An indole derivative, a synthetic method and application thereof

Abstract

This invention provides an indole derivative and its solvent-controlled synthetic method and application, belonging to the field of organic compound synthesis technology. The invention provides two novel compounds: 1'-benzyl-2-methoxyspiro[imidazo[1,5-] a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-trione and N -(1-benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H Indole-1-formamide can serve as a key intermediate in the chemical and pharmaceutical industries, showing broad application prospects. The synthetic methods for the two types of indole derivatives mentioned above are based on... N -Methoxy-1 H Indole-1-formamide was used as the starting material, and then added... N Benzylmaleimide, a solid base and a metal catalyst are added, and then the appropriate reaction solvent is added. The reaction temperature and reaction time are controlled. After the reaction is completed by TLC monitoring, the reaction is quenched with saturated sodium chloride solution. Then, the product is obtained by extraction, filtration, drying, diatomaceous earth filtration and recrystallization. The synthesis method is simple, the raw materials are cheap and readily available, and the product yield is high.

Description

Technical Field

[0001] This invention belongs to the field of organic compound synthesis technology, specifically relating to an indole derivative and its solvent-controlled synthesis method and application. Background Technology

[0002] Indole and its derivatives, as core nitrogen heterocyclic skeletons, are widely found in alkaloids, drug molecules, organic optoelectronic materials, and agrochemicals. Their structural diversity directly determines their biological activity and optoelectronic properties. Currently, more than 30% of drugs commonly used clinically for antitumor, anti-inflammatory, and antibacterial purposes contain an indole core structure, such as sumatriptan, tadalafil, and indomethacin. In the field of materials science, indole derivatives, due to their conjugation effect and solvation colorimetric properties, have become key components in fluorescent probes, organic light-emitting diodes (OLEDs), and dye-sensitized solar cells.

[0003] Novel substituted spiro[pyrrolidine-thiazole-oxindole] derivatives possess numerous biological activities and show broad clinical application prospects in psychotropic, cardiovascular disease treatment, and antibacterial drugs. The synthesis of these compounds represents a highly efficient drug design approach based on the pyridine parent ring core structure, attracting significant attention from researchers.

[0004] Spirocyclic pyrrolidine drugs, such as rhinoplastin, sitafloxacin, and lidapasvir, possess a wide range of pharmaceutical activities. However, traditional synthetic methods for these indole derivatives, mainly involving Fischer synthesis, Bartoli reaction, Sklaup synthesis, Bischler–Möhlau reaction, and transition metal-catalyzed cyclization, have several drawbacks, which are detailed below: (a) Fischer synthesis: the most widely used method, but it requires strong acid / high temperature, the substrate is easily decomposed, the regioselectivity is poor, it is difficult to synthesize polysubstituted or fused-ring indoles, and it is impossible to achieve the directional generation of different structural derivatives from the same substrate.

[0005] (ii) Bartoli reaction: It is specifically used for the synthesis of 7-substituted indoles, depends on Grignard reagents and ortho-substituted nitrobenzenes, has a narrow substrate range, poor functional group compatibility, and harsh conditions.

[0006] (III) Transition metal catalysis (Pd, Rh, Ir, Cu catalysis): Improved regioselectivity, but requires noble metal catalysts, complex ligands, high cost, difficult post-processing, and it is difficult to construct two or more novel skeletal derivatives through a single reaction system.

[0007] (iv) Conventional substituent modification: concentrated at C2, C3, and N1 positions, selective functionalization at C4–C7 positions is difficult, and the synthetic routes for novel fused ring, spiro ring, and heterocyclic fused ring indole derivatives are long, with low overall yield and difficult isomer separation.

[0008] Solvent effects are crucial for controlling reaction pathways, regioselectivity, stereoselectivity, and product distribution. Recent studies have confirmed that solvent polarity (dielectric constant, dipole moment), hydrogen bond donor / acceptor ability, and solvation directly influence intermediate stability, reaction activation energy, and transition state configuration. Polar aprotic solvents (DMF, DMSO, acetonitrile), polar protic solvents (methanol, ethanol, isopropanol), nonpolar solvents (toluene, dichloromethane, cyclohexane), and mixed solvents can lead to the formation of structurally distinct products from the same substrate via different cyclization, addition, and rearrangement pathways. However, current solvent synthesis techniques primarily focus on substituent modification of known indole derivatives, failing to achieve solvent-controlled directed synthesis of novel indole derivatives with different skeletons. Furthermore, a single reaction system can only synthesize one structure, making it impossible to efficiently construct two entirely new indole derivatives with different skeletons through solvent switching. The lack of systematic research on the relationship between solvent effects and product structures results in unclear synthetic mechanisms, poor reproducibility, and difficulties in large-scale application. Currently, solvent-free control strategies are lacking in synthetic methods that achieve the directed generation of two new types of indole derivatives under the same substrate and reaction conditions (except for the solvent), but also lack highly selective, high-yield, mild, and substrate-wide synthetic methods.

[0009] Therefore, traditional synthetic methods cannot meet the requirements of efficiency and cost in the synthesis of novel substituted spiro[pyrrolidine-thiazole-oxindole] derivatives, and the potential of solvent-controlled synthesis has not been fully explored. How to apply solvent-controlled methods to the synthesis and application of novel substituted spiro[pyrrolidine-thiazole-oxindole] derivatives has become an urgent technical problem to be solved. Summary of the Invention

[0010] The present invention aims to solve the above-mentioned technical problems, thereby providing a synthesis method and application of indole derivatives and their solvent-controlled synthesis, which provides a synthesis method that is simple, uses inexpensive and readily available raw materials, and has a high product yield.

[0011] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides two classes of indole derivatives having the compound structures shown in formula <Ⅰ> or <Ⅱ>: ; .

[0012] The two types of indole derivatives provided by this invention, the Chinese name of compound of formula <Ⅰ> is: 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H)-Triketones, which insert an imidazo[1,5-]indole ring into their structure, enhance the interaction between the compound and specific proteins. Therefore, compared with traditional spirocyclic pyrrolidine compounds, 1'-benzyl-2-methoxyspiro[imidazo[1,5-] a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H Triones have better potential for biological and pharmaceutical activity.

[0013] The Chinese name of the compound of formula <II> is: N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H -Indole-1-carboxamide, this compound has an indole ring, formamide, substituted maleimide and other structures in its structure. It has easily activated, highly controllable and easy addition substitution reaction sites, and is a building block with multiple good reaction sites.

[0014] The two novel compounds of this invention, 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-triketone and N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H -Indole-1-carboxamide can be used as a key intermediate in the chemical and pharmaceutical industries and has broad application prospects.

[0015] A second objective of this invention is to provide a method for synthesizing the two types of indole derivatives described above, which is based on... N -Methoxy-1 H -Indole-1-carboxamide as the starting material, added N Benzylmaleimide, a solid base and a metal catalyst were added, and then the appropriate reaction solvent was added. The reaction temperature and reaction time were controlled. After the reaction was completed by TLC monitoring, the reaction was quenched with saturated sodium chloride solution. Then, the reaction was extracted, filtered, dried, filtered with diatomaceous earth and recrystallized to obtain the above two types of indole derivatives.

[0016] Furthermore, the solid alkali is cesium carbonate, and the metal catalyst is dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer.

[0017] Furthermore, the reaction solvent for the compound shown in Formula is trifluoroethanol, and the reaction solvent for the compound shown in Formula <II> is dichloromethane.

[0018] Furthermore, the aforementioned N -Methoxy-1 H -Indole-1-formamide andN The molar ratio of β-benzylmaleimide is 1:0.8 to 2.5, preferably 1:1.2.

[0019] Furthermore, the solid alkali and N -Methoxy-1 H The molar ratio of indole-1-carboxamide is 1:0.5 to 5.0, preferably 1:2.

[0020] Furthermore, the reaction temperature is 20-25℃; the reaction time is 12 hours. It should also be noted that the reaction of this invention does not require a special reaction atmosphere; an air atmosphere is sufficient.

[0021] Furthermore, the amount of the metal catalyst used is N -Methoxy-1 H The amount of indole-1-carboxamide added is 0.5-10% by molar mass, preferably 1.0%; the amount of the reaction solvent added is... N -Methoxy-1 H -10 times the weight of indole-1-carboxamide.

[0022] Furthermore, the solvent used for recrystallization is a mixture of ethyl acetate and petroleum ether, with a volume ratio of ethyl acetate to petroleum ether of 1:2 to 10, preferably 1:5.

[0023] This invention utilizes the aforementioned solvent control method to successfully synthesize two different types of substituted pyridine compounds. This is achieved through the following method: using... N -Methoxy-1 H -indole-1-formamide and N Using benzylmaleimide as a starting material, by changing the solvent during synthesis while keeping other conditions unchanged, the reaction can yield 1'-benzyl-2-methoxyspiro[imidazo[1,5-] a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-trione (compound of formula ) and N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H -Indole-1-carboxamide (formula <II> compound) are two types of indole compounds.

[0024] The synthesis method of this invention is simple, the raw materials are inexpensive and readily available, and the product yields are as follows: 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-Triketone: 86.7%, N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H-pyrrole-3-yl)-1 H -Indole-1-carboxamide: 84.1%, purity ≥99.5%, which can meet the requirements of large-scale industrial production, and at the same time expand the application of indole derivatives in chemical and pharmaceutical intermediates and end products.

[0025] A third objective of this invention is to provide applications of the two types of indole derivatives synthesized by the methods described above in the preparation of chemical raw materials, pharmaceutical intermediates, and related end products.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention, through solvent control, allows for the preparation of the two new compounds mentioned above through a "one-pot" reaction, using the same reaction substrate and conditions but only changing the solvent in the reaction system.

[0027] 2. The novel substituted spiro[imidazo[1,5-]pyrrolidine] compound of the present invention is 1'-benzyl-2-methoxyspiro[imidazo[1,5-] a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-Triketone, with a reaction yield as high as 86.7%, this compound possesses multiple biologically active structures such as spirocyclic, imidazole, indole, and pyrrolidine. Compared with traditional spirocyclic pyrrolidine compounds, 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H Triones have better potential for biological and pharmaceutical activity.

[0028] 3. The present invention described N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H -Indole-1-carboxamide, with a reaction yield of 84.1%, has multiple easily activated, highly controllable, and easily addition-substitution reaction sites in its structure. It is a building block with multiple good reaction sites and has good potential for use as an intermediate in drug synthesis and organic research. Attached Figure Description

[0029] Figure 1 The present invention relates to 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-trione 1 H NMR spectrum.

[0030] Figure 2 The present invention relates to 1'-benzyl-2-methoxyspiro[imidazo[1,5- a[Indole-1,3'-pyrrolidine]-2',3,5'(2 H )-trione 13 C10 NMR spectrum.

[0031] Figure 3 The present invention relates to 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H IR spectrum of )-trione.

[0032] Figure 4 The present invention relates to 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H HRMs spectrum of )-trione.

[0033] Figure 5 For the present invention N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H -indole-1-carboxamide 1 H NMR spectrum.

[0034] Figure 6 For the present invention N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H -indole-1-carboxamide 13 C10 NMR spectrum.

[0035] Figure 7 For the present invention N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H IR spectrum of indole-1-formamide.

[0036] Figure 8 For the present invention N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H HRM spectrum of indole-1-carboxamide.

[0037] Figure 9 1'-Benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H Molecular docking diagram and docking effect of )-trione.

[0038] Figure 10 for N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H Molecular docking diagram and docking effect of indole-1-carboxamide. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0040] Example 1

[0041] 1'-Benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H Synthesis and characterization of )-triones: Add to a 100 mL round-bottom flask N -Methoxy-1 H -Indole-1-carboxamide (3.8g, 20mmol) and N Benzylmaleimide (4.0 g, 21 mmol) was added with a solid base of Cs₂CO₃ (3.3 g, 10 mmol) and a metal catalyst of C. 20 H 30 Cl4Rh2 (150 mg, 0.24 mmol) was added to 38 g of trifluoroethanol as solvent. A magnetic stir bar was added, and the reaction temperature was controlled at 25 °C. The mixture was stirred thoroughly, and the reaction was carried out in a sealed environment for 12 h without any special gas atmosphere. After the reaction was detected by TLC, 150 mL of saturated brine was added to quench the reaction. The mixture was extracted three times with ethyl acetate (100 mL * 3), the organic phase was washed with water, separated, dried over anhydrous magnesium sulfate, filtered, and the solvent was recovered by vacuum distillation. 20 mL of ethyl acetate was added to dissolve the mixture completely. The solution was filtered through a filter lined with diatomaceous earth, and the diatomaceous earth was washed with ethyl acetate (10 mL * 3). The ethyl acetate was combined and concentrated to about 20 mL by vacuum distillation. 100 mL of petroleum ether was added, and the mixture was placed in a refrigerator overnight. The solution was then filtered and dried to obtain 1'-benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H 6.5g of )-trione, purity 99.5%, yield 86.7%. Melting point is 172.3 – 174.2 °C.

[0042] 1H NMR (600MHz, CDCl3) δ = 7.98 (d, J = 8.1 Hz, 1H, ArH), 7.57 (d, J =7.9 Hz, 1H, ArH), 7.34 (m, 7H, ArH), 6.35 (s, 1H, CH), 4.80 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.43 (d, J = 18.6 Hz, 1H, CH), 3.22 (d, J = 18.6 Hz, 1H, CH); 13 C10 NMR (150MHz, CD13) δ = 171.97, 171.45, 151.69, 134.93, 132.87, 132.29, 131.32, 128.86, 128.69, 128.39, 125.06, 123.92, 121.79, 113.21, 99.66, 66.40, 66.16, 43.48, 37.77; IR (KBr): 3012, 2953, 1777, 1712, 1452, 1436, 1398, 1355, 1317, 1169,1015, 748, 705, 631 cm -1 ; HRMS (TOF EMS) + ): m / z calcd. For C 21 H 13 N3O4[M+H] + , 376.1292; found,376.1292.

[0043] Example 2

[0044] N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H Synthesis and characterization of indole-1-carboxamide: Add to a 100 mL round-bottom flask N -Methoxy-1 H -Indole-1-carboxamide (3.8g, 20mmol) and NBenzylmaleimide (4.0 g, 21 mmol) was added with a solid base of Cs₂CO₃ (3.3 g, 10 mmol) and a metal catalyst of C. 20 H 30 Cl4Rh2 (150 mg, 0.24 mmol) was added to 38 g of dichloromethane solvent. A magnetic stir bar was added, and the reaction temperature was controlled at 25 °C. The mixture was stirred thoroughly, and the reaction was carried out in a sealed environment for 12 h without any special gas atmosphere. After the reaction was completed by TLC, 150 mL of saturated brine was added to quench the reaction. The mixture was extracted three times with ethyl acetate (100 mL * 3), washed with water, separated, dried over anhydrous magnesium sulfate, filtered, and the solvent was recovered by vacuum distillation. 20 mL of ethyl acetate was added to dissolve the mixture completely. The solution was filtered through a filter lined with diatomaceous earth, and the diatomaceous earth was washed with ethyl acetate (10 mL * 3). The ethyl acetate was combined and concentrated to about 20 mL by vacuum distillation. 100 mL of petroleum ether was added, and the mixture was placed in a refrigerator overnight. The solution was then filtered and dried to obtain the final product. N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H 5.8 g of indole-1-carboxamide, purity 99.5%, yield 84.1%. Melting point 163.2 – 164.7 °C.

[0045] 1 H NMR (600MHz, CDCl3) δ = 8.16 (d, J = 8.4 Hz, 2H, ArH), 7.59 (d, J =7.8 Hz, 1H, ArH), 7.46 (d, J = 3.8 Hz, 1H, ArH), 7.39 – 7.26 (m, 7H), 6.71(d, J = 3.8 Hz, 1H, CH), 6.55 (s, 1H, NH), 4.69 (s, 2H, CH2); 13 C10 NMR (150MHz, CD13) δ = 171.12, 167.54, 147.66, 138.68, 135.92, 135.22, 130.37, 128.78, 128.35, 128.02, 125.42, 123.77, 123.07, 121.62, 114.70, 109.94, 104.30, 41.63; IR (KBr): 3268, 3143, 3037, 2945, 1706, 1642, 1586, 1559, 1452, 1433,1411, 1353, 1244, 1200, 830, 758, 725, 697, 625 cm -1 ; HRMS (TOF EMS) + ): m / z calcd. For C 20 H 15 N3O3[M+H] + , 346.1186; found,346.1187.

[0046] The application prospects of the two types of indole derivatives of this invention are introduced as follows: Application Example 1: 1'-Benzyl-2-methoxyspiro[imidazo[1,5- a [Indole-1,3'-pyrrolidine]-2',3,5'(2 H Application of )-trione (compound of formula ) I. Molecular docking and result analysis (a) Connection method 1. For small molecules, SwissTargetPrediction was used to predict the Top 10 targets and identify the receptor protein with the highest binding probability. The PDB file of the receptor protein was downloaded from the RCSB PDB website, and relevant molecular docking experiments were performed.

[0047] 2. The docking receptor protein in this study was 2W96. Verified by the RCSB PDB, this structure is the crystal structure of the human CDK4-CycD1 (cyclin-dependent kinase 4 - cyclin D1) complex, a classic target for anti-tumor drugs. This complex primarily regulates the G1 / S phase transition of the cell cycle; abnormal activation of this pathway is a core factor in the occurrence and proliferation of various tumors and is currently a popular research target for anti-tumor targeted drugs.

[0048] 3. Ligand small molecules: SMILES: O=C (N1OC) N2C3=CC=CC=C3C=C2C14CC (N (CC5=CC=CC=C5) C4=O)=O. The molecule contains heterocyclic, carbonyl, benzyl, and fused-ring aromatic structures, and belongs to typical drug-like small molecules.

[0049] 4. Docking Tools and Scoring Rules: The CB-Dock2 platform is used, equipped with the AutoDock Vina scoring system. The Vina Score represents the binding free energy, with the unit being kcal / mol. The more negative the value, the stronger the affinity between the small molecule and the protein. The industry-standard judgment is: a score ≤-5.0 kcal / mol is considered an effective binding, and ≤-7.0 kcal / mol is considered a strong binding.

[0050] 5. Docking mode: Blind docking of the entire protein, without pre-setting active sites, the algorithm automatically identifies all potential binding pockets on the protein surface.

[0051] (II) Docking Results Using the CDK4-CycD1 complex with PDB ID 2W96 as the target, a full-pocket blind docking was performed to detect the first 5 potential binding pockets. The relevant information of the first 5 binding pockets is shown in Table 1 below.

[0052] Table 1. Docking results of the first 5 potential binding pockets with the small molecule.

[0053] Molecular docking diagrams and docking effects of spirocyclic molecules are shown in [reference needed]. Figure 9 The C1 pocket (cavity volume 3794 ų, center coordinates (-1.964, -1.306, 83.809), size 35×33×30 ų) is the core pocket for ATP binding and the optimal binding site for small molecules. Target small molecules are stably embedded in the C1 pocket through a multi-hydrogen bond network (with residues such as K40, E144, and N145), hydrophobic / π-π stacking (with residues such as V96, I147, and W150), and electrostatic assistance (with residues such as R101) (Table 2).

[0054] Table 2 Key interacting residues and their interaction types in the core binding pocket (C1)

[0055] The free energy of small molecule binding to target protein was predicted to be -9.0 kcal / mol using AutoDock Vina. The molecule has a compact conformation and high specificity, which provides a structural basis for it to become a potential CDK4 inhibitor (Table 3).

[0056] Table 3. Core Scoring and Conclusion of Docking Effectiveness

[0057] The above analysis shows that the small molecule is a drug-like compound containing diimide and benzyl heterocycle, most likely a CDK, PARP, or HDAC inhibitor. Its main pharmacological effects are anti-tumor, anti-inflammatory, immunomodulatory, and central nervous system regulation. Potential indications include various solid tumors, autoimmune diseases, and neuropsychiatric disorders. The overall skeleton is safe and has no obvious toxic groups, making it suitable for further drug development.

[0058] II. Analysis of Drug Biological Activity (1) In vitro enzyme level IC 50 2.30 μM (IC) 50 The half-maximal inhibitory concentration (MCI) refers to the drug concentration required to inhibit 50% of a compound's biological activity. The lower the MCI, the stronger the inhibitory effect. Experimental system: Recombinant human CDK4-Cyclin D1 kinase complex was used to screen for in vitro kinase activity using Rb protein fragments as substrates. The small molecules to be tested were prepared into a series of gradient concentration solutions with dimethyl sulfoxide (DMSO). Blank control group, negative control group and positive drug control group were set up.

[0059] IC 50 The results at 2.30 μM showed that this small molecule exhibited moderate to high inhibitory activity against the target enzyme, stably binding to the CDK4 catalytic pocket and blocking kinase function. The target binding effect was clear, and it was not a weakly active compound.

[0060] (2) Cellular level IC 50 6.80 μM Experimental cell line: human breast cancer cells MCF-7 (HR positive, CDK4 pathway highly dependent classic tumor cells, the mainstream evaluation model for CDK4 / 6 inhibitors). After culture, the cell proliferation was detected by CCK-8 cell proliferation assay reagent, and the absorbance of each well was measured by microplate reader. The cell proliferation inhibition rate corresponding to different drug concentrations was calculated.

[0061] IC 50 6.80 μM, cellular level IC50 50 The levels were slightly higher than in vitro enzyme levels, which is normal and mainly influenced by factors such as cell membrane penetration, intracellular metabolism, and non-specific protein binding. The compound maintained a stable inhibitory effect on tumor cell proliferation within the cells, and the cell activity results were reliable.

[0062] The in vitro enzyme activity reaches the micromolar level (μM), which is considered moderate to high activity, and it has the potential to be further optimized into a nanomolar level lead compound. In vitro evaluation has been completed, demonstrating clear target binding and stable cellular inhibitory effects. It is a high-quality CDK4-targeted antitumor lead compound. Further studies can include kinase profile screening, cytotoxicity evaluation, physicochemical property analysis, and in vivo animal experiments to further validate its druggability and safety characteristics. It is suitable for subsequent structural optimization and in-depth pharmacological research.

[0063] Application Example 2: N -(1-Benzyl-2,5-dioxo-2,5-dihydro-1 H -pyrrole-3-yl)-1 H Application of -indole-1-carboxamide (compound of formula <II>) I. Molecular docking and result analysis (a) Connection method 1. For small molecules, SwissTargetPrediction was used to predict the Top 10 targets and identify the receptor protein with the highest binding probability. The PDB file of the receptor protein was downloaded from the RCSB PDB website, and relevant molecular docking experiments were performed.

[0064] 2. Receptor protein 7RA5, verified by RCSB PDB, is a co-crystal structure of human CDK2 (Cyclin-dependent kinase 2) and its inhibitor. It is a classic target of anti-tumor drugs and mainly regulates the G1 / S phase transition of the cell cycle. Abnormally high expression can drive the unlimited proliferation of tumor cells.

[0065] 3. Ligand small molecules: SMILES: O=C (NC1=CC (N (CC2=CC=CC=C2) C1=O)=O) N (C=C3)C4=C3C=CC=C4, containing diimide, benzyl and heterocyclic structures, belonging to drug-like small molecule compounds.

[0066] 4. Docking tools and scoring rules: CB-Dock2 is equipped with the AutoDock Vina scoring system. The Vina Score is the binding free energy (kcal / mol). The more negative the value, the stronger the binding affinity. The industry-standard judgment is: a score ≤ -5.0 kcal / mol indicates that the small molecule has an effective binding ability with the protein, and -7.0 and above indicates a strong binding.

[0067] 5. Docking mode: Blind docking of the entire protein is adopted, with no preset active sites. The algorithm autonomously identifies potential binding pockets.

[0068] (II) Docking Results Using human CDK2 (Cyclin-dependent kinase 2) with PDB ID 7RA5 as the target, a full-pocket blind docking was performed to detect the top five potential binding pockets. Pockets C1 and C5 had the best binding scores (-7.6 kcal / mol), indicating they are the major binding sites for this small molecule; C2, C3, and C4 were secondary binding pockets. The relevant information for the five binding pockets is shown in Table 4 below.

[0069] Table 4. Docking results of the first 5 potential binding pockets with the small molecule.

[0070] C1 is the core active pocket for ATP binding of the CDK2 protein, a key site for its pharmacological action. This pocket has a volume of 1711 ų, with a central coordinate of (28.929, 36.347, -45.752), and a docking box size of 23 × 23 × 23 ų, providing ample space to accommodate the urea group, polycyclic aromatic core, and all other structural components of this small molecule. The largest hydrophobic cavity is the typical kinase hinge region active pocket of CDK2. The urea group (-NC (=O) N-) of the small molecule forms multiple hydrogen bonds with conserved amino acids in the CDK2 hinge region (such as Gln, Leu, and Phe). The large aromatic rings on both sides of the molecule are embedded in the hydrophobic region of the pocket, forming π-π stacking and hydrophobic interactions, occupying the ATP binding site and competitively blocking ATP binding to CDK2. This is the classic mode of action for kinase inhibitors and also the main binding site for natural inhibitors.

[0071] Table 5. Key interacting residues and their interaction types in the core binding pocket (C1)

[0072] The free energy of small molecule binding to target protein was predicted to be -7.6 kcal / mol using AutoDock Vina, indicating that the small molecule can bind to the ATP core active pocket (C1) of CDK2, competitively inhibit CDK2 kinase activity, and block cell cycle signal transduction (Table 6).

[0073] Table 6. Core Scoring and Conclusion of Docking Effectiveness

[0074] The above analysis shows that the target of this small molecule compound is CDK2 (Cyclin-dependent kinase 2), which is a candidate inhibitor of cell cycle kinase. Its main pharmacological effects are to inhibit tumor cell proliferation, arrest the cell cycle, and induce tumor cell apoptosis. Potential indications cover various types of malignant tumors with active proliferation. The protein sequence has four chain breaks, but these do not affect the core binding region. The small molecule can bind stably in the active pocket, and the overall binding characteristics are good with no obvious structural conflicts. It has the potential to further conduct in vitro activity verification and drug development optimization.

[0075] II. Analysis of Drug Biological Activity (1) In vitro enzyme level IC 50 18.62 μM (IC) 50 This refers to the half-maximal inhibitory concentration (MCC): the concentration of a drug required to inhibit 50% of its biological activity. The lower the value, the stronger the inhibitory effect of the compound. Experimental system: Recombinant human CDK2 (Cyclin-dependent kinase 2) kinase protein was used to conduct in vitro kinase activity screening experiments. The small molecules to be tested were prepared into a series of gradient concentration solutions with dimethyl sulfoxide (DMSO). Blank control group, negative control group and positive drug control group were set up.

[0076] IC 50 The result of 18.62 μM indicates that this small molecule can bind to the CDK2 ATP catalytic pocket and exhibits moderate to low inhibitory activity against the target enzyme. It can only partially block the catalytic function of CDK2 kinase, and its target binding ability is average, resulting in limited inhibitory effect.

[0077] (2) Cellular level IC 50 42.35 μM Cell selection and culture: Human cervical cancer HeLa cells (CDK2 pathway-dependent tumor cells, the mainstream evaluation model for CDK2 inhibitors) were used. Cell viability was detected using the CCK-8 assay, and the cell proliferation inhibition rate at different drug concentrations was calculated. The dose-response curve was fitted using software to calculate the cell-level IC50. 50 value.

[0078] IC 50 42.35 μM, cellular level IC50 50 The levels were significantly higher than those of in vitro enzymes, with a large range of differences. Due to factors such as cell membrane penetration, intracellular metabolism, and non-specific binding, the inhibitory effect of the compound in the tumor cell microenvironment was further weakened, resulting in a weaker overall cell inhibitory effect.

[0079] The compound exhibits both in vitro enzyme activity and cellular activity in the micromolar range, but its concentration values ​​are relatively high, resulting in an overall assessment of moderate to low activity. While the small molecule can bind to CDK2 and produce an inhibitory effect, further research should prioritize molecular structure optimization and structure-activity relationship studies, modifying functional groups to enhance target binding ability and cell penetration. Simultaneously, supplementary experiments such as kinase spectrum screening, normal cell toxicity evaluation, and physicochemical property analysis are needed. This compound currently possesses only drug development potential and basic research value.

Claims

1. Two types of indole derivatives, characterized in that, Compounds having the structure shown in formula <Ⅰ> or formula <Ⅱ>: ； 。 2. The method for synthesizing the two types of indole derivatives according to claim 1, characterized in that, Therefore N -Methoxy-1 H -Indole-1-carboxamide as the starting material, added N Benzylmaleimide, a solid base and a metal catalyst were added, and then the appropriate reaction solvent was added. The reaction temperature and reaction time were controlled. After the reaction was completed by TLC monitoring, the reaction was quenched with saturated sodium chloride solution. Then, the reaction was extracted, filtered, dried, filtered with diatomaceous earth and recrystallized to obtain the above two types of indole derivatives.

3. The synthesis method according to claim 2, characterized in that, The solid alkali is cesium carbonate, and the metal catalyst is dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer.

4. The synthesis method according to claim 2 or 3, characterized in that, The reaction solvent for the compound shown in Formula is trifluoroethanol, and the reaction solvent for the compound shown in Formula <II> is dichloromethane.

5. The synthesis method according to claim 2 or 3, characterized in that, The N -Methoxy-1 H -Indole-1-formamide and N The molar ratio of β-benzylmaleimide is 1:0.8 to 2.5, preferably 1:1.

2.

6. The synthesis method according to claim 2 or 3, characterized in that, The solid alkali and N -Methoxy-1 H The molar ratio of indole-1-carboxamide is 1:0.5 to 5.0, preferably 1:

2.

7. The synthesis method according to claim 2 or 3, characterized in that, The reaction temperature is 20-25℃; the reaction time is 12 h.

8. The method according to claim 2 or 3, characterized in that, The amount of the metal catalyst used is N -Methoxy-1 H The amount of indole-1-carboxamide added is 0.5-10% by molar mass, preferably 1.0%; the amount of the reaction solvent added is... N -Methoxy-1 H -10 times the weight of indole-1-carboxamide.

9. The synthesis method according to claim 2 or 3, characterized in that, The solvent used for recrystallization is a mixture of ethyl acetate and petroleum ether, with a volume ratio of ethyl acetate to petroleum ether of 1:2 to 10, preferably 1:

5.

10. The application of the two types of indole derivatives synthesized by the method of claims 1 or 2-9 in the preparation of chemical raw materials, pharmaceutical intermediates and related end products.

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