A method for designing compounds based on v-atpase enzyme a subunit, vanillin derivatives and applications

Abstract

The application discloses a compound design method based on V-ATPase enzyme A subunit, a vanillin derivative and application, and is the first time to carry out receptor structure-based drug design on the allosteric site of vacuolar-type H+ ATPases (V-ATPases) enzyme A subunit, and a lead compound with V-ATPase enzyme inhibitory activity and insecticidal activity containing a vanillin fragment is screened, and on this basis, a preparation method of the vanillin derivative and application of the vanillin derivative as V-ATPase enzyme inhibition and in insecticidal aspects are further provided. The vanillin derivative of the application shows good V-ATPase enzyme inhibitory activity, V-ATPase enzyme A subunit binding capacity and insecticidal activity.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical technology and pesticide technology, and relates to a compound design method based on the V-ATPase enzyme A subunit, vanillin derivatives and their applications. Background Technology

[0002] In recent years, due to the irrational application of many pesticides, agricultural pests have developed significantly increased resistance to existing commercial pesticides, resulting in a marked decrease in their control efficacy. Therefore, it is particularly important to develop highly efficient and green pesticides with novel target sites, unique mechanisms of action, and no cross-resistance with existing pesticides.

[0003] bubbly type H + V-ATPase, also known as vesicular adenosine triphosphatase (V-ATPase), is a large membrane-embedded enzyme complex that uses the rotational motion generated by ATP hydrolysis to pump hydrogen ions across the membrane. V-ATPase is a major proton pump responsible for acidification and maintaining the pH of cell compartments. V-ATPase is a highly conserved multi-subunit gyrase, widely distributed in all eukaryotes, playing an irreplaceable role in physiological processes as a proton pump in both membrane and intramembrane systems. In the medical field, V-ATPase dysfunction has been reported to be associated with various diseases, such as cancer, osteoporosis, male infertility, and renal acidosis (ACS Chem. Biol. 2022, 17(3), 619-628). In the pesticide field, the plant insecticide *Cephalotaxus fortunei* may bind to certain sites on the H subunit of V-ATPase, inhibiting holoenzyme assembly and thus affecting enzyme activity. Insects are unable to perform normal functions, leading to death (Toxins 2016, 8(3), 79). Therefore, V-ATPase can serve as a potential target for novel bactericides and insecticides. Summary of the Invention

[0004] This invention provides a compound design method based on the V-ATPase enzyme A subunit, vanillin derivatives, and their applications. It is the first invention to target the allosteric site of the V-ATPase enzyme A subunit using receptor-based drug design, screening for lead compounds containing vanillin fragments with both V-ATPase enzyme inhibitory and insecticidal activities. Furthermore, it provides a method for preparing vanillin derivatives and their applications as V-ATPase enzyme inhibitors and in insecticidal applications. The vanillin derivatives of this patent exhibit excellent V-ATPase enzyme inhibitory activity, V-ATPase enzyme A subunit binding capacity, and insecticidal activity.

[0005] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0006] A compound design method based on the V-ATPase A subunit, which designs compounds based on receptor structures targeting the allosteric site of the V-ATPase A subunit, includes the following steps:

[0007] Step 1, Preparation of virtual screening ligands: Screening of small molecule compound libraries according to drug-like and pesticide-like rules and 2D-to-3D structural transformation;

[0008] Step 2, Preparation of virtual screening receptors: Construct the 3D structure of V-ATPase enzyme A subunit protein using homology modeling method, and optimize the structure using molecular dynamics method;

[0009] Step 3, Virtual screening based on molecular docking: Targeting the allosteric site of the V-ATPase A subunit, using the 3D structure of the V-ATPase A subunit protein obtained in Step 2, and considering receptor flexibility, virtual screening is performed using molecular docking.

[0010] Step 4: Evaluation of the activity of screened molecules: including evaluation of V-ATPase inhibitory activity and insecticidal activity.

[0011] A compound based on the V-ATPase enzyme A subunit, said compound being obtained using the compound design method based on the V-ATPase enzyme A subunit described in this invention, specifically comprising at least one of the following molecular structures;

[0012]

[0013]

[0014] The compound based on the V-ATPase A subunit described in this invention is used for the preparation of V-ATPase inhibitors.

[0015] The compound based on the V-ATPase A subunit described in this invention is used in the preparation of insecticides.

[0016] A vanillin derivative, wherein the vanillin derivative is designed and synthesized using the compound AN-465 / 41851572 of the present invention as a lead compound, and the vanillin derivative has a compound or a pharmaceutically acceptable salt thereof having the structure shown in the following general formula.

[0017]

[0018] Among them, R 1 It is selected from at least one of hydrogen, halogen, cyano, nitro, trifluoromethyl and trifluoromethoxy;

[0019] R 2Selected from N-substituted aminomethyl, N-substituted carbamoyl, O-substituted oxymethyl, O-substituted oxycarbonyl, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, substituted or unsubstituted benzyl, nitrogen-containing heterocyclic substituted methyl containing 2-10 carbon atoms, oxygen-containing heterocyclic substituted methyl containing 2-10 carbon atoms, and sulfur-containing heterocyclic substituted methyl containing 2-10 carbon atoms;

[0020] The substituents in the aforementioned N-substituted aminomethyl, N-substituted carbamoyl, O-substituted oxymethyl, O-substituted oxycarbonyl, and substituted benzyl groups are each independently selected from at least one of hydrogen, halogen, cyano, nitro, trifluoromethyl, trifluoromethoxy, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, nitrogen-containing heterocycles containing 2-10 carbon atoms, oxygen-containing heterocycles containing 2-10 carbon atoms, and sulfur-containing heterocycles containing 2-10 carbon atoms;

[0021] R 3 It is selected from at least one of hydrogen, halogen, cyano, nitro, trifluoromethyl, trifluoromethoxy, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, nitrogen-containing heterocycles containing 2-10 carbon atoms, oxygen-containing heterocycles containing 2-10 carbon atoms, and sulfur-containing heterocycles containing 2-10 carbon atoms.

[0022] Optionally, the R 1 It is chlorine;

[0023] R 2 It is selected from at least one of N-anilinomethyl, N-((4-ethynylphenyl)amino)methyl, N-((4-methoxyphenyl)amino)methyl, N-(isobutylamino)methyl, N-(sec-butylamino)methyl, N-(cyclopentyl)amino)methyl, N-((4-fluorophenyl)amino)methyl, N-((2,4-difluorophenyl)amino)methyl, N-((benzothiazol-2-yl)amino)methyl, 4-(pentylamino)methyl, N,N-((dimethyl)amino)formyl, N-((p-tolyl)amino)formyl, ethoxycarbonyl, butoxycarbonyl, ((but-3-yn-1-yl)oxy)carbonyl, ((2,3,5,6-tetrafluorophenyl)oxy)carbonyl, (o-tolyl)carbonyl, cyclohexyloxycarbonyl, and ((4-methoxybenzyl)oxy)carbonyl.

[0024] Alternatively, the compounds listed in the table below can be used;

[0025]

[0026]

[0027]

[0028] The application of any of the vanillin derivatives described in this invention in the preparation of V-ATPase inhibitors.

[0029] The vanillin derivative described in any one of the present invention is used in the preparation of insecticides.

[0030] Optionally, the insecticide is used to control at least one of aphids and armyworms.

[0031] The advantages of this invention are:

[0032] This invention represents the first drug design based on receptor structure targeting the allosteric site of the A subunit of vacuolar-type H+-ATPases (V-ATPases). Lead compounds containing vanillin fragments with both V-ATPase inhibitory and insecticidal activity were screened. Furthermore, methods for preparing vanillin derivatives and their applications as V-ATPase inhibitors and insecticides are provided. The vanillin derivatives of this invention exhibit excellent V-ATPase inhibitory activity, V-ATPase A subunit binding capacity, and insecticidal activity. Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 The V-ATPase inhibitory activity and insecticidal activity of 31 small molecule compounds obtained by virtual screening in this invention are shown. Note: The inhibitory effect of the 200 μM compound on V-ATPase and the lethality of the 15 mg / mL compound on armyworms are shown. BA1, bafimycin A1 (3 μM); PSP, coumarin P (200 μM anti-V-ATPase, 1 mg / mL anti-armyworm). Detailed Implementation

[0035] The following examples can provide a better understanding of the invention, but they do not limit the scope of the invention.

[0036] The allosteric site of the V-ATPase A subunit of the present invention, taking the protein sequence of the V-ATPase A subunit of *Armworm of Orientia* (NCBI accession number: KAJ8720449.1) as an example, is composed of amino acid residues such as Leu43, Arg45, Val52, Lys85, Leu87, Ser88, Glu90, Ile103, Val125, Ser127, Arg171, Tyr202, Ser204, Leu206, Gln207, Val208, Trp209, and Pro210.

[0037] This invention relates to receptor-based drug design targeting the allosteric site of the V-ATPase A subunit, comprising the following steps:

[0038] Step 1: Preparation of virtual screening ligands;

[0039] Step 2: Preparation of virtual recipient screening;

[0040] Step 3: Virtual screening based on molecular docking

[0041] Step 4: Activity evaluation of screened molecules

[0042] Furthermore, the key to step 1, the preparation of virtual screening ligands, lies in screening the small molecule compound library according to drug-like and pesticide-like rules and performing 2D-to-3D structural transformation.

[0043] Step 2, the preparation of virtual screening receptors, is key to constructing the 3D structure of the V-ATPase A subunit protein of *Armoria spp.* using homology modeling and optimizing the structure using molecular dynamics.

[0044] Step 3, virtual screening based on molecular docking, is key to targeting the allosteric site of the V-ATPase A subunit. Using the V-ATPase A subunit structure obtained in step 2, and considering receptor flexibility, virtual screening is performed using molecular docking.

[0045] The structures of the 31 molecules screened are as follows:

[0046]

[0047]

[0048] Step 4: Evaluation of the activity of the screened molecules, mainly including evaluation of V-ATPase inhibitory activity and insecticidal activity.

[0049] This invention uses the virtual screening compound AN-465 / 41851572 as a lead, and includes, but is not limited to, preparing vanillin derivatives through a three-step reaction mainly consisting of aromatic halogenation, Williamson etherification, and imine condensation, using vanillin as a raw material.

[0050] The vanillin derivative is a compound having the structure shown in the following general formula:

[0051]

[0052] Among them, R 1 It is selected from at least one of hydrogen, halogen, cyano, nitro, trifluoromethyl and trifluoromethoxy;

[0053] R 2 Selected from N-substituted aminomethyl, N-substituted carbamoyl, O-substituted oxymethyl, O-substituted oxycarbonyl, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, substituted or unsubstituted benzyl, nitrogen-containing heterocyclic substituted methyl containing 2-10 carbon atoms, oxygen-containing heterocyclic substituted methyl containing 2-10 carbon atoms, and sulfur-containing heterocyclic substituted methyl containing 2-10 carbon atoms;

[0054] The substituents in the aforementioned N-substituted aminomethyl, N-substituted carbamoyl, O-substituted oxymethyl, O-substituted oxycarbonyl, and substituted benzyl groups are each independently selected from one or more of the following: hydrogen, halogen, cyano, nitro, trifluoromethyl, trifluoromethoxy, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, nitrogen-containing heterocycles containing 2-10 carbon atoms, oxygen-containing heterocycles containing 2-10 carbon atoms, and sulfur-containing heterocycles containing 2-10 carbon atoms.

[0055] R 3 It is selected from one or more of hydrogen, halogen, cyano, nitro, trifluoromethyl, trifluoromethoxy, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, nitrogen-containing heterocycles containing 2-10 carbon atoms, oxygen-containing heterocycles containing 2-10 carbon atoms, and sulfur-containing heterocycles containing 2-10 carbon atoms.

[0056] Preferably, R 1 Selected from chlorine;

[0057] R 2Selected from N-anilinemethyl, N-((4-ethynylphenyl)amino)methyl, N-((4-methoxyphenyl)amino)methyl, N-(isobutylamino)methyl, N-(sec-butylamino)methyl, N-(cyclopentyl)amino)methyl, N-((4-fluorophenyl)amino)methyl, N-((2,4-difluorophenyl)amino)methyl, N-((benzothiazol-2-yl)amino)methyl, 4-(pentylamino)methyl, N,N-((dimethyl)amino)formyl, N-((p-tolyl)amino)formyl, ethoxycarbonyl, butoxycarbonyl, ((but-3-yn-1-yl)oxy)carbonyl, ((2,3,5,6-tetrafluorophenyl)oxy)carbonyl, (o-tolyl)carbonyl, cyclohexyloxycarbonyl, ((4-methoxybenzyl)oxy)carbonyl

[0058] The compound is one of the compounds shown in the following formula:

[0059] Table 1. Vanillin derivatives shown in general formulas

[0060]

[0061]

[0062]

[0063] The compounds of the general formula of this invention have V-ATPase inhibitory activity and can be used as V-ATPase inhibitors.

[0064] The compounds of the general formula of this invention have activity against armyworms and cereal aphids.

[0065] The compounds of the general formula of this invention can be used directly as insecticides, or they can be combined with agriculturally acceptable carriers and other insecticides such as pyridaben and bromuconazole to form compound insecticides. Some of these compositions exhibit synergistic effects, while others exhibit additive effects.

[0066] Example 1: Drug design based on receptor structure targeting the allosteric site of the V-ATPase A subunit.

[0067] Drug design based on receptor structure targeting the allosteric site of the V-ATPase A subunit includes the following steps:

[0068] Step 1: Preparation of virtual screening ligands;

[0069] Step 2: Preparation of virtual recipient screening;

[0070] Step 3: Virtual screening based on molecular docking;

[0071] Step 4: Activity evaluation of screened molecules;

[0072] Step 1. Preparation of virtually screened ligands, the key of which lies in screening the small-molecule compound library with drug-likeness rules and pesticide-likeness rules and performing 2D-to-3D structure conversion. The specific method is: subjecting the SPECS compound library to drug-likeness rules (-4 < log P < 4, 0 < MW < 500) and pesticide-likeness rules (number of hydrogen bond acceptors (HBA) ≤ 6, number of hydrogen bond donors (HBD) ≤ 2, number of rotatable bonds (ROB) ≤ 9); then converting the small molecules into 3D structures using the Ligand Structure Preparation method in SYBYL X-2.1 software, and performing energy minimization on the small molecules with the Tripos force field.

[0073] Step 2. Preparation of virtually screened receptors, the key of which lies in constructing the 3D structure of the Mythimna separata V-ATPase subunit A protein by a homology modeling method, and performing structure optimization using a molecular dynamics method. The specific method is: using the crystal structure of bovine V-ATPase subunit A (PDB ID: 7KHR) as a template, constructing the 3D structure of the Mythimna separata V-ATPase subunit A protein (NCBI accession number: KAJ8720449.1) sequence via SWISS-MODEL, designated as MsVatpA; then using AMBER18 software, the AMBER99bsc1 force field and the SPC water model to construct the simulation system; in the simulation system, the target protein is placed at the center of the water box, and the minimum distance from the surface of the water box is To be consistent with the solution environment, metal cations and corresponding anions are added to keep the solution environment neutral; the simulation process adopts periodic boundary conditions, electrostatic and van der Waals interactions are calculated by the PME and Cut-off methods respectively, and the cut-off distances are both All chemical bonds are constrained by the LINCS algorithm; wherein the integration time step is 1 to 3 fs; the specific process is: first, using the steepest descent algorithm to perform energy minimization on the simulation system; then adopting the v-rescale thermostat to control the equilibrium temperature of the system, and the berendsen pressure coupling to equilibrate the pressure; finally, performing molecular dynamics simulation on the system for a duration of no less than 500 ps in the md integrator; after the dynamics simulation, extracting one average structure at an interval of 500 ps from the last 2500 ps of the equilibrium system, which are respectively designated as: MsVatpA-MD1, MsVatpA-MD2, MsVatpA-MD3, MsVatpA-MD4, and MsVatpA-MD5; performing energy minimization based on the Tripos force field on the above 5 structures.

[0074] Step 3: The key to virtual screening based on molecular docking is to target the allosteric site of the V-ATPase enzyme A subunit. Using the V-ATPase enzyme A subunit structure obtained in Step 2, and considering the receptor flexibility, virtual screening is performed using molecular docking. The specific method is as follows: Using the five structures of the V-ATPase enzyme A subunit obtained through molecular dynamics optimization in step 2—MsVatpA-MD1, MsVatpA-MD2, MsVatpA-MD3, MsVatpA-MD4, and MsVatpA-MD5—as acceptors, the allosteric site of the A subunit is defined as the docking pocket. Small molecules obtained in step 1 are used as docking screening targets, and surfdock docking screening is performed. In surfdock docking, the number of additional initial conformations for each molecule is set to 4, and other parameters are left as default. Next, molecules with a docking score (total score) greater than 8.0 based on each acceptor structure are unioned. The molecules in the unioned set are then further docked using the MsVatpA structure as the acceptor and the allosteric site of the A subunit as the docking pocket, based on Autodock Vina. In Autodock Vina docking, the docking box size is 19.5 x 18.0 x 18.0A, global search delimitation is set to 20, 12 CPUs are used, the maximum number of binding modes is 20, the maximum energy difference between modes is 4 kcal / mol, and the scoring function weights and terms are set to default values; molecules with docking scores (binding energies) below -6.0 kcal / mol are selected as a new molecular set; from the aforementioned molecular set, 31 molecules are selected based on the principles of high structural variability, low synthesis difficulty, and availability.

[0075] The structures of the 31 selected molecules are as follows:

[0076]

[0077]

[0078] Step 4: Activity evaluation of screened molecules, mainly including V-ATPase inhibitory activity evaluation and insecticidal activity evaluation. Evaluation results are shown in [link to relevant documentation]. Figure 1 .

[0079] Figure 1 The results firstly demonstrate that the 31 screened compounds exhibit significant V-ATPase inhibitory activity. In particular, compounds AN-465 / 41851572, AG-690 / 12135130, AJ-292 / 14556185, and AG-205 / 12424041 demonstrate even superior V-ATPase inhibitory activity. Further calculations of the IC50 values... 50The concentrations were 53.04, 99.86, 92.12, and 57.56 μM, respectively; secondly, all 31 screened compounds showed significant insecticidal activity, with compound AN-465 / 41851572 exhibiting the best insecticidal activity.

[0080] Example 2: Synthesis of vanillin derivatives

[0081] This invention uses the virtual screening compound AN-465 / 41851572 as a lead, and includes, but is not limited to, preparing vanillin derivatives through a three-step reaction mainly consisting of aromatic halogenation, Williamson etherification, and imine condensation, using vanillin as a raw material.

[0082]

[0083] Synthetic routes for vanillin derivatives IA-1-6, IB-1-6, IIB-1-2, and IIC-1-7.

[0084] Synthesis of intermediates:

[0085] Preparation of intermediate 1: Vanillin (76.1 mg, 0.5 mmol) and NCS (79.8 mg, 0.6 mmol) were dissolved in 2 mL of chloroform. Dimethyl sulfoxide (7 μL, 0.1 mmol) was added at room temperature (25 °C) and the mixture was stirred for 12 h. After the reaction was completed, the mixture was concentrated under vacuum and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:1) to obtain intermediate compound 1 (84.6 mg, 91%).

[0086] Preparation of intermediates 2A and 2B: Compound 1 (1 mmol) or vanillin (1 mmol) was added to a 100 mL dry round-bottom flask, followed by anhydrous potassium carbonate (1.5 mmol), which was then dissolved in 20 mL acetonitrile. Benzyl bromide (1.1 mmol) was then added, and the mixture was heated under reflux for 6 h. The reaction was then monitored by TLC. After the reaction was complete and cooled to room temperature, the inorganic powder was filtered off, the mixture was concentrated under reduced pressure, extracted with water and dichloromethane, washed with brine, dried over anhydrous sodium sulfate, and then concentrated. Compounds 2A and 2B were separated by rapid column chromatography.

[0087] Table 2. Yields, properties, and melting points of intermediates 2A and 2B

[0088] 2A 87 Yellow solid 75.8-77.6 2B 86 Yellow solid 76.2-78.3

[0089] Synthesis of intermediate 2C: The synthesis method is the same as that for intermediate 2A.

[0090] Synthesis of Intermediate 3: In a 500 mL reaction flask, aldehyde 2B (20.66 mmol), NaH2PO4 (6.20 mmol), and H2O2 (35%, 2.3 mL, 21.69 mmol) were added, followed by 120 mL of MeCN / H2O (5:1, v / v). The mixture was stirred, and then 30 mL of an aqueous solution of NaOClO (28.92 mmol) was added dropwise. The mixture was cooled with water and maintained at 10 °C. The mixture was stirred at room temperature for 90 min, and then Na2SO3 (3.2 mmol) was added. The mixture was stirred for another 5 min to decompose excess H2O2. The mixture was diluted with an aqueous solution of NaCl and extracted with ethyl acetate (3 × 30 mL). The organic extract was separated, washed with an aqueous solution of NaCl (×2), and extracted with an aqueous solution of NaHCO3 (×3). The basic extract was separated, acidified (concentrated HCl), and extracted with ethyl acetate (×2). The organic extract was dried over Na2SO4 and concentrated, yielding a white solid after crystallization in hexane.

[0091] Synthesis of target compound IA-1-6

[0092] Synthesis of N-(4-(benzyloxy)-3-methoxybenzyl)aniline (IA-1).

[0093] Compound 2A was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Aniline (1.1 mmol) was then added, followed by sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow solid; yield 82%; melting point 83.2–85.1 °C. 1 H NMR(400MHz,Chloroform-d)δ7.46(d,J=7.5Hz,2H),7.41-7.35(m,2H),7.35-7.28(m,1H),7.24-7.15(m,2H),6.9 5(s,1H),6.86(s,2H),6.78-6.71(m,1H),6.66(d,J=8.0Hz,2H),5.16(s,2H),4.25(s,2H),3.89(d,J=1.2Hz,3H); 13C NMR(101MHz,Chloroform-d)δ150.00,148.30,147.53,137.35,132.67,129.37,128 .65,127.93,127.38,119.82,117.76,114.30,113.03,111.54,71.27,56.13,48.40.

[0094] Synthesis of N-(4-(benzyloxy)-3-methoxybenzyl)ethylamine (IA-2)

[0095] Compound 2A was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Then, 1.1 mmol of 4-ethynylaniline was added, followed by 1.4 mmol of sodium triacetoxyborohydride. After stirring for one minute, 1 mmol of glacial acetic acid was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow solid; yield 69%; melting point 68.5–70.1 °C. 1 H NMR(400MHz,Chloroform-d)δ7.39-7.28(m,5H),6.89-6.83(m,2H),6.80(dd,J=8.2,2.0Hz,1H) ,6.65-6.61(m,2H),6.58(d,J=8.7Hz,2H),5.13(s,2H),4.66(s,1H),4.27(s,2H),3.86(s,3H); 13 C NMR(101MHz,Chloroform-d)δ151.25,150.60,149.98,147.70,137.04,133.82,133.73, 130.93,128.62,127.96,127.35,119.66,114.46,112.45,111.21,71.13,56.07,47.36.

[0096] Synthesis of N-(4-(benzyloxy)-3-methoxybenzyl)-O-methylhydroxylamine (IA-3).

[0097] Compound 2A was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. O-methylhydroxylamine (1.1 mmol) was then added, followed by sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellowish-brown solid; yield 75%; melting point 63.2–65.4 °C. 1 H NMR(400MHz,Chloroform-d)δ7.47-7.41(m,2H),7.40-7.34(m,2H),7.34-7.27(m,1H),6.94(d,J=1.4Hz,1H),6 .84(d,J=1.0Hz,2H),6.82-6.76(m,2H),6.66-6.59(m,2H),5.15(s,2H),4.20(s,2H),3.88(s,3H),3.75(s,3H); 13 CNMR(101MHz,Chloroform-d)δ152.30,149.82,147.34,142.46,137.25,132.77,128.58 ,127.85,127.30,119.73,114.91,114.28,114.06,111.41,71.12,56.01,55.83,49.26.

[0098] Synthesis of N-(4-(benzyloxy)-3-methoxybenzyl)-2-methyl-1-propanamine (IA-4).

[0099] Compound 2A was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Isobutylamine (1.1 mmol) was then added, followed by sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow liquid; yield 77%. 1H NMR(400MHz,Chloroform-d)δ7.47-7.41(m,2H),7.40-7.33(m,2H),7.32-7.27(m,1H),6.95(d,J=1.9Hz,1H),6.86-6.75( m,2H),5.14(s,2H),3.90(s,3H),3.72(s,2H),2.44(d,J=6.8Hz,2H),1.79(hept,J=6.7Hz,1H),0.93(s,3H),0.92(s,3H); 13 CNMR(101MHz,Chloroform-d)δ149.71,147.16,137.37,133.70,128.57,127.8 2,127.32,120.22,113.97,111.94,71.16,57.37,56.03,53.79,28.26,20.77.

[0100] Synthesis of N-(4-(benzyloxy)-3-methoxybenzyl)but-2-amine (IA-5).

[0101] Compound 2A was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Then, sec-butylamine (1.1 mmol) was added, followed by sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow solid; yield 78%; melting point 68.5–70.0 °C. 1 H NMR(400MHz,Chloroform-d)δ7.43(d,J=7.0Hz,2H),7.35(dd,J=8.2,6.5Hz,2H ),7.32-7.27(m,1H),6.92(d,J=1.9Hz,1H),6.85-6.75(m,2H),5.14(s,2H),3.9 0(s,3H),3.80-3.65(m,2H),2.61(dq,J=12.6,6.0Hz,1H),1.60-1.47(m,1H),1 .37(dq,J=13.1,7.2Hz,1H),1.08(dd,J=6.3,1.1Hz,3H),0.90(t,J=7.4Hz,3H); 13C NMR(101MHz,Chloroform-d)δ149.72,147.12,137.36,133.98,128.55,127.81,1 27.31,120.24,114.07,112.04,71.15,56.04,54.00,51.17,29.45,19.74,10.35.

[0102] Synthesis of N-(4-(benzyloxy)-3-methoxybenzyl)cyclopentanamine (IA-6).

[0103] Compound 2A was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Cyclopentanylamine (1.1 mmol) was then added, followed by sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow liquid; yield 81%. 1 H NMR(400MHz,Chloroform-d)δ7.46-7.40(m,2H),7.39-7.32(m,2H),7.32-7.27( m,1H),6.91(d,J=1.9Hz,1H),6.85-6.75(m,2H),5.13(s,2H),3.90(s,3H),3.70( s,2H),3.11(p,J=6.8Hz,1H),1.93-1.80(m,2H),1.69(ddq,J=7.4,5.5,2.7Hz,2H ),1.54(dddd,J=11.8,9.9,6.2Hz,2H),1.38(dddd,J=14.1,12.4,6.6,1.8Hz,2H); 13 C NMR(101MHz,Chloroform-d)δ149.68,147.16,137.33,133.61,128.54,127.8 0,127.29,120.36,114.01,112.15,71.13,59.16,56.03,52.47,33.01,24.15.

[0104] Synthesis of target compounds IB-1 to IB-6

[0105] Synthesis of N-(4-(benzyloxy)-3-chloro-5-methoxybenzyl)aniline (IB-1).

[0106] Compound 2B was added to a 50 mL round-bottom flask, dissolved in 15 mL of dichloromethane, followed by the addition of aniline (1.1 mmol), then sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow solid; yield 90%; melting point 75.1–78.5 °C. 1 H NMR(400MHz,Chloroform-d)δ7.57-7.52(m,2H),7.42-7.31(m,3H),7.24-7.17(m,2H),7.01(d,J=1.9Hz,1H),6.86 (d,J=2.0Hz,1H),6.76(tt,J=7.3,1.1Hz,1H),6.64(dt,J=7.8,1.1Hz,2H),5.04(s,2H),4.26(s,2H),3.84(s,3H); 13 C NMR(101MHz,Chloroform-d)δ154.27,147.99,143.39,137.30,136.39,129.43,128 .79,128.57,128.43,128.20,120.66,118.03,113.08,110.10,75.00,56.25,48.08;

[0107] Synthesis of N-(4-(benzyloxy)-3-chloro-5-methoxybenzyl)-4-fluoroaniline (IB-2).

[0108] Compound 2B was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Then, 1.1 mmol of 4-fluoroaniline was added, followed by 1.4 mmol of sodium triacetoxyborohydride. After stirring for one minute, 1 mmol of glacial acetic acid was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellowish-brown solid; yield 85%; melting point 75.1–79.2 °C. 1 H NMR(400MHz,Chloroform-d)δ7.58-7.49(m,2H),7.44-7.29(m,3H),6.99(d,J=1.9H z,1H),6.93-6.80(m,3H),6.59-6.50(m,2H),5.03(s,2H),4.22(s,2H),3.84(s,3H); 13CNMR(101MHz,Chloroform-d)δ156.21(d,J=235.5Hz),154.33,144.30,143.48,137.30,136.14,128.86, 128.59,128.45,128.23,120.69,115.87(d,J=22.4Hz),113.94(d,J=7.4Hz),110.10,75.02,56.28,48.69

[0109] Synthesis of N-(4-(benzyloxy)-3-chloro-5-methoxybenzyl)-2,4-difluoroaniline (IB-3).

[0110] Compound 2B was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Then, 1.1 mmol of 2,4-difluoroaniline was added, followed by 1.4 mmol of sodium triacetoxyborohydride. After stirring for one minute, 1 mmol of glacial acetic acid was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow solid; yield 82%; melting point 74.5–75.1 °C. 1 H NMR(400MHz,Chloroform-d)δ7.56-7.49(m,2H),7.41-7.29(m,3H),6.99(d,J=1.9Hz,1H),6.89-6.76(m, 2H),6.76-6.68(m,1H),6.54(td,J=9.3,5.4Hz,1H),5.03(s,2H),4.26(s,2H),4.16(s,1H),3.84(s,3H); 13 C NMR(101MHz,Chloroform-d)δ157.07-152.69(m),154.39,150.95(dd,J=241.8,11.5Hz),143.56,137.26,135.68,132.94(d,J=11.6Hz),128.94,1 28.58,128.45,128.24,120.57,112.48(dd,J=8.8,4.4Hz),110.82(dd,J =21.6,3.8Hz),109.88,103.66(dd,J=26.7,22.7Hz),75.02,56.28,48.06

[0111] Synthesis of N-(4-(benzyloxy)-3-chloro-5-methoxybenzyl)benzothiazol-2-amine (IB-4).

[0112] Compound 2B was added to a 50 mL round-bottom flask, dissolved in 15 mL of dichloromethane, followed by the addition of benzothiazolamide (1.1 mmol), then sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a white solid; yield 80%; melting point 83.3–91.1 °C. 1 H NMR(400MHz,Chloroform-d)δ7.60(d,J=7.9Hz,1H),7.57-7.45(m,3H),7.41-7.28(m,4H),7.17-7.07(m ,1H),7.01(d,J=2.0Hz,1H),6.87(d,J=2.1Hz,1H),5.89(s,1H),5.02(s,2H),4.58(s,2H),3.82(s,3H); 13 Synthesis of N-(4-(benzyloxy)-3-chloro-5-methoxybenzyl)cyclopentanamine (IB-5) by C NMR (101 MHz, Chloroform-d) δ 167.13, 154.41, 152.32, 143.97, 137.18, 134.28, 130.68, 128.99, 128.61, 128.47, 128.28, 126.23, 122.07, 121.12, 121.04, 119.27, 110.42, 75.05, 56.31, 48.89.

[0113] Compound 2B was added to a 50 mL round-bottom flask and dissolved in 15 mL of dichloromethane. Cyclopentanylamine (1.1 mmol) was then added, followed by sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a yellow liquid; yield 80%. 1 H NMR(400MHz,Chloroform-d)δ7.54-7.47(m,2H),7.39-7.30(m,3H),6.96(dd,J=10.7,2.0Hz,2H),4.99(s,2H),3.85( s,3H),3.77(s,2H),3.17(p,J=6.9Hz,1H),1.89(qd,J=9.3,7.9,4.2Hz,2H),1.73(t,J=4.2Hz,2H),1.60-1.43(m,4H); 13C NMR(101MHz,Chloroform-d)δ154.20,143.84,137.26,128.62,128.55,128.4 4,128.21,123.31,122.27,111.76,74.96,58.78,56.30,51.25,31.91,24.20

[0114] Synthesis of N-(4-(benzyloxy)-3-chloro-5-methoxybenzyl)pent-1-amine (IB-6).

[0115] Compound 2B was added to a 50 mL round-bottom flask, dissolved in 15 mL of dichloromethane, followed by the addition of pentylamine (1.1 mmol), then sodium triacetoxyborohydride (1.4 mmol). After stirring for one minute, glacial acetic acid (1 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was detected by TLC. After completion, the reaction was quenched with 5% sodium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and separated by rapid column chromatography to obtain a white solid; yield 80%; melting point 51.2–52.6 °C. 1 H NMR(400MHz,Chloroform-d)δ7.56-7.48(m,2H),7.35(dt,J=13.7,6.8Hz,3H),6.95(s,1H),6.89(s,1H),5.01(s,2H),3. 86(s,3H),3.74(s,2H),2.63(t,J=7.4Hz,2H),1.55(t,J=7.3Hz,2H),1.32(dq,J=7.3,3.3Hz,4H),0.90(t,J=6.7Hz,3H); 13 CNMR(101MHz,Chloroform-d)δ154.14,143.41,137.31,135.99,128.56,128.49,128. 42,128.18,121.71,111.13,74.96,56.28,53.16,49.12,29.55,29.27,22.66,14.15.

[0116] Example 4. Synthesis of target compounds IIB-1 to IIB-2

[0117] Synthesis of 4-benzyloxy-3-chloro-5-methoxy-N,N-dimethylbenzamide (ⅠⅠB-1).

[0118] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, dimethylamine (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; the differential concentration was 75%; the melting point was 99.6–101.5 °C. 1 H NMR(400MHz,Chloroform-d)δ7.54-7.48(m,2H),7.40-7.29(m,3H),7.02(d,J=1. 8Hz,1H),6.92(d,J=1.9Hz,1H),5.05(s,2H),3.87(s,3H),3.04(d,J=31.2Hz,6H); 13 C NMR (101MHz, Chloroform-d) δ170.11,154.16,145.33,136.93,132.55,128.56,128.44,128.31,120.71,110.33,75.05,56.31,39.78,35.60.

[0119] Synthesis of 4-benzyloxy-3-chloro-5-methoxy-N-(p-tolyl)benzamide (ⅠⅠB-2).

[0120] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane. The mixture was stirred at room temperature for a few minutes, and then p-toluidine (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 86%; melting point 145.5–150.6 °C. 1 HNMR(400MHz,Chloroform-d)δ7.70(s,1H),7.50(td,J=6.8,6.3,1.9Hz,4H),7 .45-7.29(m,5H),7.18(d,J=8.2Hz,2H),5.12(s,2H),3.93(s,3H),2.35(s,3H); 13 C NMR(101MHz,Chloroform-d)δ164.93,154.37,147.11,136.76,135.18,134.66,131.45, 129.78,128.76,128.68,128.53,128.47,120.45,119.98,110.73,75.15,56.44,21.07.

[0121] Synthesis of target compounds IIC-1 to IIC-7

[0122] Synthesis of ethyl 4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-1).

[0123] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, ethanol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 86%; melting point 57.3–58.2 °C. 1 H NMR(400MHz,Chloroform-d)δ7.70(d,J=1.9Hz,1H),7.50(td,J=3.9,3.0,1.6Hz,3H),7. 42-7.29(m,3H),5.12(s,2H),4.37(q,J=7.1Hz,2H),3.92(s,3H),1.39(t,J=7.1Hz,3H); 13 C NMR (101MHz, Chloroform-d) δ165.44,153.76,148.11,136.82,128.69,128.59,128.47,128.39,126.65,123.81,112.02,75.08,61.49,56.37,14.44.

[0124] Synthesis of butyl 4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-2).

[0125] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, butanol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 86%; melting point 81.2–83.8 °C. 1 H NMR(400MHz,Chloroform-d)δ7.69(d,J=1.8Hz,1H),7.54-7.46(m,3H),7.41-7.29(m,3H),5.12(s,2H ), 4.31 (t, J = 6.7Hz, 2H), 3.92 (s, 3H), 1.80-1.68 (m, 2H), 1.47 (h, J = 7.4Hz, 2H), 0.99 (t, J = 7.4Hz, 3H); 13C NMR(101MHz,Chloroform-d)δ165.52,153.77,148.11,136.82,128.70,128.60,12 8.49,128.40,126.69,123.77,112.05,75.09,65.38,56.37,30.87,19.37,13.89.

[0126] Synthesis of butyl 3-yn-1-yl-4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-3).

[0127] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, propynyl alcohol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a yellow solid; yield 76%; melting point 78.4–80.5 °C. 1 H NMR(400MHz,Chloroform-d)δ7.71(d,J=2.1Hz,1H),7.54-7.47(m,3H),7.42-7.30(m,3H),5.13 (s,2H),4.42(t,J=6.8Hz,2H),3.92(s,3H),2.67(td,J=6.9,2.7Hz,2H),2.05(t,J=2.7Hz,1H); 13 Synthesis of 2,3,5,6-tetrafluorophenyl 4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-4) by C NMR (101MHz, Chloroform-d) δ 165.11, 153.77, 148.35, 136.76, 128.75, 128.57, 128.47, 128.40, 126.02, 123.95, 112.11, 79.95, 75.08, 70.27, 63.01, 56.36, 19.18

[0128] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, 2,3,5,6-phenol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 76%; melting point 131.7–134.3 °C. 1H NMR(400MHz,Chloroform-d)δ7.90(d,J=2.0Hz,1H),7.62(d,J=2.0Hz,1H),7.56-7.4 9(m,2H),7.43-7.32(m,3H),7.06(tt,J=10.0,7.0Hz,1H),5.20(s,2H),3.96(s,3H); 13 C NMR (101MHz, Chloroform-d) δ 161.52, 154.06, 149.72, 147.45 (d, J = 4.1Hz), 144.96, 142.08, 139. 58,136.56,129.27,128.63,128.58,125.22,123.00,112.84,103.60(t,J=22.8Hz),75.30,56.52.

[0129] Synthesis of o-tolyl-4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-5).

[0130] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, o-methylphenol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 88%; melting point 146.3–152.8 °C. 1 H NMR(400MHz,Chloroform-d)δ7.93(d,J=1.9Hz,1H),7.67(d,J=2.0Hz,1H),7.59-7.52(m,2H),7.44-7.34(m,3H),7.30( td,J=7.3,1.9Hz,2H),7.22(td,J=7.4,1.5Hz,1H),7.13(dd,J=7.8,1.4Hz,1H),5.19(s,2H),3.96(s,3H),2.25(s,3H); 13 C NMR(101MHz,Chloroform-d)δ163.70,153.93,149.49,148.79,136.72,131.35,130.34,128.95, 128.60,128.52,128.46,127.16,126.37,125.50,124.46,121.97,112.48,75.17,56.43,16.36.

[0131] Synthesis of cyclohexyl 4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-6).

[0132] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, cyclohexanol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 76%; melting point 81.8–82.1 °C. 1 H NMR(400MHz,Chloroform-d)δ7.70(d,J=1.9Hz,1H),7.51(dt,J=4.2,1.7Hz,3H),7.41-7.30(m,3H),5.12(s,2H),5.00(tt,J=8.7,3. 9Hz,1H),3.92(s,3H),2.03-1.88(m,2H),1.80(ddd,J=14.1,6.5,3.9Hz,2H),1.59(ddt,J=12.5,9.1,4.6Hz,3H),1.54-1.18(m,4H); 13 C NMR(101MHz,Chloroform-d)δ164.76,153.69,147.96,136.79,128.57,128.44,12 8.35,127.15,123.85,123.68,112.02,75.04,73.74,56.32,31.70,25.50,23.81.

[0133] Synthesis of 4-methoxybenzyl-4-benzyloxy-3-chloro-5-methoxybenzoate (ⅠⅠC-7).

[0134] Compound 3 (0.3 mmol), EDCI (0.45 mmol), and DMAP (0.06 mmol) were added to 20 mL of dichloromethane and stirred at room temperature for a few minutes. Then, 4-methoxyphenol (0.45 mmol) was added. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, packed into a column, and subjected to silica gel column chromatography. The solution was concentrated under reduced pressure to give a white solid; yield 78%; melting point 55.4–55.8 °C. 1 H NMR (400MHz, Chloroform-d) δ7.70 (dd, J = 2.0, 1.2Hz, 1H), 7.55-7.47 (m, 3H), 7.42-7. 29(m,5H),6.92(d,J=8.7Hz,2H),5.28(s,2H),5.12(s,2H),3.90(s,3H),3.82(s,3H); 13C NMR(101MHz,Chloroform-d)δ165.39,159.91,153.78,148.24,136.79,130.37,128.74,128 .61,128.49,128.42,128.00,126.43,123.95,114.16,112.16,75.10,67.09,56.40,55.44.

[0135] The vanillin series derivatives synthesized in the embodiments of this invention all have good crystal form and purity. The melting points of all compounds have been determined, and their structures have been verified. 1 H NMR and 13 Confirmed by C NMR.

[0136] Example 3: Determination of insecticidal activity

[0137] Armyworm activity test

[0138] The insecticidal activity of compounds against third-instar larvae of armyworms was determined using the leaf-loaded method. Fresh wheat leaves (5 mm × 5 mm) were immersed in an acetone solution containing the test compounds. After the acetone evaporated, the loaded leaves were placed in 24-well cell culture plates to feed third-instar larvae that had been starved for 12 hours. Each compound treatment was repeated three times, with each cell culture plate constituting one replicate, for a total of 72 tested third-instar armyworm larvae. Flufenoxuron and PSP treatments served as positive controls, and acetone treatment served as a negative control. The cell culture plates were then wrapped in a damp cloth and placed in a controlled environment at 25°C and 70% relative humidity. Mortality was recorded after 48 hours. The median lethal concentration (LC50) was further determined. 50 To determine the toxicity of the corresponding compound.

[0139] Table 3. LC50 of the target compound against 3rd instar armyworms. 50 value

[0140]

[0141] As can be seen from the activity data in Table 3, the vanillin derivatives exhibit good insecticidal activity against armyworms. In particular, compounds IB-1 and IIB-3 show excellent insecticidal activity, with LC50 values ​​of [missing data]. 50 The concentrations were 0.93 and 0.68 mg / mL, respectively.

[0142] Rice constrictor aphid activity test

[0143] The test compound was dissolved in acetone and diluted in Tween water containing 0.1% Tween 80 to a final acetone concentration of 0.1%. Wheat leaves infested with wingless adult aphids were immersed in the compound solution for 3 seconds. Any remaining droplets on the leaves were blotted dry with filter paper, and then placed in petri dishes containing moistened filter paper. Thiamethoxam was used as the positive control, and distilled water containing 0.1% Tween 80 and 0.1% acetone was used as the negative control. Thiamethoxam was used as the positive control in 30 wingless adult aphids per treatment, repeated three times. Mortality was measured after 24 and 48 hours. The toxicity of the most active compounds was further determined, and LC50 was calculated using SPSS software. 50 And 95% confidence limit.

[0144] Corrected mortality rate (%) = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) × 100%;

[0145] Table 4. LC-100 results of the target compound against *Aphidius gracilis* 50 value

[0146]

[0147] As can be seen from the activity data in Table 4, the vanillin derivatives exhibit good aphid-killing activity. In particular, compound IB-1 demonstrates excellent insecticidal activity, with an LC50 of [missing data]. 50 It was 7.83 mg / L, which is close to that of the commercial pesticide thiamethoxam.

[0148] Insecticidal activity studies showed that some compounds exhibited good insecticidal activity against armyworms and cereal aphids, with compounds IB-1 and IB-6 showing the best activity. The LC50 of the stomach poison of IB-1 against 3rd instar armyworm larvae was also investigated. 50 The concentration was 0.93 mg / mL, and its insecticidal activity was higher than that of the control agent, natural product PSP (LC). 50 =1.57 mg / mL), and lead compound AN-465 / 41851572 (LC50). 50 Compared to 21.28 mg / mL, the activity was increased by 22.88 times. LC50 of IB-1 and IB-6 against wingless adult *Aphis graminearum*. 50 The concentrations were 7.83 mg / L and 7.92 mg / L, respectively, with aphid-killing activity approximately 5.4 times higher than that of PSP (42.53 mg / L) and more than 13 times higher than that of the lead compound 41851572 (104.53 mg / L).

[0149] Example 4: V-ATPase inhibition assay

[0150] The procedure for V-ATPase inhibition assay is as follows:

[0151] Buffer preparation

[0152] Ringer Solution: 25mmol / L HEPES, 150mmol / L NaCl, 3.5mmol / L KCl, 1.8mmol / LNa HCO3, 1.0mmol / L MgSO4, 1.7mmol / L CaCl2, 5mmol / L Glucose, pH=7.1.

[0153] Lysis Buffer-PIC: 5mmol / L Na-HEPES, 2mmol / L EGTA, 10mmol / L MercaptoEtOH, Ph=7.1.

[0154] Lysis Buffer + PIC: 5 mmol / L Na-HEPES, 2 mmol / L EGTA, 10 mmol / L MercaptoEtOH, PIC (Protease Inhibitor Cocktail), pH = 7.1; PO Buffer: 160 mmol / L Tris-Mes, pH = 6.9; MV Buffer: 30 mmol / L MgCl2, 0.8 mmol / L Orthovanadate, boiled at 98℃ for 3 min; KN Buffer: 160 mmol / L KCl, 4 mmol / L NaN3 (10×KN: 2.6 mg / mL); 8 mmol / L Tris-ATP: prepared to 8 mmol / L with dd H2O, aliquoted and stored at -20℃; Bafilomycin A1: diluted to 48 μmol / L with DMSO (positive control).

[0155] Sodium molybdate solution: Add 3 parts of 200 mmol / L sodium molybdate solution to 6 parts of ddH2O, and then add 1 part of freshly prepared 24% sulfuric acid.

[0156] Malachite Green: 18.5 mg of malachite green is dissolved in 25 mL of 1% polyvinyl alcohol.

[0157] Enzyme solution preparation

[0158] Determination of the effect of the target compound on V-ATPase activity: Sixth-instar, non-starved armyworms were selected, dissected, and the midgut was removed. The contents and peritrophic membrane were discarded. Malpighian tubules and food residue were removed in pre-chilled Ringer Solution. The posterior midgut was collected and placed in pre-chilled low-adsorption EP tubes, with ten midguts per tube. The tubes were rapidly frozen in liquid nitrogen and then stored at -80°C. Before use, 20 μL of Lysis buffer-PIC was added, and the mixture was thoroughly homogenized on ice using a micro-homogenizing stick. The mixture was centrifuged at 20,000 rpm, 4°C, for 20 min. The supernatant was discarded, and 200 μL of Lysis buffer-PIC was added to resuspend the precipitate. The mixture was centrifuged at 20,000 rpm, 4°C, for 20 min. After discarding the supernatant, the precipitate was dissolved in 1 mL of Lysis buffer+PIC, and then aliquoted into 150 μL units in 1.5 mL EP tubes and stored at -20°C until use.

[0159] Enzymatic reaction

[0160] The specific operating procedure is as follows: Take the enzyme solution obtained in the above steps and dilute it 6-fold with 750 μL Lysis buffer + PIC. Add 50 μL Buffer PO, 20 μL MV, and 20 μL KN to 1.5 mL EP tubes, followed by 40 μL of the above enzyme solution, 10 μL DMSO (negative control), or the test compound (treatment group), or Bafilomycin A1 (positive control), with each treatment repeated three times. After incubating at 30°C for 30 min, add 20 μL of substrate Tris-ATP (8 mmol / L) to each centrifuge tube sequentially, and incubate at 30°C for 1 h to terminate the reaction. The experiment is set to start at 0 as a blank control, i.e., add 20 μL of substrate Tris-ATP (8 mmol / L) after 1 h of incubation, and immediately terminate the reaction.

[0161] Determination of inorganic phosphorus content: (1) Take 100 μL of sodium molybdate solution into a 1.5 mL centrifuge tube for later use; (2) Add 50 μL of 20% TCA solution to the sample tube of the enzyme-catalyzed reaction and react at 70℃ for 15 s to terminate the reaction; (3) Centrifuge the sample tube at 12000 rpm for 1 min; (4) Then take 30 μL of supernatant, add it to the centrifuge tube containing 100 μL of sodium molybdate solution to dissolve and mix well, react for 15 s and then add 30 μL of malachite green solution and mix well; (5) After reacting for 2 min, add 200 μL of 7.8% sulfuric acid and mix well; (6) After incubating at room temperature for 70 min, measure its absorbance at a wavelength of 625 nm.

[0162] The inhibition rate of enzyme activity was calculated as follows: the inhibition rate of the test compound against V-ATPase (%) = (ΔODn - ΔODi) / ΔODn × 100%. ΔODn = absorbance value of negative control - absorbance value of blank control; ΔODi = absorbance value of treatment group - absorbance value of blank control. The experiment was repeated three times, and the average value was taken.

[0163] V-ATPase inhibition test results:

[0164] Table 5. IC50 of the target compound against V-ATPase 50

[0165]

[0166]

[0167] The results in Table 5 demonstrate that the vanillin derivatives exhibit good V-ATPase inhibitory activity, particularly IIC-5 and IIC-6, which show even higher V-ATPase inhibitory activity at IC50. 50 The values ​​were 77.43 and 73.71 μM, respectively.

[0168] The binding affinity of compounds IB-1 and IB-6, which exhibited high inhibitory activity against V-ATPase and insecticidal activity against 3rd instar armyworm larvae, to the V-ATPase A subunit was determined by isothermal titration calorimetry (ITC).

[0169] The binding affinity of the compound to the V-ATPase A subunit:

[0170] Table 6. Thermodynamic parameters and dissociation constants of compounds binding to the V-ATPase A subunit.

[0171] 41851572 1.000E-9 0.567 100.00 507.7 IB-1 8.037E-7 0.797 100.00 452.1 IB-6 7.112E-8 0.644 100.00 472.2

[0172] The results are shown in Table 6. Both IB-1 (Kd = 0.8037 μM) and IB-6 (Kd = 0.0711 μM) exhibited strong binding abilities, with IB-6 showing a stronger binding ability than IB-1, consistent with their inhibitory activity against V-ATPase. Thermodynamic parameters of IB-1 and IB-6 indicate that the binding of both compounds to the V-ATPase A subunit is an entropy-driven process, possibly due to hydrophobic interactions, consistent with the behavior of the lead compound 41851572. Protein binding data show that the optimized compounds still act on the V-ATPase A subunit.

[0173] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A vanillin derivative, characterized in that, The vanillin derivative has the structure shown in the table below; 。 2. The use of the vanillin derivative of claim 1 in the preparation of V-ATPase inhibitors.

3. Application of vanillin derivatives in the preparation of V-ATPase inhibitors, wherein the vanillin derivatives are shown in the table below: 。 4. The use of vanillin derivatives in the preparation of insecticides targeting V-ATPase, wherein the insecticides are used to control at least one of aphids and armyworms; The vanillin derivatives are shown in the table below: 。

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