcholinesterase were tested using Ellman's method (Biochemical Pharmacology 1961, 7, 88-95). The results were analyzed using IC50. 50 Values ​​are expressed as positive controls, with Donepezil and Tacrine used as positive controls. All tests were performed on a PowerWave XS2 full-wavelength microplate reader at 37°C. Data analysis was performed using Origin software.

[0099] Experimental steps:

[0100] (1) Preparation of drug solution:

[0101] Weigh a certain amount of each sample to be analyzed and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 10 mM concentration. Store it in a -20°C freezer. Before use, dilute it with phosphate buffer (0.1 mol / L, pH=8.0) to the required concentration so that the final concentration of DMSO is less than or equal to 0.5% (v / v).

[0102] (2) Preparation of enzyme stock solution:

[0103] Acetylcholinesterase (EC 3.1.1.7, from electric ell.) and butyrylcholinesterase (EC 3.1.1.8, from equine serum) were purchased from Sigma. Weigh out a certain amount of acetylcholinesterase or butyrylcholinesterase and dilute it with deionized water to the appropriate activity range.

[0104] (3) Preparation of substrate stock solution:

[0105] Acetylthiocholine (ATC) and butylthiocholine (BTC) were purchased from Sigma. A certain amount of ATC or BTC was weighed and prepared into a 0.01 mol / L solution with phosphate buffer (0.1 mol / L, pH 8.0), and stored in the dark at 4°C.

[0106] (4) Preparation of the colorimetric reagent stock solution:

[0107] The colorimetric reagent 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) was purchased from Sigma-Aldrich. A certain amount of DTNB was weighed and prepared into a 0.01 mol / L solution with phosphate buffer (0.1 mol / L, pH 8.0), and stored in the dark at 4°C.

[0108] (5) Enzyme inhibition test

[0109] The compound dissolved in DMSO was diluted to different concentrations with PB (phosphate buffer) solution and then added to 48-well plates (100 μL per well). The plates were then incubated with 100 μL of AChE or BuChE at 37°C for 20 minutes. After 20 minutes, 100 μL of substrate (iodothioacetylcholine or S-iodobutyrylthiocholine) and 100 μL of PB solution were added to the 48-well plates. Then, 100 μL of chromogenic material (5,5-dithiobis-(2-nitrobenzoic acid, DTNB, Ellman's reagent) was added and incubated for 5 minutes. The absorbance was then measured at 419 nm using a multi-mode microplate reader.

[0110] (6) Calculation of results:

[0111] IC 50 Calculation of the value: The change in absorbance (slope) measured without the addition of inhibitor is taken as 100 activity units (Acontrol). Relative enzyme activity = (absorbance change with inhibitor / absorbance change without inhibitor) × 100. When the relative enzyme activity reaches 50, it is the IC50 of the inhibitor. 50 value.

[0112] The experimental results are the average of three independent experiments.

[0113] (7) Experimental results:

[0114] Table 1. Inhibitory activities of the compounds against acetylcholinesterase and butyrylcholinesterase

[0115]

[0116] a Selectivity for acetylcholinesterase = IC 50 (Butyrylcholinesterase) / IC 50 (acetylcholinesterase)

[0117] (8) Results and Discussion: Table 1 shows that the synthesized compounds all exhibited good inhibitory activity against acetylcholinesterase, ranging from 0.15 to 45.33 nM, while their inhibitory activity against butyrylcholinesterase was weaker. The selectivity for acetylcholinesterase was high, ranging from 194.8 to 5666.6. This suggests that compounds f1-n3 have the potential to be developed into acetylcholinesterase inhibitors and further into AD treatment drugs. Among the above compounds, n2 showed the best acetylcholinesterase inhibitory activity (IC50). 50 It has a molecular weight of 0.15 ± 0.01 nM and the best acetylcholinesterase inhibitory selectivity (5666.6), making it a potential drug for the treatment of AD.

[0118] Example 14

[0119] Molecular docking

[0120] use Discovery Studio 2017 R2 (DS, BIOVIA Software, Inc., San Diego, CA, USA The software performs the interaction between the ligand and receptor. The X-ray crystal structure of the AChE protein was obtained from the PDB database (ID: 4EY7). First, we removed the original ligands and water molecules from the protein structure, then added hydrogen atoms and a CHARMm force field to complete the protein preparation. Subsequently, the prepared protein was obtained using a protein preparation program. The ligands were treated with a fully minimized small molecule scheme. The ligands were then attached to the active site of the protein using the CDOCKER program.

[0121] Discussion of Results: Figure 1 As shown, n2 can bind to the PAS and CAS of AChE, exhibiting good binding ability, with a CDOCKER_INTERACTION_ENERGY value of 59.0024. Docking results show that the quinoline and indole rings form six π-π stacking interactions (distances = 4.33–5.67 Å) with the key amino acid residues Tyr341, Phe338, Typ337, and Trp86. Furthermore, the carbonyl oxygen on the quinoline ring interacts with Tyr124 via hydrogen bonding (distance = 2.20 Å). The oxygen atom on the amide interacts with the key amino acid Phe295 via hydrogen bonding (distance = 2.59 Å). The fluorine atom on the benzene ring interacts with the key amino acid Ser293 via hydrogen bonding (distance = 2.21 Å). Overall, n2 demonstrates a strong and stable interaction with AChE in molecular docking.

[0122] Example 15

[0123] Inhibition of H2O2-induced cytotoxicity

[0124] Mouse neuronal cell line PC12 was routinely cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C, under saturated humidity, in a carbon dioxide incubator containing 5% CO2 and 95% air. Cells in the logarithmic growth phase were harvested, digested with 0.25% trypsin solution, and then resuspended in complete medium. Cells were counted using a cell counting chamber under a microscope, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at 100 μL / well in 96-well cell culture plates and cultured overnight to allow cell adhesion. The experiment included a test sample group, a blank group, and a solvent control group. The culture medium in the 96-well cell culture plates was discarded, and the test sample group solution was added. The blank group received complete culture medium without the compound, and the solvent control group received DMSO at the same concentration. After a 30-minute pre-incubation, 150 μM hydrogen peroxide was added. The model group did not receive the test compound; 140 μM hydrogen peroxide was added directly. After 24 hours of incubation, the cells were protected from light, and 20 μL of 5 mg / mL MTT stock solution was added to each well, and the cells were cultured for 4 hours. The culture medium in the 96-well cell culture plates was discarded, and 100 μL of DMSO was added to each well. The plates were shaken to completely dissolve the formazan in the DMSO, and the absorbance of each well was measured at 570 nm using a microplate reader. The formulated compound was used to promote cell viability.

[0125] Cell survival rate (%) = 100% The cell viability was calculated as (A test compound - A model group) / (A model group - A blank).

[0126] Discussion of Results: From Figure 2It can be seen that compound n2 has no obvious toxicity to PC12 cells at a concentration of 20 mM. Figure 3 It can be seen that compound n2 increased the survival rate of H2O2-induced PC12 cells. Compound n2 at 10 At time M, the survival rate of H2O2-induced PC12 cells was 63.44±0.3%, significantly better than that of the positive control drug quercetin. This indicates that this type of compound has a neuroprotective effect to some extent.

[0127] Example 16

[0128] Intracellular reactive oxygen species assay

[0129] Intracellular ROS concentration was measured using a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Sigma Aldrich) that can penetrate cells and be rapidly oxidized by intracellular ROS to highly fluorescent 2',7'-dichlorofluorescein (DCF). Intracellular fluorescence intensity was directly proportional to intracellular ROS levels. PC12 cells were seeded in black 96-well plates (8000 cells / well) and cultured for 24 hours in high-glucose DMEM medium (supplemented with 10% FBS, 1% penicillin, and 1% streptomycin). PC12 cells were cultured in media containing or without the test compound at concentration gradients of 10 and 30 μM. After washing PC12 cells with PBS, 150 μM H2O2 was added to each well, and the cells were incubated for 24 hours. After discarding the medium, cells were fixed with 4% paraformaldehyde for 10 minutes, followed by the addition of the fluorescent dye Hoechst 33258. The samples were incubated at 37°C in the dark for 10 minutes. Finally, we added the fluorescent dye DCFH-DA (10 μM), incubated the sample at 37°C for 10 minutes in the dark, and then imaged the sample using a laser confocal microscope.

[0130] Discussion of Results: Figure 4 and Figure 5 As shown, compared with the H2O2 group, compound n2 reduced ROS accumulation in PC12 cells. When the concentration of n2 was 30 μM, it significantly inhibited ROS accumulation, indicating that the compound has a neuroprotective effect to some extent.

[0131] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An amide-substituted tryptamine ketone derivative, characterized in that, The tryptophan ketone derivatives are n1, n2, or n3, and their structural formulas are shown below: （n1）， （n2）， （n3）。 2. A method for preparing an amide-substituted tryptamine ketone derivative as described in claim 1, characterized in that, Includes the following steps: S1. Indomethacin anhydride yields the precursor of the compound in concentrated H2SO4 in the presence of KNO3; S2. The precursor compound reacts with indigo under the protection of triethylamine and nitrogen to undergo a dehydration condensation reaction, yielding 2-nitrotryptamine ketone; S3.2-nitrotryptamine and stannous chloride dihydrate were added to an ethanol-hydrochloric acid solution and then hydrogenated to reduce the 2-aminotryptamine. S4. 2-Aminotryptamine ketone reacts with chloropropionyl chloride to give the corresponding acylated products; S5. The acylated products prepared in step S4 are reacted with pyridine, 3-fluoropyridine, and 1-methylimidazole in acetonitrile solution, respectively, and after purification, the tryptophan ketone derivatives n1, n2, or n3 of claim 1 are obtained respectively.

3. The preparation method according to claim 2, characterized in that, In step S5, the reaction temperature is 110℃ and the stirring time is 8-12 hours.

4. The use of an amide-substituted tryptamine derivative as described in claim 1 in the preparation of an acetylcholinesterase inhibitor.

5. The use of an amide-substituted tryptamine derivative as described in claim 1 in the preparation of a medicament for treating Alzheimer's disease.

6. An acetylcholinesterase inhibitor comprising a pharmaceutically effective dose of the tryptophan derivative as described in claim 1.

7. A medicament for treating Alzheimer's disease, comprising a pharmaceutically effective dose of the tryptophan derivative as described in claim 1.

8. The drug as described in claim 6 or 7, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

9. The medicament as described in claim 8, characterized in that, The pharmaceutically acceptable carriers include carriers that function as stabilizers, colorants, diluents, and sustained-release agents, or a combination thereof.

10. The drug as claimed in claim 6 or 7, characterized in that, The drug is any one of the following: injection, tablet, pill, capsule, suspension, or emulsion.

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