A method for preparing a highly effective and selective antagonist BQ-788

Abstract

The present invention provides a method for preparing a highly efficient and selective antagonist BQ-788, and relates to the technical field of preparation of antagonists. This application adopts a solid-liquid combination method for synthesis. Since dimethyl dialkanoate on the side chain of tryptophan (DTrp) is difficult, liquid phase synthesis is used to obtain Fmoc-DTrp(CO2Me)-OH. During the synthesis process, p-methoxybenzyl is used to protect the carboxyl group, which can be subsequently removed by trifluoroacetic acid, which can successfully avoid the risk of reduction of the indole ring of tryptophan due to hydrogenation. Then, using the solid-phase synthesis method, fragments can be obtained quickly and efficiently, which can effectively avoid the problem of racemization in the condensation process, and urea is formed on the solid phase. The operation is simple and convenient, hydrogen and diphosgene are not used in the preparation process, production is safe, the yield and purity of the obtained product are high, and the preparation steps are relatively few.

Description

Technical Field

[0001] The present invention relates to the technical field of antagonist preparation, and in particular to a method for preparing a highly effective and selective antagonist BQ-788. Background Art

[0002] BQ-788 is a very potent and selective ETB receptor antagonist;

[0003] English name (2R)-2-[[(2R)-2-[[(2S)-2-[[(2R,6S)-2,6-dimethylpiperidine-1-carbonyl]amino]-4,4-dimethylpentanoyl]amino]-3-(1-methoxycarbonylindol-3-yl)propanoyl]amino]hexanoic acid;

[0004] Peptide sequence: cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle;

[0005] Structural formula:

[0006] ;

[0007] Salt type: trifluoroacetic acid Molecular formula: C 34 H 51 N5O7 molecular weight: 641.80CAS number: 173326-37-9Appearance: white powder,

[0008] Solubility: 100 mg can be dissolved in 1 mL DMSO (100 mg / mL). Storage conditions: 2-8°C.

[0009] This antagonist has been widely used to demonstrate the effects of endogenous or exogenous ET in vitro and in vivo. In vitro, BQ-788 potently and competitively inhibited the binding of 125I-labeled ET-1 to the ETB receptor in human Girrardi heart cells (hGH) with an IC50 of 1.2 nM, but only weakly inhibited the binding of ET receptors in the human neuroblastoma cell line SK-N-MC (IC50 1300 nM). In isolated rabbit pulmonary arteries, BQ-788 exhibited no agonist activity up to 10 μM and competitively inhibited vasoconstriction induced by an ETB-selective agonist (pA2, 8.4). BQ-788 also inhibited several biological activities of ET-1, such as bronchoconstriction, cell proliferation, and clearance of infused ET-1, further confirming that BQ-788 is a potent and selective ETB receptor antagonist.

[0010] Currently, the preparation of the synthetic antagonist BQ-788 involves many steps, complex post-processing, and low yield. Therefore, the development of a synthetic, highly efficient and selective antagonist BQ-788 is of great significance.

[0011] The significant steric hindrance of the raw material methylleucine (tBuAla) makes it difficult to form urea with cis-2,6-dimethylpiperidine. Furthermore, the indole ring on the tryptophan (DTrp) side chain hinders the addition of dimethyl dioxanoate. Furthermore, the multi-step hydrogenation and debenzylation process also results in reductive hydrogenation of the indole ring of tryptophan. This results in low product yield and purity, and the complex synthesis steps make the existing preparation process unfavorable for subsequent optimization and scale-up. Summary of the Invention

[0012] In order to solve the problems in the prior art of the preparation of the antagonist BQ-788 that the number of steps is large and the yield and purity are low, the present application provides a method for preparing the highly efficient and selective antagonist BQ-788.

[0013] In the first aspect, the present application provides a method for preparing a highly effective and selective antagonist BQ-788:

[0014] A method for preparing a highly effective and selective antagonist BQ-788, wherein the reactants include the following structural units:

[0015] Structure (1) Structure (2),

[0016] Wherein, A1 is an H group or a carboxyl protecting group;

[0017] The -NH2 group in structure (1) and the -NH- structure in structure (2) participate in the reaction and are connected through the carbonyl group, thereby preparing the antagonist BQ-788.

[0018] Furthermore, the method for preparing the highly effective and selective antagonist BQ-788 comprises a liquid phase reaction and a solid phase reaction. Furthermore, the carboxyl protecting group comprises any one of 4-methoxybenzyl, WangResin-, and Bzl-.

[0019] Furthermore, the reactants of structure (1) include the following structural units:

[0020] Structure (3)

[0021] Structure (4)

[0022] Structure (5),

[0023] wherein any one of A3 and A5 is an H group or a carboxyl protecting group, and any one of A2, A4, and A6 is an H group or an amine protecting group;

[0024] Among them, the amine group formed by connecting A2 or removing the protection of A2 undergoes an amidation reaction with the carboxyl group in structure (5), and the carboxyl group formed by connecting A3 or removing the protection of A3 undergoes an amidation reaction with the amine group formed by connecting A4 or removing the protection of A4 to obtain structure (1).

[0025] Furthermore, the carboxyl protecting group includes 4-methoxybenzyl, WangResin-, and Bzl-; the amine protecting group includes Fmoc- and Boc-.

[0026] Furthermore, in the structure (3), -A2 is a -Fmoc group, and -A3 is a -H group;

[0027] The preparation process of the structure (3) is as follows:

[0028] 1) Liquid phase synthesis of raw material Boc-DTrp-OPMB:

[0029] 9-11 g of Boc-DTrp-OH was dissolved in an organic solvent, 4-6 g of sodium carbonate was added, and 4-6 mL of 4-methoxybenzyl chloride was slowly added dropwise. After completion of the addition, the mixture was stirred at room temperature for 11-15 h to react. Boc-DTrp-OPMB was then isolated by vacuum rotary evaporation, extraction, washing, and purification. The reaction process is as follows:

[0030] ;

[0031] 2) Liquid phase synthesis of raw material Boc-DTrp(CO2Me)-OPMB:

[0032] 9-11 g of Boc-DTrp-OPMB was dissolved in 40-60 mL of acetonitrile, 3-5 mL of dimethyl dioxanoate and 0.5-1 g of 4-dimethylaminopyridine were added, and the mixture was stirred at 25-30° C. for 18-25 h. Boc-DTrp(CO2Me)-OPMB was then isolated by vacuum rotary evaporation, extraction, washing, and purification. The reaction process is as follows:

[0033] ;

[0034] 3) Liquid phase synthesis of raw material H-DTrp(CO2Me)-OH:

[0035] 8-9.5 g of Boc-DTrp(CO2Me)-OPMB was dissolved in 80-100 mL of cutting solution and stirred at room temperature for 1.5-3 h. After removing the cutting solution by rotary evaporation, water and acetonitrile were added and freeze-dried to obtain H-DTrp(CO2Me)-OH. The reaction process is as follows:

[0036] ;

[0037] 4) Liquid phase synthesis of raw material Fmoc-DTrp(CO2Me)-OH:

[0038] Dissolve 5-7.5g H-DTrp(CO2Me)-OH.TFA in 90mL water / tetrahydrofuran (1:2), add 2.5-4g sodium carbonate, then add 5-7g Fmoc-OSu (9-fluorenylmethyl-N-succinimidyl carbonate), and stir at room temperature for 10-15h. Then, separate and obtain structure 2, namely Fmoc-DTrp(CO2Me)-OH, by vacuum rotary evaporation, extraction, washing, and purification. The reaction process is as follows:

[0039] .

[0040] Furthermore, the preparation process of the structure (1) is as follows:

[0041] Solid phase synthesis was used; wherein the -A4 group is -H, the -A5 group is -WangResin;

[0042] The specific reactions are as follows:

[0043] 1) The resin is fully swollen

[0044] 16-18.5 g of Fmoc-DNle-WangResin was soaked in N,N-dimethylformamide to swell. After vacuum filtration to remove N,N-dimethylformamide, 200-300 mL of 20% piperidine / DMF solution was added and nitrogen was blown for 0.2-0.8 h to remove the Fmoc protecting group. The resin was then washed with N,N-dimethylformamide to obtain resin A.

[0045] 2) Connecting Fmoc-DTrp(CO2Me)-OH

[0046] Using Fmoc-DNle-WangResin resin as the base, weigh 1.3 times the amount of Fmoc-DTrp(CO2Me)-OH and 1.3 times the amount of HOBt, dissolve them in 200 mL of N,N-dimethylformamide, cool to -10°C, add 1.3 times the amount of DIC, activate for 10 minutes, then add to resin A, and couple at room temperature for 1-2 hours. After testing, the amino group has been completely coupled. After coupling, wash with N,N-dimethylformamide and drain; then add 200-300 mL of 20% piperidine / DMF solution to remove Fmoc, deprotect with nitrogen for 0.1-0.8 hours, and then wash with N,N-dimethylformamide to obtain resin DTrp(CO2Me)DNle-WangResin;

[0047] 3) Connecting Fmoc-tBuAla-OH:

[0048] Using DTrp(CO2Me)DNle-WangResin as the base, weigh 1.2-1.8 times the amount of Fmoc-tBuAla-OH and 1.2-1.8 times the amount of HOBt and dissolve them in 200 mL of N,N-dimethylformamide. Cool to -10 degrees Celsius, add 1.5 times the amount of DIC to activate for 10 minutes, then add to the resin. Couple at room temperature for 1-2 hours. Kaiser assay shows colorless. After coupling, wash with N,N-dimethylformamide solution and drain.

[0049] 4) Removal of Fmoc protecting group:

[0050] Vacuum filter off N,N-dimethylformamide, wash with N,N-dimethylformamide solution, drain, add 200-280 mL of 20% piperidine / DMF solution, deprotect with nitrogen for 0.2-0.8 h, and then wash with N,N-dimethylformamide to obtain the resin tBuAla-DTrp(CO2Me)-DNle-WangResin;

[0051] The specific reactions are as follows:

[0052] .

[0053] Furthermore, the preparation method of the antagonist BQ-788 includes the following preparation process:

[0054] 1): 2,6-dimethylpiperidine forms urea:

[0055] Based on the resin tBuAla-DTrp(CO2Me)-DNle-WangResin, weigh 1.2-1.8 times the amount of 2,6-methylpiperidine and dissolve it in acetonitrile. Add 2.8-3.5 times the amount of triethylamine, then add 2.5-3 times the amount of N,N'-succinimidyl carbonate DSC, stir at room temperature for 8-15 hours, then concentrate, add dichloromethane to dissolve, then add to the resin, add 2.8-3.2 times the amount of N,N-diisopropylethylamine, react for 10-15 hours, and it is colorless after Kaiser detection. After the coupling is completed, add N,N-dimethylformamide to wash and drain to obtain cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle-WangResin;

[0056] 2): Cutting steps

[0057] Disperse and suspend 20-23g of cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle-WangResin in 200-250mL of liquid F and shake at room temperature for 2-3h to obtain a resin suspension;

[0058] The specific ratio of F liquid is: TFA (trifluoroacetic acid) 95wt%, TIS (triisopropylsilane) 2.5wt% and water 2.5wt%.

[0059] The resin suspension was filtered, washed with liquid F and methyl tert-butyl ether, and centrifuged to obtain a crude product. After purification, the resin cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle was obtained. The reaction process was as follows;

[0060] .

[0061] Furthermore, the purity of the antagonist BQ-788 obtained by the preparation method is greater than 90%, and the total synthesis yield is greater than 20%.

[0062] Furthermore, the purification process is to first dissolve with acetonitrile / water, filter with a 0.35-0.55 μm mixed fiber membrane, and then purify with a chromatographic column.

[0063] Beneficial effects: 1. The present application adopts a solid-liquid combination method for synthesis. Since it is difficult to produce dimethyl dialkanoate on the side chain of tryptophan (DTrp), liquid phase synthesis is used to obtain Fmoc-DTrp(CO2Me)-OH. During the synthesis process, p-methoxybenzyl is used to protect the carboxyl group, which can be subsequently removed by trifluoroacetic acid. This can successfully avoid the risk of reduction of the indole ring of tryptophan due to hydrogenation, thereby improving the purity and yield. Then, the solid phase synthesis method is used to quickly and efficiently obtain fragments, effectively avoiding the problem of racemization in the condensation process, and forming urea on the solid phase. The operation is simple and convenient. No hydrogen and diphosgene are used in the preparation process, the production is safe, the yield and purity of the obtained product are high, and the preparation steps are fewer. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 , mass spectrum of Boc-DTrp(CO2Me)-OPMB prepared in Example 1 of the present application;

[0065] Figure 2 , the chromatogram of Fmoc-DTrp(CO2Me)-OH in Example 1 of the present application;

[0066] Figure 3 , mass spectrum of the antagonist BQ-788 prepared in Example 1 of the present application;

[0067] Figure 4 , chromatogram of the antagonist BQ-788 prepared in Example 1 of the present application. DETAILED DESCRIPTION

[0068] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to specific embodiments.

[0069] Example 1, a method for preparing a highly effective and selective antagonist BQ-788, comprising the following preparation process:

[0070] Step S1: Preparation of structure (1) tBuAla-DTrp(CO2Me)-DNle-WangResin, including the following preparation process:

[0071] The preparation of structure (3) Fmoc-DTrp(CO2Me)-OH is as follows:

[0072] Formula 1:

[0073] ;

[0074] Formula 2:

[0075] ;

[0076] Formula 3:

[0077] ;

[0078] Formula 4:

[0079] .

[0080] Formula 1: Liquid phase synthesis of raw material Boc-DTrp-OPMB, using raw materials as shown in Table 1, the preparation process is as follows:

[0081] Table 1. List of raw materials used in the preparation of Boc-DTrp-OPMB

[0082] ;

[0083] (1) Dissolve 10.088 g of Boc-DTrp-OH in 50 mL of N,N-dimethylformamide (DMF), add 4.216 g of sodium carbonate, slowly add 5 mL of 4-methoxybenzyl chloride (PMBCL) in a flask, and stir at room temperature for 12 h after the addition is complete.

[0084] (2) After the reaction, N,N-dimethylformamide (DMF) was removed by rotary evaporation under reduced pressure, 5% phosphoric acid aqueous solution was added, and the aqueous phase was extracted with 100 mL of ethyl acetate. The organic phase was washed with water and saturated brine in sequence, and the ethyl acetate was removed by rotary evaporation under reduced pressure. Then, Boc-DTrp-OPMB was separated by silica gel column chromatography to obtain 10.3 g.

[0085] (3) Yield: 73.0%;

[0086] (4) Mass spectrum: 424.49 (425.31 (M+1), 369.19 (M-56+1), 447.19 (M+23)).

[0087] Formula 2: Liquid phase synthesis of raw material Boc-DTrp (CO2Me) -OPMB, using raw materials as shown in Table 2, the preparation process is as follows:

[0088] Table 2. List of raw materials used in the preparation of Boc-DTrp(CO2Me)-OPMB

[0089] ;

[0090] (1) Dissolve 10.3 g of Boc-DTrp-OPMB in 50 mL of acetonitrile (CH3CN), add 3.6 mL of dimethyl dioxanoate and 0.74 g of 4-dimethylaminopyridine (DMAP), and stir at 30 °C for 20 h.

[0091] (2) After the reaction, acetonitrile was removed by rotary evaporation under reduced pressure, 5% aqueous phosphoric acid solution was added, and the aqueous phase was extracted with 100 mL of ethyl acetate. The organic phase was washed with water and saturated brine in sequence, and ethyl acetate was removed by rotary evaporation under reduced pressure. Then, Boc-DTrp(CO2Me)-OPMB was separated by silica gel column chromatography to obtain 8.5 g.

[0092] (3) Yield: 72.6%;

[0093] (4) Mass spectrum: 482.53 (483.32 (M+1), 383.3 (M-100+1), 427.19 (M-56+1)). The mass spectrum is as follows Figure 1 .

[0094] Formula 3: Liquid phase synthesis raw material H-DTrp (CO2Me) -OH uses raw materials as shown in Table 3, and the preparation process is as follows:

[0095] Table 3. List of raw materials used in the preparation of H-DTrp(CO2Me)-OH

[0096] ;

[0097] (1) Dissolve 8.5 g of Boc-DTrp(CO2Me)-OPMB in 90 mL of liquid E and stir at room temperature for 2 h.

[0098] (2) The cutting solution was removed by rotary evaporation, and water and acetonitrile were added and freeze-dried to obtain 6.5 g of H-DTrp(CO2Me)-OH.

[0099] (3) Yield: 98.5%.

[0100] (4) Mass spectrum: 262.26 (263.28 (M+1)) (MS604941YF-01-14).

[0101] Formula 4: Liquid phase synthesis raw material Fmoc-DTrp(CO2Me)-OH, using raw materials as shown in Table 4, the preparation process is as follows:

[0102] Table 4. List of raw materials used in the preparation of Fmoc-DTrp(CO2Me)-OH

[0103] ;

[0104] (1) Dissolve 6.5 g of H-DTrp(CO2Me)-OH.TFA in 90 mL of water / tetrahydrofuran (volume ratio 1:2), add 3.48 g of sodium carbonate, and then add 6.09 g of Fmoc-OSu (9-fluorenylmethyl-N-succinimidyl carbonate), and stir at room temperature for 12 h.

[0105] (2) After the reaction, tetrahydrofuran was removed by rotary evaporation under reduced pressure, 5% phosphoric acid aqueous solution was added, and the aqueous phase was extracted with 100 mL of ethyl acetate. The organic phase was washed with water and saturated brine in sequence, and the ethyl acetate was removed by rotary evaporation under reduced pressure. Then, Fmoc-DTrp(CO2Me)-OH was separated by silica gel column chromatography, 7.5 g. The chromatogram is shown in FIG. Figure 2 .

[0106] (3) Yield: 94.4%, total yield of liquid phase synthesis: 49.3%

[0107] (4) Purity: 98.5%, chromatogram as shown Figure 3 .

[0108] (5) Mass spectrum: 484.26 (485.31 (M+1), 969.55 (2M+1)) (MS604941YF-01-15).

[0109] Step S2: Structure (3), structure (4) and structure (5) react to generate structure (1). The reaction process is as follows:

[0110] Formula 5:

[0111] ;

[0112] Formula 6-1:

[0113] ;

[0114] Formula 6-2:

[0115] ;

[0116] The preparation process of formula 5 is as follows:

[0117] (1) Full swelling of the resin and removal of Fmoc

[0118] 17.8 g of Fmoc-DNle-WangResin with a degree of substitution of 0.563 mol / g was weighed and placed in a polypeptide solid phase reactor. 250 mL of N,N-dimethylformamide (DMF) was added for soaking and swelling for 1 h. The N,N-dimethylformamide was vacuum filtered off, and the mixture was washed three times with 250 mL of N,N-dimethylformamide solution. The N,N-dimethylformamide was drained, and 250 mL of 20% piperidine / DMF solution (20% Pip / DMF) was added. Deprotection was carried out under nitrogen bubbling for 0.5 h, and the mixture was washed five times with 250 mL of N,N-dimethylformamide to obtain the resin DNle-WangResin.

[0119] Kaiser test

[0120] Preparation of detection reagents: 1) Kaiser detection solution A: 20% ethanol + 80% phenol; 2) Kaiser detection solution B: redistilled pyridine; 3) Kaiser detection solution C: 5% ninhydrin in ethanol. Detection procedure: Place a small amount of resin in a test tube and wash twice with ethanol. Add two drops each of Kaiser detection solution A, Kaiser detection solution B, and Kaiser detection solution C. Heat to 110°C for 2 minutes. Observe the resin color; the color development result should be a transparent dark blue. After removing the Fmoc protecting group during solid-phase synthesis, the resin will be observed as an opaque dark blue. If the amino acid is fully coupled during solid-phase synthesis and there are no free amino groups, the detection result should be yellow or colorless.

[0121] (2) Connection structure (3) Fmoc-DTrp(CO2Me)-OH:

[0122] Based on the resin DNle-WangResin, 1.3 times the amount of Fmoc-DTrp(CO2Me)-OH and 1.3 times the amount of HOBt were weighed and dissolved in 200 mL of N,N-dimethylformamide. The mixture was cooled to -10°C, and 1.3 times the amount of DIC was added for activation for 10 min. The mixture was then added to the resin and coupled at room temperature for 1-2 h. The mixture was colorless after Kaiser detection. After the coupling, 300 mL of N,N-dimethylformamide was added and washed three times with the mixture and dried. The N,N-dimethylformamide was vacuum filtered off, and the mixture was washed three times with 300 mL of N,N-dimethylformamide solution. The N,N-dimethylformamide was dried, and 250 mL of 20% Pip / N,N-dimethylformamide was added. The mixture was deprotected under nitrogen for 0.5 h to remove the Fmoc protecting group. The mixture was washed five times with 300 mL of N,N-dimethylformamide to obtain DTrp(CO2Me)-DNle-WangResin.

[0123] The preparation process of formula 6-1 is as follows:

[0124] (1) Connecting Fmoc-tBuAla-OH

[0125] Based on DTrp(CO2Me)-DNle-WangResin, weigh 1.5 times the amount of Fmoc-tBuAla-OH and 1.5 times the amount of HOBt and dissolve them in 200 mL of N,N-dimethylformamide. Cool to -10°C, add 1.5 times the amount of DIC for activation for 10 minutes, and then add to the resin. Coupling at room temperature for 1-2 hours, Kaiser assay indicates colorless. After coupling, add 300 mL of N,N-dimethylformamide and wash three times. Vacuum filter the N,N-dimethylformamide, then wash three times with 300 mL of N,N-dimethylformamide solution. Drain the N,N-dimethylformamide, add 250 mL of 20% Pip / N,N-dimethylformamide, deprotect under nitrogen for 0.5 hours, and wash five times with 300 mL of N,N-dimethylformamide.

[0126] The preparation process of formula 6-2 is as follows:

[0127] (1) 2,6-Dimethylpiperidine forms urea

[0128] Taking the resin tBuAla-DTrp(CO2Me)-DNle-WangResin as the benchmark, weigh 1.5 times the amount of 2,6-dimethylpiperidine and dissolve it in acetonitrile, add 3 times the amount of triethylamine, then add 2.5 times the amount of DSC, stir at room temperature for 12 hours, then concentrate, add 100 mL of dichloromethane to dissolve, add to the resin tBuAla-DTrp(CO2Me)-DNle-WangResin, add 3 times the amount of N,N-diisopropylethylamine and react for 12 hours. It is colorless after Kaiser detection. After the coupling is completed, add 300 mL of N,N-dimethylformamide, wash three times and dry.

[0129] (2) Resin washing

[0130] The mixture was washed once with 300 mL of methanol, twice with 300 mL of dichloromethane, and once with 300 mL of methanol, and finally dried under vacuum to obtain 21.912 g of cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle-WangResin.

[0131] (3) Cutting steps

[0132] 1) Disperse and suspend 21.9 g of resin in 220 mL of liquid F and shake at room temperature for 2.5 h.

[0133] 2) Filter the resin suspension, wash the resin twice with 20 mL of liquid and once with 20 mL of methyl tert-butyl ether. Add 1200 mL of methyl tert-butyl ether to the container, then add the filtrate to the methyl tert-butyl ether. After thorough stirring, transfer the mixture evenly to a centrifuge cup, balance the balance, let it stand for 10 minutes, centrifuge at 3500 rpm for 2 minutes, and discard the supernatant.

[0134] 4) Wash the crude product, add 500 mL of methyl tert-butyl ether, stir evenly, balance the balance, centrifuge at 3500 rpm for 2 min, discard the supernatant, wash three more times using the same method, and centrifuge. Transfer the precipitate to a drying oven and drain to obtain 6.4 g of crude cis-2,6-Dimethyl-piperidine-CO-tBuAla-DTrp(CO2Me)-DNle. Purity: 79.4% (HPLC 604941C), mass spectrum: 641.8 (642.52 (M+1)) (MS 604941C). The mass spectrum is shown in the figure below. Figure 4 .

[0135] 5) Preparation and purification of crude product

[0136] 1) Sample pretreatment: The sample was dissolved in 50% acetonitrile / water and filtered through a 0.45 μm mixed fiber membrane.

[0137] 2) Purification preparation method:

[0138] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06);

[0139] Mobile phase A: 0.1% TFA in water

[0140] Mobile phase B: acetonitrile

[0141] Loading flow rate: 60 mL / min Elution flow rate: 60 mL / min Detection wavelength: 220 nm

[0142] Elution gradient: 60-90% in 60 minutes

[0143] For details of the purification preparation spectrum, please see the purification record 6049412311142123111422

[0144] After preparation and purification, the product was collected and freeze-dried to obtain 6049413.0g

[0145] Purity: 98.1% (HPLC: 60494123100031Final)

[0146] Mass spectrum: 641.8 (642.65 (M+1)) (MS: 604941)

[0147] Solid phase synthesis yield: 46.9%, total synthesis yield: 23.1%.

[0148] Example 2, a method for preparing a highly effective and selective antagonist BQ-788, differs from Example 1 in that the preparation process of structure (3) Fmoc-DTrp(CO2Me)-OH is different, including reaction A1, reaction A2, reaction A3 and reaction A4. The specific preparation process is as follows:

[0149] Formula 7:

[0150] ;

[0151] Reaction A1:

[0152] 1.0 equivalent of Boc-DTrp-OH (as a reference substrate) was dissolved in anhydrous dichloromethane / N,N-dimethylformamide to form an 8 mL / mmol solution. The solution was cooled to 0°C in an ice bath, and 1.5 equivalents of HOBt and 1.5 equivalents of EDC·HCl were added sequentially. The solution was stirred at 0°C for 30 minutes (to activate the active ester). 1.8 equivalents of benzyl alcohol were slowly added dropwise, maintaining the temperature at 0°C. 1.2 equivalents of triethylamine (TEA) were added to neutralize the hydrochloric acid generated during the reaction. The ice bath was removed, the solution was warmed to room temperature and stirred for 20 hours, and then quenched with dilute hydrochloric acid (1 M). The pH was adjusted to neutral. The solution was extracted with ethyl acetate (3 × 20 mL), and the organic phases were combined. The product was washed sequentially with saturated sodium bicarbonate (to neutralize the acid) and brine, dried over anhydrous sodium sulfate, and then separated by silica gel column chromatography to obtain Boc-DTrp-OBzl in an 80% yield.

[0153] Reaction A2:

[0154] Dissolve 1.0 equivalent of Boc-DTrp-OBzl (substrate) in 15 mL of anhydrous tetrahydrofuran and cool to 0°C in an ice bath. Add 0.2 equivalents of 4-dimethylaminopyridine and stir for 5 minutes to achieve uniform dispersion. Slowly add 3 equivalents of dimethyl carbonate (DMDC) (dissolved in a small amount of anhydrous solvent) dropwise via a constant pressure dropping funnel, maintaining 0°C. Remove the ice bath, warm to room temperature, and continue stirring for 6-12 hours (reaction endpoint confirmed by TLC). Add 10 mL of saturated sodium bicarbonate solution to quench unreacted dimethyl carbonate. Wash with 0.1 M dilute hydrochloric acid and saturated brine, sequentially, to remove 4-dimethylaminopyridine and residual reagents. Dry the organic phase over anhydrous sodium sulfate and separate by silica gel column chromatography to obtain Boc-DTrp(CO2Me)-OBzl in a 70% yield.

[0155] Reaction A3:

[0156] Boc-DTrp(CO2Me)-OBzl was added to a round-bottom flask, followed by 10 mL of 4M HCl / dioxane solution. The mixture was cooled to 0°C in an ice bath and stirred for 10 minutes to allow the substrate to disperse evenly. The ice bath was removed, the mixture was warmed to room temperature (25°C), and stirring was continued for 4 hours. The reaction mixture was slowly poured into an ice-water mixture (20 mL) and rapidly quenched. Saturated sodium bicarbonate solution was slowly added dropwise to pH 7. The aqueous phase was extracted with ethyl acetate (3 × 20 mL). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was then removed by rotary evaporation. H-DTrp(CO2Me)-OBzl was then purified by silica gel column chromatography to obtain H-DTrp(CO2Me)-OBzl in a 78% yield.

[0157] Reaction A4: The reaction process is as follows:

[0158] ;

[0159] 1) Fmoc protection of the amino group: Dissolve 1.0 equivalent of H-DTrp(CO2Me)-OBzl (reference substrate) in 12 mL of anhydrous dichloromethane, cool to 0°C in an ice bath, add 3.0 equivalents of NMM dropwise, and stir for 5 minutes. Slowly add 1.5 equivalents of Fmoc-Cl solution (1.5 equivalents of Fmoc-Cl dissolved in 2 mL of dichloromethane) dropwise, maintaining 0°C. Remove the ice bath and stir at room temperature for 4 hours (TLC shows disappearance of the starting material). Adjust the pH to an acidic pH of 3 by adding 1 M HCl. Extract with dichloromethane (3 × 20 mL). Combine the organic phases, wash sequentially with saturated sodium bicarbonate and brine, dry over anhydrous sodium sulfate, concentrate, and rotary evaporate to obtain the crude product. This is then separated by silica gel column chromatography to afford Fmoc-DTrp(CO2Me)-OBzl.

[0160] 2) Benzyl Ester Hydrolysis: Dissolve 1.0 equivalent of Fmoc-DTrp(CO2Me)-OBzl in 20 mL of tetrahydrofuran and add 0.2 g of Pd-C catalyst. Evacuate the mixture, then inject H2 (repeat three times), maintaining the H2 pressure at 2 atm. Stir at room temperature at 25°C for 5 h (TLC indicates complete hydrolysis of the benzyl ester). Remove the Pd-C by filtration through celite, and wash the catalyst with tetrahydrofuran. Rotary evaporation removes the tetrahydrofuran to obtain the crude product. Acidify the mixture to pH 2 by adding 0.1 M HCl (10 mL). Extract with ethyl acetate (3 × 20 mL). Combine the organic phases, dry over anhydrous sodium sulfate, and separate by silica gel column chromatography to obtain Fmoc-DTrp(CO2Me)-OH.

[0161] Comparative Example 1, a method for preparing a highly effective and selective antagonist BQ-788, using a liquid phase preparation method, comprising the following preparation steps:

[0162] Step S1: Preparation of H-DTrp(CO2Me)-OBzl. The reaction process is shown in Formula 7 and the preparation process is the same as that of the preparation of H-DTrp(CO2Me)-OBzl in Example 2:

[0163] Formula 7:

[0164] ;

[0165] Step S2: Preparation of cis-2,6-Dimethylpiperidine-CO-tBuAla-OH. The reaction process is shown in Formula 8.

[0166] Formula 8:

[0167] ;

[0168] The preparation process is as follows:

[0169] Reaction B1:

[0170] 1) Benzyl esterification reaction (Boc-tBuAla-OH→Boc-tBuAla-OBzl)

[0171] Boc-tBuAla-OH (1 mmol) and cesium carbonate (1.5 mmol) were added to a flask, followed by anhydrous N,N-dimethylformamide (5 mL). Stirring was initiated until the solid was completely dissolved (approximately 10 minutes). Heat in an oil bath at 50°C (cooled with a reflux condenser). Benzyl bromide (1.2 mmol) was slowly added dropwise. Maintain a nitrogen atmosphere and stir for 6 hours. TLC monitoring was performed (developing solvent: ethyl acetate / petroleum ether = 1:2, UV or iodine color development). After cooling to room temperature, the reaction solution was poured into 50 mL of ice water and stirred for 10 minutes. Extraction was performed with ethyl acetate (3 × 20 mL). The organic phases were combined and washed sequentially with saturated sodium bicarbonate (10 mL), water (10 mL), and saturated brine (10 mL). The mixture was then dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield a crude white solid.

[0172] 2) Boc deprotection (Boc-tBuAla-OBzl → H-tBuAla-OBzl):

[0173] Boc-tBuAla-OBzl (1 mmol) was dissolved in 4 M HCl / dioxane (5 mL), cooled to 0°C in an ice bath, slowly warmed to room temperature, and stirred for 1-2 hours. The reaction was monitored by TLC (developing solvent: dichloromethane / MeOH = 10:1, ninhydrin was used for color development to confirm exposure of the amino group).

[0174] Concentrate under reduced pressure to remove most of the solvent, dissolve the residue in 10 mL of dichloromethane, and slowly add saturated sodium bicarbonate solution dropwise to pH 8 (to neutralize excess HCl). Separate the layers, back-extract the aqueous phase with dichloromethane (2 × 10 mL), and combine the organic phases. Dry over anhydrous sodium sulfate and concentrate to obtain a crude white solid. This can be further purified by recrystallization (e.g., ether / n-hexane).

[0175] Reaction B2:

[0176] 1) Formation of isocyanate (H-tBuAla-OBzl→R-NCO)

[0177] H-tBuAla-OBzl (1 mmol) was dissolved in anhydrous dichloromethane (5 mL), cooled to 0°C in an ice bath, and diphosgene (0.6 mmol, dissolved in 5 mL dichloromethane) was slowly added dropwise. After the addition was complete, the ice bath was removed, and the temperature was raised to room temperature (25°C). The reaction was stirred for 2 hours and monitored by TLC (developing solvent: dichloromethane / MeOH = 10:1, ninhydrin color development was used to confirm the disappearance of the amino group).

[0178] The reaction solution was quenched with ice water (10 mL), separated, and the organic phase was retained; the aqueous phase was back-extracted with dichloromethane (2×10 mL), and the organic phases were combined; dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the isocyanate intermediate.

[0179] 2) Urea bond formation (R-NCO + cis-2,6-dimethylpiperidine → target product)

[0180] The isocyanate intermediate was dissolved in anhydrous dichloromethane (5 mL), and then cis-2,6-dimethylpiperidine (1.2 mmol) and triethylamine (2 mmol) were added. The reaction was stirred at room temperature (25°C) for 20 hours and monitored by TLC (developing solvent: EtOAc / Hexane = 1:1, UV or iodine color development). The reaction solution was then washed with 1M HCl (10 mL) to remove unreacted amine. The organic phase was washed with saturated sodium bicarbonate (10 mL) and saturated brine (10 mL) in sequence, then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product.

[0181] Reaction B3:

[0182] Dissolve cis-2,6-Dimethylpiperidine-CO-tBuAla-OBzl (1 mmol) in 20 mL of anhydrous methanol and add it to a pressure-resistant three-necked flask. Add Pd / C catalyst (50 mg, 10% loading) and stir to suspend evenly. Replace the gas in the reactor with hydrogen three times, maintaining a hydrogen pressure of 1 atm. Stir the reaction at room temperature (25°C) for 12 hours and monitor by TLC (developing solvent: dichloromethane / MeOH = 5:1, UV or iodine color development, observe the disappearance of benzyl ester).

[0183] After the reaction is complete, the hydrogen atmosphere is turned off and the atmosphere in the reactor is replaced with nitrogen. The Pd / C catalyst is removed by filtration through a pad of celite, and the filter cake is washed with methanol (3 x 10 mL). The combined filtrates are concentrated to dryness under reduced pressure to obtain a crude white solid product.

[0184] The crude product was dissolved in 10 mL of water, 1 M HCl was added dropwise to pH = 2-3, and stirred for 30 minutes; hydrophobic impurities were removed by extraction with ethyl acetate (3 × 15 mL), and the aqueous phase was adjusted to pH = 7 with NaOH, extracted again with ethyl acetate, and dried and concentrated.

[0185] Step S3: Preparation of cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle. The reaction process is shown in Formula 9.

[0186] Formula 9:

[0187] ;

[0188] The preparation process is as follows:

[0189] Reaction C1:

[0190] Amide bond coupling (EDC·HCl / HOBt mediated)

[0191] cis-2,6-Dimethylpiperidine-CO-tBuAla-OH (1 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL) and added to a round-bottom flask equipped with a magnetic stirrer. EDC·HCl (1.5 mmol), HOBt (1.5 mmol), and N,N-diisopropylethylamine (3 mmol) were then added in sequence to form a mixed solution, which was stirred at room temperature for 30 minutes. H-DTrp(CO2Me)-OBzl (1.2 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL) and slowly added dropwise to the mixture; the reaction was stirred at room temperature (25°C) for 24 hours and monitored by TLC (developing solvent: dichloromethane / MeOH = 5:1, UV or ninhydrin color development); the reaction solution was then adjusted to pH = 3 with 1 M HCl, extracted with ethyl acetate (3 × 30 mL), and the organic phases were combined; then washed with saturated sodium bicarbonate, water, and saturated brine in sequence; dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product to obtain the intermediate.

[0192] 2) Benzyl ester hydrogenolysis (Pd / C / H2 catalysis)

[0193] Hydrogenolysis reaction:

[0194] The intermediate from step 1) (1 mmol) was dissolved in deoxygenated methanol (20 mL), and Pd / C (50 mg) was added. H₂ (1 atm) was introduced and stirred at room temperature for 12 hours. TLC was monitored (developing solvent: dichloromethane / MeOH = 5:1, disappearance of the benzyl ester spot). The Pd / C was removed by filtration (diatomaceous earth pad), and the filter cake was washed with methanol (3 × 10 mL). The combined filtrates were concentrated to dryness under reduced pressure. The crude product was dissolved in water (10 mL), and 1 M HCl was added dropwise to a pH of 2-3. The mixture was stirred for 30 minutes. The hydrophobic impurities were removed by extraction with ethyl acetate, and the aqueous phase was adjusted to pH 7. The mixture was extracted again, dried, and concentrated to obtain the product as a white solid.

[0195] Reaction C2:

[0196] 1) Dissolution and activation:

[0197] The carboxylic acid cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-OH (1 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL) and added to a round-bottom flask equipped with a magnetic stirrer. HBTU (1.2 mmol) and N,N-diisopropylethylamine (DIPEA) (3 mmol) were then added and stirred at room temperature (25°C) for 30 minutes to generate an active ester intermediate.

[0198] H-DNle-OBzl·HCl (1.2 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (1.5 mmol) was added and stirred for 5 minutes to free the amino group (-NH2).

[0199] 2) Coupling reaction:

[0200] Slowly add the free amine solution dropwise to the active ester solution, stir at room temperature for 12-24 hours, and monitor by TLC (developing solvent: dichloromethane / MeOH = 5:1, UV or ninhydrin color development, observe the disappearance of the carboxylic acid raw material).

[0201] 3) Post-processing

[0202] The reaction solution was poured into 50 mL of ice water and stirred for 10 minutes to precipitate a solid or emulsion. The mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed sequentially with 1 M HCl (10 mL, to remove excess N,N-diisopropylethylamine), saturated sodium bicarbonate (10 mL, to neutralize the acid), and saturated brine (10 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow oily or solid crude product. The product was then concentrated to obtain a white solid product with a yield of 73%.

[0203] Reaction C3:

[0204] 1) Dissolve the substrate cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle-OBzl (1 mmol) in 20 mL of anhydrous methanol and add the mixture to a pressure-resistant three-necked flask. Then, add Pd / C catalyst (50 mg, 10% loading) and stir to form a uniform suspension. Replace the atmosphere in the reactor with hydrogen three times, maintaining a hydrogen pressure of 1 atm. Stir the reaction at room temperature (25°C) for 12 hours and monitor by TLC (developing solvent: dichloromethane / MeOH = 5:1, UV or iodine color development, observing the disappearance of the benzyl ester spot).

[0205] 2) After the reaction is complete, the hydrogen gas is turned off and the gas in the reactor is replaced with nitrogen. The mixture is filtered through a celite pad to remove the Pd / C catalyst. The filter cake is washed with methanol (3 × 10 mL). The filtrates are combined and concentrated to dryness under reduced pressure to obtain a crude white solid product.

[0206] 3) Wash the crude product, add 500 mL of methyl tert-butyl ether and stir evenly, balance the balance, centrifuge at 3500 rpm for 2 min, discard the supernatant, and wash and centrifuge three more times using the same method; transfer the precipitate to a drying oven and drain to obtain the crude product cis-2,6-Dimethyl-piperidine-CO-tBuAla-DTrp(CO2Me)-DNle.

[0207] 4) Preparation and purification of crude product

[0208] 1) Sample pretreatment: The sample was dissolved in 50% acetonitrile / water and filtered through a 0.45 μm mixed fiber membrane.

[0209] 2) Purification preparation method:

[0210] Chromatographic column: Huapu C18 10μm 100A 50*450mm (P06)

[0211] Mobile phase A: 0.1% TFA in water

[0212] Mobile phase B: acetonitrile

[0213] Loading flow rate: 60 mL / min Elution flow rate: 60 mL / min Detection wavelength: 220 nm

[0214] Elution gradient: 60-90% in 60 minutes.

[0215] Comparative Example 2, a method for preparing a highly effective and selective antagonist BQ-788, differs from Example 1 in that Fmoc-tBuAla-OH in Formula 6-1 is replaced by an equimolar amount of tBuAla-OBzl; Formula 6-1 is eliminated, tBuAla-OBzl and cis-2,6-Dimethylpiperidine are added simultaneously, and the -Bzl group is removed after reacting for 12 hours (the process is the same as reaction C3), followed by addition of dichloromethane for dissolution, and addition of resin tBuAla-DTrp(CO2Me)-DNle-WangResin for reaction. The specific reaction equation is as follows:

[0216] Formula 10-1:

[0217] ;

[0218] The purity and yield of each step in the Examples and Comparative Examples are shown in Tables 5 and 6:

[0219] Single-step yield = theoretical / actual × 100%;

[0220] Total yield (%) = (yield 1 × yield 2 ×… × yield n) × 100%.

[0221] Table 5. Purity, yield and total reaction steps of Example 1, Example 2 and Comparative Example 2

[0222] .

[0223] Table 6. Purity, yield and total reaction steps of Comparative Example 1

[0224] .

[0225] In Example 1 of this application, the liquid-phase synthesis started with a p-methoxybenzyl group attached to the carboxyl group of Boc-DTrp-OH. The Trp side chain was then attached to dimethyl dioxanoate in the presence of 4-dimethylaminopyridine to yield Boc-DTrp(CO2Me)-OPMB. Trifluoroacetic acid was then used to simultaneously remove the Boc residue from the amino group and the PMB residue from the carboxyl group. Fmoc-OSu (9-fluorenylmethyl-N-succinimidyl carbonate) was then used under alkaline conditions to yield Fmoc-DTrp(CO2Me)-OH, which served as the starting material for solid-phase synthesis. The overall yield of the liquid-phase synthesis was 49.3%. The solid-phase synthesis used Fmoc-DNle-WangResin as the starting material. Protected amino acids were coupled sequentially from the carbon terminus to the nitrogen terminus using solid-phase synthesis methods to yield fragments. After Fmoc removal, the urea product was formed using N,N'-succinimidyl carbonate and cis-2,6-dimethylpiperidine. Finally, the final product was cleaved and purified. After preparation and purification, 3.0 g of the final product was obtained, with a solid phase synthesis yield of 46.9% and an overall yield of 23.11%.

[0226] Compared with Example 1, the total yield and purity of the liquid phase in Example 2 are lower because the indole ring of tryptophan is also reduced and hydrogenated during the hydrogenation debenzylation process in step A4, resulting in more by-products and a decrease in yield.

[0227] Compared with Example 1, the steric hindrance of methylleucine (tBuAla) in reaction B2 in comparative example 1 is greater, making it more difficult to form urea with cis-2,6-dimethylpiperidine. In addition, the hydrogenation debenzylation process in reactions C1 and C3 also causes the indole ring of tryptophan to be reductively hydrogenated, further resulting in lower product purity and yield.

[0228] Compared with Example 1, the steric hindrance in reaction D1 in Comparative Example 2 is larger, which makes it more difficult to form urea with cis-2,6-dimethylpiperidine, resulting in lower yield and purity. It also shows that the connection of methylleucine (tBuAla) to the DTrp(CO2Me)-DNle-WangResin group is conducive to the formation of urea with cis-2,6-dimethylpiperidine. This may be because the carboxyl amidation reaction of methylleucine (tBuAla) produces fewer by-products. In addition, after the reaction of methylleucine (tBuAla) with DTrp(CO2Me)-DNle-WangResin, the DTrp(CO2Me)-DNle-WangResin group may be conducive to the removal of the H group of the amine on methylleucine (tBuAla), making it easier to form urea with cis-2,6-dimethylpiperidine.

[0229] The equipment used in this application is detailed in Table 7, and the budget of Example 1 of this application is detailed in Tables 8 and 9.

[0230] Table 7. Equipment List

[0231] .

[0232] Table 8. Budget list of Example 1 of this application

[0233] .

[0234] Table 9. Budget list of Example 1 of this application

[0235] .

[0236] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing a highly effective and selective antagonist BQ-788, characterized in that: The reactants include the following building blocks: Structure (1) Structure (2), Among them, A1 is WangResin; Among them, the -NH2 group in structure (1) and the -NH- structure in structure (2) participate in the reaction and are connected through the carbonyl group, and the reaction prepares the antagonist BQ-788; The reactants of structure (1) include the following structural units: Structure (3) Structure (4), Structure (5), The amine group formed by connecting A2 or removing the protection of A2 undergoes an amidation reaction with the carboxyl group in structure (5), and the carboxyl group formed by connecting A3 or removing the protection of A3 undergoes an amidation reaction with the amine group formed by connecting A4 or removing the protection of A4 to obtain structure (1); In the structure (3), -A2 is an -Fmoc group, and -A3 is an -H group; The preparation process of the structure (3) is as follows: 1) Liquid phase synthesis of raw material Boc-DTrp-OPMB: 9-11 g of Boc-DTrp-OH was dissolved in an organic solvent, 4-6 g of sodium carbonate was added, and 4-6 mL of 4-methoxybenzyl chloride was slowly added dropwise. After completion of the addition, the mixture was stirred at room temperature for 11-15 h to react. Boc-DTrp-OPMB was then isolated by vacuum rotary evaporation, extraction, washing, and purification. The reaction process is as follows: ； 2) Liquid phase synthesis of raw material Boc-DTrp(CO2Me)-OPMB: 9-11 g of Boc-DTrp-OPMB was dissolved in 40-60 mL of acetonitrile, 3-5 mL of dimethyl dioxanoate and 0.5-1 g of 4-dimethylaminopyridine were added, and the mixture was stirred at 25-30° C. for 18-25 h. Boc-DTrp(CO2Me)-OPMB was then isolated by vacuum rotary evaporation, extraction, washing, and purification. The reaction process is as follows: ； 3) Liquid phase synthesis of raw material H-DTrp(CO2Me)-OH: 8-9.5 g of Boc-DTrp(CO2Me)-OPMB was dissolved in 80-100 mL of cutting solution and stirred at room temperature for 1.5-3 h. After removing the cutting solution by rotary evaporation, water and acetonitrile were added and freeze-dried to obtain H-DTrp(CO2Me)-OH. The reaction process is as follows: ； 4) Liquid phase synthesis of raw material Fmoc-DTrp(CO2Me)-OH: 5-7.5 g of H-DTrp(CO2Me)-OH obtained in step 3) is dissolved in a mixture of 90 mL of water and tetrahydrofuran, 2.5-4 g of sodium carbonate is added, and then 5-7 g of 9-fluorenylmethyl-N-succinimidyl carbonate is added, and stirred at room temperature for 10-15 h; then, after vacuum rotary evaporation, extraction, washing, and purification, structure (2) is separated and obtained, namely Fmoc-DTrp(CO2Me)-OH. The reaction process is as follows: ； The preparation process of the structure (1) is as follows: Solid phase synthesis was adopted; wherein, the -A4 group was -H, and the -A5 group was -WangResin; The specific reactions are as follows: 1) The resin is fully swollen 16-18.5 g of Fmoc-DNle-WangResin was soaked in N,N-dimethylformamide to swell. After vacuum filtration to remove N,N-dimethylformamide, 200-300 mL of 20% piperidine / DMF solution was added and deprotected by nitrogen bubbling for 0.2-0.8 h to remove the Fmoc protecting group. The resin was then washed with N,N-dimethylformamide to obtain resin A. 2) Connecting Fmoc-DTrp(CO2Me)-OH Taking Fmoc-DNle-WangResin resin as the base, weigh 1.3 times the amount of Fmoc-DTrp(CO2Me)-OH and 1.3 times the amount of HOBt and dissolve them in 200 mL of N,N-dimethylformamide. Cool to -10°C, add 1.3 times the amount of DIC, activate for 10 minutes, and then add to resin A. Couple at room temperature for 1-2 hours. After testing, the amino group has been completely coupled. After the coupling is completed, wash with N,N-dimethylformamide and dry. Then, add 200-300 mL of 20% piperidine / DMF solution to remove Fmoc, deprotect with nitrogen for 0.1-0.8 hours, and then wash with N,N-dimethylformamide to obtain resin DTrp(CO2Me)DNle-WangResin. 3) Connecting Fmoc-tBuAla-OH: Using DTrp(CO2Me)DNle-WangResin as the base, weigh 1.2-1.8 times the amount of Fmoc-tBuAla-OH and 1.2-1.8 times the amount of HOBt and dissolve them in 200 mL of N,N-dimethylformamide. Cool to -10 degrees Celsius, add 1.5 times the amount of DIC to activate for 10 minutes, then add to the resin. Couple at room temperature for 1-2 hours. Kaiser assay shows colorless. After coupling, wash with N,N-dimethylformamide solution and drain. 4) Removal of Fmoc protecting group: The N,N-dimethylformamide was removed by vacuum filtration, and the mixture was washed with N,N-dimethylformamide solution and dried. 200-280 mL of 20% piperidine / DMF solution was added, and nitrogen was blown for deprotection for 0.2-0.8 h. The mixture was then washed with N,N-dimethylformamide to obtain the resin tBuAla-DTrp(CO2Me)-DNle-WangResin. The specific reactions are as follows: 。 2. The method for preparing a highly effective and selective antagonist BQ-788 according to claim 1, characterized in that: The preparation method of the antagonist BQ-788 includes the following preparation process: 1): 2,6-dimethylpiperidine forms urea: Taking the resin tBuAla-DTrp(CO2Me)-DNle-WangResin as the base, weigh 1.2-1.8 times the amount of 2,6-methylpiperidine and dissolve it in acetonitrile. Add 2.8-3.5 times the amount of triethylamine, then add 2.5-3 times the amount of N,N'-succinimidyl carbonate DSC, stir at room temperature for 8-15 hours, then concentrate, add dichloromethane to dissolve, then add to the resin, add 2.8-3.2 times the amount of N,N-diisopropylethylamine, react for 10-15 hours, and it is colorless after Kaiser detection. After the coupling is completed, add N,N-dimethylformamide to wash and drain to obtain cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle-WangResin; 2): Cutting steps Disperse and suspend 20-23g of cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle-WangResin in 200-250mL of liquid F and shake at room temperature for 2-3h to obtain a resin suspension, wherein the ratio of liquid F is: 95wt% of trifluoroacetic acid, 2.5wt% of triisopropylsilane and 2.5wt% of water; The resin suspension was filtered, washed with liquid F and methyl tert-butyl ether, and centrifuged to obtain a crude product. After purification, the resin cis-2,6-Dimethylpiperidine-CO-tBuAla-DTrp(CO2Me)-DNle was obtained. The reaction process was as follows: 。 3. The method for preparing a highly effective and selective antagonist BQ-788 according to claim 1, characterized in that: The purity of the antagonist BQ-788 obtained by the preparation method is greater than 90%, and the total synthesis yield is greater than 20%.

4. The method for preparing a highly effective and selective antagonist BQ-788 according to claim 2, characterized in that: The purification process is to first dissolve it with acetonitrile / water, filter it with a 0.35-0.55 μm mixed fiber membrane, and then purify it with a chromatographic column.

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