A tetrapeptide compound TP-3, preparation method and use thereof

Abstract

The application discloses a tetrapeptide compound and a preparation method and application thereof. The tetrapeptide compound is TP shown in formula 1, and the preparation method comprises the following steps: firstly, an intermediate compound is prepared; then, the intermediate compound and a catalyst Pd / C are added into a solvent, hydrogen gas with a pressure of 1-30 Mpa is introduced into the solvent to react, and a final product tetrapeptide TP is obtained. The compound can be used in an Aldol reaction of a ketone and an aromatic aldehyde, or used in catalyzing asymmetric conjugate addition reaction of an aliphatic aldehyde and a maleimide. The tetrapeptide compound can efficiently and asymmetrically catalyze the conjugate addition reaction of the aliphatic aldehyde and the maleimide, the yield of the reaction reaches 98 %, and the highest enantioselectivity reaches 99 %.

Description

Technical Field

[0001] This invention relates to a tetrapeptide compound, its preparation method, and its uses. More specifically, this invention relates to a tetrapeptide compound, its preparation method, and its uses, particularly a compound that can be used to catalyze the asymmetric conjugate addition reaction of aliphatic aldehydes and maleimides, along with its preparation method and uses. Background Technology

[0002] Optically active compounds are a ubiquitous phenomenon in nature, playing an irreplaceable role in the life processes of cells and organisms. Many drugs exhibit optical activity, and chirality is a crucial element of drug development. Catalyzing asymmetric reactions with chiral catalysts is the most economical and environmentally friendly method for synthesizing chiral compounds. Asymmetric catalysis encompasses three areas: chiral ligand-metal complex catalysts, small organic molecule catalysts, and bioenzyme catalysis. Due to its environmental friendliness and good economic performance, bioenzyme catalysis has always been a hot research area in asymmetric catalysis; however, the complexity of enzyme protein structures, their susceptibility to inactivation, and their specificity limit their rapid application. Peptides are a novel class of organic catalysts designed and synthesized according to enzyme catalytic mechanisms. They possess numerous advantages, including simple structure, ease of synthesis, broad substrate range, adaptability to various reaction types, mild reaction conditions, and structural stability. They are excellent mimics of natural enzymes and a key research direction in chemical biology.

[0003] Existing reports have shown that artificially synthesized short peptides can effectively replace natural proteases and mimic enzyme catalysis of a series of chemical reactions. They are widely used in catalysis of many asymmetric reactions such as Aldol, Michael, Stetter, azidation, Strecker, Baylis-Hillman, allyl substitution, hydrocyanation, epoxidation, hydrogenation, and acylation, synthesizing a variety of optically active compounds. This effectively overcomes the shortcomings of protease-catalyzed reactions in terms of substrate specificity and single reaction type, expands the types of reactions and substrates, is suitable for various reaction conditions, and basically maintains the advantages of green and economical biocatalysis [(1) Liu Aixiang, Fu Yao, Liu Lei, Guo Qingxiang, Organic Chemistry , 2007, 27 , 1195-1219. (2) Davie, EAC;Mennen, SM; Xu, Y.; Miller, SJ Chem. Rev. 2007, 107 , 5759-5812. (3)Wennemers, H. Chem. Commun. ,2011, 47 , 12036–12041. (4) Lewandowski, B.;Wennemers, H. Curr. Opin. Chem. Biol. 2014, 22, 40–46. (5) Kelly, DR;Roberts, SM Biopolymers 2006, 84 , 74–89. (6) Akagawa, K.; Kudo, K. Acc. Chem. Res. 2017, 50 , 2429-2439. (7) Ball, ZT Acc. Chem. Res. 2012, 46 [560-570.]. Therefore, the discovery of novel and highly efficient short peptide catalysts to mimic protease-catalyzed organic chemical reactions has always been a hot topic in the field of chiral compound synthesis.

[0004] Under asymmetric catalytic conditions, the nucleophile and N Chiral 3-substituted succinimides, synthesized via asymmetric conjugation of maleimides, can be readily converted into a series of chiral pyrrole compounds through reduction. Chiral pyrrole compounds form the core skeleton of many bioactive compounds, and the asymmetric synthesis of chiral pyrrole compounds has always been a core research topic in organic chemistry and medicinal chemistry. Furthermore, chiral 3-substituted succinimides can be readily converted into chiral succinic acids via hydrolysis, which are important physiologically active compounds and building blocks for chiral synthesis. Aldehydes, as nucleophiles, are asymmetric conjugation products of maleimides, possessing an additional effective group that readily derives into other functional groups, making this reaction of significant application value. Therefore, the continuous development of highly efficient chiral catalysts for catalyzing the asymmetric conjugation of aliphatic aldehydes with maleimides is a goal pursued by many research institutions. In summary, the catalysts successfully applied to this reaction are mostly chiral organic catalysts, and the vast majority are primary amine thiourea or squaramide catalysts derived from chiral cyclohexanediamine, achieving ideal yields and enantioselectivity [(8) Xue, F.; Liu, L.; Zhang, S.; Duan, W.; Wang, W.]. Chem. Eur. J. 2010, 16 , 7979-7982; (9) Ma, Z.-W.;Liu, Y.-X.; Zhang, W.-J.; Tao, Y.; Zhu, Y.; Tao, J.-C.; Tang, M.-S. Eur. J. Org. Chem. 2011, 6747-6754. (10) Orlandi, S.; Pozzi, G.; Ghisetti, M.; Benaglia, M. New J. Chem. 2013, 37, 4140-4147. (11) Yu, F.; Jin, Z.; Huang, H.; Ye, T.; Liang, X.; Ye, J. Org. Biomol. Chem. 2010, 8 , 4767-4774. (12) Bai, J.-F.; Peng, L.; Wang, L.-L.; Wang, L.-X.; Xu, X.-Y. Tetrahedron 2010, 66 , 8928-8932. (13) Ma, Z.-W.; Liu, Y.-X.; Li, P.-L.; Ren, H.; Zhu, Y.; Tao, J.-C.; Tetrahedron: Asymmetry 2011, 22 , 1740-1748. (14) Ma, Z.-W.; Liu, X.-F.; Liu,J.-T.; Liu, Z.-J.; Tao, J.-C. Tetrahedron Letters 2017, 58 [4487-4490.]. Cinchona bark thiourea and 2-chlorophenylglycine, as a highly efficient dual-catalyst system, also achieved ideal yields and enantioselectivity [(15) Muramulla, S.; Ma, J.-A.; Zhao, JC-G.]. Adv. Synth. Catal. 2013, 355 [1260-1264]. Chiral primary amines derived from cinchona alkaloids exhibit excellent catalytic yields and enantioselectivity under the addition of triphenylphosphine [(16) Yang, W.; Jiang, K.-Z.; Lu, X.; Yang, H.-M.; Li, L.; Lu,Y.; Xu, L.-W.]. Chem. Asian J. 2013, 8 [1182-1190]. Under the condition of adding alkali metals, it was found that β-phenylalanine, aspartic acid-α-tert-butyl ester, and threonine and isoleucine with tert-butyl side chain protection all achieved good asymmetric catalytic effects in this reaction [(17) Kokotos, CG]. Org. Lett. 2013, 15 , 2406-2409. (18)Nugent, TC; Sadiq, A.; Bibi, A.; Heine, T.; Liu L.; Vankova, N.; Bassil,BS Chem. Eur. J. 2012, 18[4088-4098]. There is also a report of a dipeptide Ala-Ala catalyzing the reaction of propionaldehyde with maleimide, with the product having an ee value of only 38% [(19) Zhao, G.-L.; Xu, Y.; Sundén, H.; Eriksson, L.; Sayah, M.; Córdova, A.]. Chem. Commun. [2007, 734–735.]. Therefore, developing efficient peptide catalysts for this reaction is a challenging task. Summary of the Invention

[0005] This invention provides a compound that overcomes the shortcomings of the prior art and can efficiently and asymmetrically catalyze the conjugate addition reaction of aliphatic aldehydes and maleimides.

[0006] The tetrapeptide compound of this invention is TP, as shown in Formula 1.

[0007] Wherein: R1 and R2 are any one of the following: straight-chain alkyl, branched alkyl, cycloalkyl, hydroxy-substituted alkyl, mercapto-substituted alkyl, methylthio-substituted alkyl, amino-substituted alkyl, guanidinyl-substituted alkyl, aryl, arylmethyl or heteroatom aryl from C1 to C6.

[0008] Preferably, in the tetrapeptide compound of the present invention, R1 and R2 are any one of straight-chain alkyl, branched-chain alkyl, cyclohexyl, phenyl, and benzyl groups from C1 to C4.

[0009] More specifically, the tetrapeptide compound of the present invention has R1= i -Pr, R2= i -Bu, that is, its amino acid sequence is H2N-D-Val-Pro-Gly-D-Leu-OH, named TP-1.

[0010] Alternatively, the tetrapeptide compound of the present invention, wherein R1= t -Bu, R2= i -Bu, that is, its amino acid sequence is H2N-D-Tle-Pro-Gly-D-Leu-OH, named TP-2.

[0011] Alternatively, the tetrapeptide compound of the present invention, wherein R1 = Ph, R2 = i -Bu, that is, its amino acid sequence is H2N-D-Phg-Pro-Gly-D-Leu-OH, named TP-3.

[0012] Alternatively, the tetrapeptide compound of the present invention, wherein R1= c -hex, R2= i-Bu, that is, its amino acid sequence is H2N-D-Chg-Pro-Gly-D-Leu-OH, named TP-4.

[0013] Alternatively, the tetrapeptide compound of the present invention, wherein R1 = Bn, R2 = i -Bu, that is, its amino acid sequence is H2N-D-Phe-Pro-Gly-D-Leu-OH, named TP-5.

[0014] Alternatively, the tetrapeptide compound of the present invention, wherein R1=Bn, R2=Bn, that is, its amino acid sequence is H2N-D-Phe-Pro-Gly-D-Phe-OH, is named TP-6.

[0015] The method for preparing the tetrapeptide compound of the present invention comprises the reaction mechanism shown in Formulas 2, 3, 4 and 5, namely:

[0016] Glycine 1, amino acid 2, condensing agent C-1 and additive A-1 were added to solvent S-1 and stirred to react, yielding product 3. Then, product 3 and deprotecting agent D-1 were added to solvent S-2 and stirred to react, yielding product 4. Product 4, proline 5, condensing agent C-2 and additive A-2 were added to solvent S-3 and stirred to react, yielding product 6. Then, product 6 and deprotecting agent D-2 were added to solvent S-4 and stirred to react, yielding product 7. Product 7, amino acid 8, condensing agent C-3 and additive A-3 were added to solvent S-5 and stirred to react, yielding product 9. Then, product 9 and deprotecting agent D-3 were added to solvent S-6 and stirred to react, yielding product 10. Product 10 and catalyst Pd / C were added to solvent S-7, and hydrogen gas at a pressure of 1-30 MPa was introduced to react and obtain the final product tetrapeptide TP, wherein: R3 is an arylmethyl group; R4, R5, and R6 are any one of Boc (tert-butyloxyformyl), Cbz (benzyloxyformyl), or Fmoc (fluorenyloxyformyl). The condensing reagents C-1, C-2 and C-3 are any one of carbodiimide, alkoxyformyl chloride or R7OCOCl, where R7 is any straight-chain alkyl or branched alkyl from C1 to C4. Additives A-1, A-2, and A-3 refer to R8R9R 10 N or NMM (N-methylmorpholine) or TMEDA (4-dimethylaminopyridine) or HOBt (1-hydroxybenzotriazole), R8, R9, R 10 It is any straight-chain alkyl or branched alkyl group from C1 to C4; Solvents S-1, S-2, S-3, S-4, S-5, and S-6 are any one or a combination of several of toluene, benzene, dichloromethane, dichloroethane, diethyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, methyl tert-butyl ether, hexaoxide, ethyl acetate, methyl acetate, acetonitrile, or propionitrile; solvent S-7 is any one or a combination of several of methanol, ethanol, propanol, or butanol. The deprotecting agents D-1, D-2 and D-3 are any one of methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, trichloroacetic acid, hydrochloric acid, sulfuric acid, piperidine, morpholine, tetrahydropyrrole, dihydropyrrole, pyrrole, diethylamine, dipropylamine, dibutylamine, diisopropylamine or diisobutylamine.

[0017] Preferably, the method for preparing the compound according to the present invention includes: R3 is benzyl; R4, R5, and R6 refer to Boc or Cbz; Condensing agents C-1, C-2, and C-3 are DCC (N,N'-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide) or alkoxyformyl chloride R7OCOCl, where R7 is Et or i -Pr or i -Bu; Additives A-1, A-2 and A-3 are any one of TEA (triethylamine), DIPEA (diisopropylethylamine), NMM or HOBt; Solvents S-1, S-2, S-3, S-4, S-5 and S-6 are dichloromethane or tetrahydrofuran, and solvent S-7 is methanol or ethanol; The deprotecting agents D-1, D-2 and D-3 are methanesulfonic acid, trifluoromethanesulfonic acid or trifluoroacetic acid.

[0018] Preferably, the method for preparing the compound according to the present invention includes: R3 is benzyl; R4, R5, and R6 are Boc; Condensing reagents C-1, C-2, and C-3 are DCC or DIC or alkoxyformyl chloride R7OCOCl, and R7 is Et or i -Pr or i -Bu; Additives A-1, A-2, and A-3 refer to any one of TEA, DIPEA, NMM, or HOBt. Solvents S-1, S-2, S-3, S-4, S-5, and S-6 refer to dichloromethane or tetrahydrofuran, while solvent S-7 refers to methanol. Deprotectants D-1, D-2, and D-3 refer to trifluoroacetic acid; The hydrogen pressure is 8 MPa.

[0019] The compounds described in this invention can be used for the Aldol reaction of ketones with aromatic aldehydes, or for catalyzing the asymmetric conjugate addition reaction of aliphatic aldehydes with maleimides as shown in Formula 6. That is: the aliphatic aldehyde 11 shown in Formula 6,

[0020] Maleimide 12 and tetrapeptide TP were added to a reaction vessel containing solvent S-8 and stirred to obtain the product of the conjugated addition reaction, 3-substituted succinimide. R )-P, where: R 11 It is hydrogen or any straight-chain alkyl or branched-chain alkyl, phenyl or benzyl, C1 to C6, R 12 It is any straight-chain alkyl or branched-chain alkyl or phenyl or benzyl from C1 to C6, or -R 11 -R 12 - is cyclohexyl or cyclopentyl, R 13 It is hydrogen or phenyl or substituted phenyl or straight-chain alkyl or cycloalkyl or arylmethyl; The tetrapeptide TP is a tetrapeptide containing two terminal D-type-α-primary amino acids, as shown in Formula 1. In the above reaction, solvent S-8 refers to any one or more of the following: dichloromethane, chloroform, dichloroethane, ethyl acetate, methyl acetate, toluene, benzene, xylene, diethyl ether, tetrahydrofuran, ethylene glycol dimethyl ether, methyl tert-butyl ether, acetonitrile, propionitrile, ethanol, methanol, propanol, and butanol.

[0021] Preferably: R 11 It is hydrogen, methyl, or ethyl, R 12 It is methyl, ethyl, isopropyl, or n-butyl, or -R 11 -R 12 - is cyclohexyl or cyclopentyl, R 13 It is hydrogen or methyl or cyclohexyl or benzyl or phenyl or substituted phenyl, and substituted phenyl refers to p-methylphenyl or p-chlorophenyl or p-bromophenyl or p-fluorophenyl or p-nitrophenyl or p-methoxyphenyl; The tetrapeptide TP is any one of the aforementioned TP-1 to TP-6; Solvent S-8 is any one of dichloromethane, acetonitrile, ethanol, tetrahydrofuran, dimethyl sulfoxide, and toluene.

[0022] More preferably, the tetrapeptide TP is TP-3; and the solvent S-8 is acetonitrile.

[0023] The intermediate compounds used in this invention to prepare compound TP of formula 1 are as follows: as shown in formula 3.6, formula 3.7, formula 4.9, or formula 4.10.

[0024] The compounds of this invention can catalyze the conjugated addition reaction of aliphatic aldehydes and maleimides with high yield and high enantioselectivity in an asymmetric catalytic process. According to relevant experiments, the tetrapeptide compounds of this invention can efficiently and asymmetrically catalyze the conjugated addition reaction of aliphatic aldehydes and maleimides, achieving a yield of 98% and an enantioselectivity of up to 99%, demonstrating promising application prospects. Detailed Implementation

[0025] The present invention will be explained below with reference to the embodiments.

[0026] I. Preparation of the tetrapeptide compound TP The reaction mechanism for preparing the tetrapeptide compound TP, as shown in Formula 1 of this invention, is described in Formulas 2 to 5 above. The preparation method is detailed in the invention summary section, and the preparation process is as follows: (a) Synthesis of intermediate compound 3 1.1 Boc-Gly-D-Leu-OBn (3a)

[0027] Referring to the above formula, add Boc-Gly-OH1 (8.75 g, 50 mmol) to 100 mL of anhydrous THF, and stir for 5 min in an ice-salt bath under argon protection to lower the temperature of the reaction solution to -15 °C. Add NMM (6.2 mL, 55 mmol), followed by slow dropwise addition of isobutyl chloroformate (7 mL, 55 mmol) to the reaction solution. After stirring for 5 min, add a solution of D-Leu-OBn2a p-toluenesulfonate (19.7 g, 50 mmol) and NMM (6.2 mL) previously dissolved in anhydrous DMF, and continue stirring at -15 °C. o The reaction was continued under C with stirring for 0.5 h. The ice-salt bath was removed, and the reaction solution was allowed to return to room temperature naturally. The reaction was allowed to proceed overnight. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and then dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and a small amount of saturated brine. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 19 g of Boc-Gly-D-Leu-OBn (3a) as a pale yellow oily liquid, with a yield of 100%. [α] D 20 = + 5.0 ( c 1.0, CHCl3); 1 HNMR (200 MHz, CDCl3) δ 7.34 – 7.28 (m, 5H), 6.94 – 6.90 (d, J= 6.8 Hz, 1 H),5.51 (m, 1 H), 5.15 (s, 2H), 4.69 – 4.64 (m, 1 H), 3.83 – 3.81 (m, 2H), 1.62– 1.51 (m, 3H), 1.44(s, 9H), 0.91 – 0.89(m, 6H); 13 C NMR (100 MHz, CDC 13 ) δ172.6, 169.4, 156.0, 135.3, 128.5, 128.4, 128.2, 128.0, 80.0, 66.9, 50.7,44.1, 41.1, 28.1, 24.7, 22.7, 21.7; ESI-MS calcd for [C 20 H 30 N2O5+ H + ] 379.2, found: 379.3.

[0028]

[0029] Alternatively, as above, add Boc-Gly-OH1 (8.75 g, 50 mmol) to 100 mL of anhydrous THF, stir for 5 min in an ice-salt bath under argon protection, and lower the temperature of the reaction solution to -15 °C. Add NMM (6.2 mL, 55 mmol), followed by slow dropwise addition of ethyl chloroformate (5.3 mL, 55 mmol) to the reaction solution. After stirring for 5 min, add a solution of D-Leu-OBn2a p-toluenesulfonate (19.7 g, 50 mmol) and NMM (6.2 mL) previously dissolved in anhydrous DMF, and continue stirring at -15 °C. o The reaction was continued under C temperature for half an hour with stirring. The ice-salt bath was removed, and the reaction solution was allowed to return to room temperature naturally. The reaction was allowed to proceed overnight. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and then dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and a small amount of saturated brine. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 17.30 g of Boc-Gly-D-Leu-OBn (3a), which was a pale yellow oily liquid with a yield of 91%.

[0030]

[0031] Alternatively, referring to the above formula, add Boc-Gly-OH1 (8.75 g, 50 mmol) to 100 mL of anhydrous THF, stir for 5 min in an ice-salt bath under argon protection, and lower the temperature of the reaction solution to -15 °C. Add NMM (6.2 mL, 55 mmol), followed by slow dropwise addition of isopropyl chloroformate (6.3 mL, 55 mmol) to the reaction solution. After stirring for 5 min, add a solution of D-Leu-OBn2a p-toluenesulfonate (19.7 g, 50 mmol) and NMM (6.2 mL) previously dissolved in anhydrous DMF, and heat at -15 °C. o The reaction was stirred for another 0.5 h at C, then the ice-salt bath was removed, and the reaction solution was allowed to return to room temperature naturally. The reaction was allowed to proceed overnight. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and then dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and a small amount of saturated brine. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 17.67 g of Boc-Gly-D-Leu-OBn (3a), which was a pale yellow oily liquid with a yield of 93%.

[0032]

[0033] Alternatively, as above, add Boc-Gly-OH (8.75 g, 50 mmol) to 100 mL of anhydrous THF, stir for 5 min in an ice-salt bath under argon protection, and lower the temperature of the reaction solution to -15 °C. Add TEA (triethylamine, 7.6 mL, 55 mmol), followed by slow dropwise addition of isobutyl chloroformate (7 mL, 55 mmol) to the reaction solution. After stirring for 5 min, add a solution of D-Leu-OBn p-toluenesulfonate (19.7 g, 50 mmol) and TEA (7.6 mL) previously dissolved in anhydrous DMF, and continue stirring at -15 °C. o The reaction was stirred for another 0.5 h at C, then the ice-salt bath was removed, and the reaction mixture was allowed to return to room temperature naturally. The reaction was allowed to proceed overnight. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and then dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and a small amount of saturated brine. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 16.48 g of Boc-Gly-D-Leu-OBn (3a), which was a pale yellow oily liquid with a yield of 87%.

[0034]

[0035] Alternatively, as above, add Boc-Gly-OH1 (8.75 g, 50 mmol) to 100 mL of anhydrous THF, stir for 5 min in an ice-salt bath under argon protection, and lower the temperature of the reaction solution to -15 °C. Add DIPEA (diisopropylethylamine, 9.6 mL, 55 mmol), followed by slow dropwise addition of isobutyl chloroformate (7 mL, 55 mmol). After stirring for 5 min, add a solution of D-Leu-OBn2a p-toluenesulfonate (19.7 g, 50 mmol) and DIPEA (9.6 mL) previously dissolved in anhydrous DMF, and continue stirring at -15 °C. o The reaction was stirred for another 0.5 h at C, then the ice-salt bath was removed, and the reaction solution was allowed to return to room temperature naturally. The reaction was allowed to proceed overnight. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and then dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and a small amount of saturated brine. After drying with anhydrous sodium sulfate, the solution was concentrated under reduced pressure to obtain 15.47 g of Boc-Gly-D-Leu-OBn (3a), which was a pale yellow oily liquid with a yield of 81%.

[0036] 1.2 Synthesis of Boc-Gly-D-Phe-OBn (3b)

[0037] Boc-Gly-OH1 (8.75 g, 50 mmol) was added to 100 mL of anhydrous THF. The mixture was stirred for 5 min in an ice-salt bath under argon protection, allowing the temperature of the reaction solution to drop to -15 °C. NMM (6.2 mL, 55 mmol) was then added, followed by the slow addition of isobutyl chloroformate (7 mL, 55 mmol) to the reaction solution. After stirring for 5 min, a solution of D-Phe-OBn2b p-toluenesulfonate (21.35 g, 50 mmol) and NMM (6.2 mL) previously dissolved in anhydrous DMF was added. The mixture was then stirred at -15 °C. o The reaction was continued under C with stirring for 0.5 h. The ice-salt bath was removed, and the reaction mixture was allowed to return to room temperature overnight. After the reaction was complete, insoluble matter was removed by filtration. The filtrate was concentrated under reduced pressure and then dissolved in ethyl acetate. The ethyl acetate phase was washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and a small amount of saturated brine. After drying with anhydrous sodium sulfate, the phase was concentrated under reduced pressure to obtain a pale yellow oil, Boc-Gly-D-Phe-OBn (3b). Column chromatography with petroleum ether and ethyl acetate yielded 19.16 g of a white solid, 93% yield. White solid, Mp 73.5-75 o C; [α] D 25 = -11.6 (c 0.5, CHCl3); 1 H NMR (600 MHz, CDCl3) δ7.35 (m, 3H), 7.30 – 7.27 (m, 1H), 7.23 – 7.18 (m, 2H), 7.13 – 6.86 (m, 2H), 6.63 (d, J = 5.7 Hz, 1H), 5.16 - 5.08 (m, 2H), 4.92 (dd, J = 6, 12 Hz, 1H), 3.86– 3.71 (m, 2H), 3.15 – 3.05 (m, 2H), 1.44 (d, J = 8.5 Hz, 9H). 13 C NMR (150MHz, CDCl3) δ 171.1, 169.1, 135.5, 135.0, 129.3, 128.6, 128.6, 128.5, 128.5,128.4, 128.2, 127.1, 77.2, 77.0, 76.8, 67.3, 53.1, 41.4, 37.9, 28.3(s), 24.8,22.8, 21.8; ESI-MS calcd for [C 23 H 28 N2O5+ H + ] 413.2, found 413.2..

[0038] (II) Synthesis of intermediate compound 4 2.1 Synthesis of NH2-Gly-D-Leu-OBn (4a)

[0039] The dipeptide Boc-Gly-D-Leu-OBn3a (19 g) obtained from the above reaction was dissolved in 50 mL of dichloromethane. The reaction solution was cooled to 0 °C, and TFA (trifluoroacetic acid, 50 mL) was slowly added dropwise. After the reaction solution was allowed to recover naturally to room temperature, it was stirred for 5 h. The reaction solution was then concentrated under reduced pressure to remove TFA. The residue was dissolved in dichloromethane, and the pH was adjusted to approximately 9.0 with 1.0 M NaOH aqueous solution at 0 °C. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed successively with a small amount of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product NH2-Gly-D-Leu-OBn (4a) as a colorless oily liquid with a yield of 13.25 g (95%). No purification was required, and it was used directly in the next reaction.

[0040]

[0041] Alternatively, as shown above, dissolve the dipeptide Boc-Gly-D-Leu-OBn 3a (19 g) obtained from the above reaction in 50 mL of dichloromethane. Cool the reaction solution to 0 °C, and slowly add methanesulfonic acid (50 mL) dropwise. After the reaction solution is allowed to recover naturally to room temperature, stir for 5 h, and then adjust the pH to approximately 9.0 with 1.0 M NaOH aqueous solution at 0 °C. Separate the organic phase, and extract the aqueous phase with dichloromethane (20 mL × 3). Combine the organic phases, wash them successively with a small amount of saturated brine, dry them with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the product NH2-Gly-D-Leu-OBn (4a) as a colorless oily liquid with a yield of 12.83 g (92%). No purification is required; it can be used directly in the next reaction.

[0042]

[0043] Alternatively, as shown above, dissolve the dipeptide Boc-Gly-D-Leu-OBn 3a (19 g) obtained from the above reaction in 50 mL of dichloromethane. Cool the reaction solution to 0 °C, and slowly add 50 mL of trifluoromethanesulfonic acid. After allowing the reaction solution to naturally recover to room temperature, stir for 5 h, and then adjust the pH to approximately 9.0 with 1.0 M NaOH aqueous solution at 0 °C. Separate the organic phase, and extract the aqueous phase with dichloromethane (20 mL × 3). Combine the organic phases, wash them successively with a small amount of saturated brine, dry them with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the product NH2-Gly-D-Leu-OBn (4a) as a colorless oily liquid with a yield of 12.7 g (91%). No purification is required; it can be used directly in the next reaction.

[0044] 2.2 Synthesis of H2N-Gly-D-Phe-OBn (4b)

[0045] The dipeptide Boc-Gly-D-Phe-OBn3b (18.2 g, 44 mmol) obtained from the above reaction was dissolved in 44 mL of dichloromethane. The reaction solution was cooled to 0 °C, and TFA (44 mL) was slowly added dropwise. After the reaction solution was allowed to recover naturally to room temperature, it was stirred for 5 h. The reaction solution was then concentrated under reduced pressure to remove TFA. The residue was dissolved in dichloromethane, and the pH was adjusted to approximately 9.0 with 1 M NaOH aqueous solution at 0 °C. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed successively with a small amount of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product NH2-Gly-D-Leu-OBn (4b) as a colorless oily liquid with a yield of 12.5 g (91%). No purification was required, and it was used directly in the next reaction.

[0046] (III) Synthesis of intermediate compound 6 3.1 Synthesis of Boc-Pro-Gly-D-Leu-OBn (6a)

[0047] Boc-Pro-OH5 (9.68 g, 45 mmol) was added to 100 mL of anhydrous THF. The mixture was stirred for 5 min in an ice-salt bath under argon protection to lower the temperature of the reaction solution to -15 °C. NMM (5.6 mL, 50 mmol) was added. Isobutyl chloroformate (6.4 mL, 50 mmol) was slowly added dropwise to the reaction solution. After stirring for 5 min, a solution of dipeptide NH2-Gly-D-Leu-OBn4a (12.56 g, 45 mmol) and NMM (5.6 mL) that had been dissolved in anhydrous THF was added. The mixture was stirred for another 30 minutes under an ice-salt bath. The ice-salt bath was then removed, and the reaction was allowed to return to room temperature. The mixture was stirred overnight until the reaction was complete. Insoluble matter was filtered off, and the filtrate was concentrated under reduced pressure. The remaining oily substance was dissolved in ethyl acetate and then washed successively with 1.0 M NaOH solution, water, 1.0 M hydrochloric acid solution, and saturated brine. The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a pale yellow oily liquid, Boc-Pro-Gly-D-Leu-OBn (6a). Purification by column chromatography with petroleum ether and ethyl acetate yielded 20.33 g of product, 95% yield. Whitesolid, Mp: 104–105 ℃; [α] D 20 = -27 ( c 1.0, CHCl3); 1¹HNMR (400 MHz, CDCl₃) δ7.38 – 7.32 (m, 5H), 7.22 (br, 1H), 7.03 (br, 1H), 5.08 – 5.07 (dd, J = 12.8 Hz, J = 21.6 Hz, 2H), 4.57 – 4.55 (m, 1H), 4.25 – 4.11 (m, 2H), 3.86 – 3.81 (dd, J = 4.8 Hz, J = 17.2 Hz, 1H), 3.50 – 3.40 (m, 2H), 2.15 – 2.11 (m, 2H), 1.92 – 1.89 (m, 2H), 1.60 – 1.73 (m, 3H), 1.44 (s, 9H), 0.92 – 0.89 (m, 6H); 13 ¹³C NMR (100 MHz, CDCl₃) δ 172.5, 169.3, 155.8, 128.5, 128.1, 128.0, 109.7, 80.8, 66.7, 60.9, 51.0, 47.4, 42.8, 40.1, 29.5, 28.3, 24.7, 24.6, 22.8, 21.5; ESI-MS calcd for [C 25 H 37 N₃O₆+ H + 476.3, found: 476.3.

[0048] 3.2 Synthesis of Boc-Pro-Gly-D-Phe-OBn (6b)

[0049] Boc-Pro-OH5 (7.53 g, 35 mmol) was added to 100 mL of anhydrous THF. The mixture was stirred for 5 min in an ice-salt bath under argon protection to lower the temperature of the reaction solution to -15 °C. NMM (4.3 mL, 38.5 mmol) was added. Isobutyl chloroformate (4.9 mL, 38.5 mmol) was slowly added dropwise to the reaction solution. After stirring for 5 min, a solution of dipeptide NH2-Gly-D-Phe-OBn4b (10.92 g, 35 mmol) and NMM (4.3 mL) that had been dissolved in anhydrous THF was added. Continue stirring under an ice-salt bath for 30 min, then remove the ice-salt bath and allow the reaction to return to room temperature naturally. Continue stirring overnight until the reaction is complete. Filter out the insoluble matter, concentrate the filtrate under reduced pressure, dissolve the remaining oily substance in ethyl acetate, and then wash successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. Dry under anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography with petroleum ether and ethyl acetate to obtain the Boc-Pro-Gly-D-Phe-OBn (6b) product, 17.1 g, yield 96%. White solid, MPa 100-102. o C;[α] D 25 = -66.0 ( c 0.5, CHCl3); 1 H NMR (600 MHz, CDCl3) δ 7.34 (d, J = 6.5 Hz, 2H),7.27 (s, 1H), 7.21 (t, J = 7.3 Hz, 2H), 7.13 – 6.89 (m, 3H), 6.54 (m, 1H), 5.12(t, J = 14.8 Hz, 2H), 4.84 (s, 1H), 4.22 (s, 1H), 3.92 (d, J = 33.6 Hz, 2H), 3.51– 3.33 (m, 2H), 3.17 - 3.05 (m, 2H), 2.23 - 2.00 (m, 2H), 1.86 (s, 2H), 1.45(s, 9H). 13C NMR (150 MHz, CDCl3) δ 172.5, 171.1, 168.8, 136.1, 135.3, 129.3,129.2, 128.6,128.5,128.4, 127.0, 80.7, 77.3, 77.0, 76.8, 67.1, 60.6, 53.6,47.32, 43.0, 37.6, 28.4, 24.6; ESI-MS calcd for [C 28 H 35 N3O6+ H + ] 510.2, found510.2..

[0050] (iv) Synthesis of intermediate compound 7 4.1 Synthesis of NH2-Pro-Gly-D-Leu-OBn (7a)

[0051] The synthesized tripeptide Boc-Pro-Gly-D-Leu-OBn6a (19 g, 40 mmol) was completely dissolved in CH2Cl2 (40 mL). TFA (40 mL) was slowly added dropwise at 0 °C, and the reaction mixture was brought back to room temperature and stirred for 5 h. The reaction mixture was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 9.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 13.54 g of colorless oily liquid NH2-Pro-Gly-D-Leu-OBn (7a), with a yield of 90%. No purification was required, and it was used directly in the next reaction.

[0052] 4.2 Synthesis of Pro-Gly-D-Phe-OBn (7b)

[0053] The synthesized tripeptide Boc-Pro-Gly-D-Phe-OBn6b (16.5 g, 32 mmol) was completely dissolved in CH2Cl2 (32 mL). TFA (32 mL) was slowly added dropwise at 0 °C, and the reaction mixture was brought back to room temperature and stirred for 5 h. The reaction mixture was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 9.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 11.8 g of a colorless oily liquid, Pro-Gly-D-Phe-OBn (7b), in 90% yield. No purification was required; it was used directly in the next reaction.

[0054] (v) Synthesis of intermediate compound 9 5.1 Synthesis of Boc-D-Val-Pro-Gly-D-Leu-OBn (9a)

[0055] HOBt (4.45 g, 33 mmol) and Boc-D-Val-OH8a (6.45 g, 30 mmol) were added to a 150 mL round-bottom flask under argon protection. 60 mL of dry THF was added, and the mixture was stirred at 0 °C. When the temperature inside the flask dropped to 0 °C, DIC (N,N'-diisopropylcarbodiimide, 5.1 mL, 33 mmol) was slowly added dropwise. After 0.5 h, the tripeptide Pro-Gly-D-Leu-OBn7a (11.28 g, 30 mmol), which had been previously dissolved in THF, was added. After stirring at room temperature for 48 h, the insoluble matter was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. After drying over anhydrous sodium sulfate, the solution was concentrated under reduced pressure and purified by column chromatography with petroleum ether and ethyl acetate to give 14.84 g of a white solid tetrapeptide Boc-D-Val-Pro-Gly-D-Leu-OBn (9a), yield 86%. o C;[α] D 20 = + 15 ( c 0.7, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 7.36 – 7.30 (m, 5H), 7.24– 7.23 (m, 1 H), 5.34 – 5.32 (d, J= 6.8 Hz, 1 H), 5.20 – 5.13 (dd, J = 12.4 Hz, J = 14.8 Hz, 2H), 4.64 – 4.59 (m, 1H), 4.50 – 4.50 (t, J = 6.0 Hz, l H), 4.20– 4.14 (dd, J = 7.2 Hz, J = 16.8 Hz, 1 H), 4.07 (m, 1 H), 3.98 – 3.94 (m, 1 H),3.65 – 3.56 (m, 2H), 2.21 – 2.16 (m, 2H), 2.09 – 1.99 (m, 2H), 1.95 – 1.90(m, 1 H), 1.76 – 1.61 (m, 3H), 1.40 (s, 9H), 1.01 – 1.00 (d, J = 6.8 Hz, 3H),0.97 – 0.95 (d, J = 6.8 Hz, 3H), 0.92 – 0.88 (m, 6H); 13 C NMR (100 MHz, CDCl3) δ172.6, 171.7, 169.0, 156.9,135.7, 128.4, 128.0, 127.8, 80.3, 66.4, 61.0,58.2, 50.7, 47.6, 43.1, 40.7, 30.1, 29.2, 28.1, 24.6, 23.4, 22.8, 21.7, 19.1,18.8; ESI-MS calcd for [C 30 H 46 N4O7+H + ] 575.3, found 575.3.。

[0056]

[0057] Alternatively, as shown in the formula above, add HOBt (4.45 g, 33 mmol) and Boc-D-Val-OH8a (6.51 g, 30 mmol) to a 150 mL round-bottom flask, under argon protection, add 60 mL of dry THF, stir at 0 °C until the temperature inside the flask drops to 0 °C, then slowly add DCC (N,N'-dicyclohexylcarbodiimide, 6.8 g, 33 mmol) dissolved in DMF. After 0.5 h, add the tripeptide Pro-Gly-D-Leu-OBn7a (11.28 g, 30 mmol) previously dissolved in THF. After stirring at room temperature for 48 h, filter off the insoluble matter, and concentrate the filtrate to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure and purified by column chromatography with petroleum ether and ethyl acetate to give 12.42 g of white solid tetrapeptide Boc-D-Val-Pro-Gly-D-Leu-OBn (9a), yield 72%.

[0058] 5.2 Synthesis of Boc-D-Tle-Pro-Gly-D-Leu-OBn (9b)

[0059] HOBt (4.45 g, 33 mmol) and Boc-D-Tle-OH8b (6.93 g, 30 mmol) were added to a 150 mL round-bottom flask under argon protection. 60 mL of dry THF was added, and the mixture was stirred at 0 °C. When the temperature inside the flask dropped to 0 °C, DIC (5.1 mL, 33 mmol) was slowly added dropwise. After 0.5 h, the tripeptide Pro-Gly-D-Leu-OBn7a (11.28 g, 30 mmol), which had been previously dissolved in THF, was added. After stirring at room temperature for 48 h, the insoluble matter was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. After drying over anhydrous sodium sulfate, the solution was concentrated under reduced pressure and purified by column chromatography with petroleum ether and ethyl acetate to give 15.02 g of white solid tetrapeptide Boc-D-Tle-Pro-Gly-D-Leu-OBn (9b), yield 85%. (White solid, MPa 168–169.4) o C; [α] D 25 = + 9.0 ( c 0.5, CHCl3); 1 H NMR (600 MHz, CDCl3) δ 7.47 (d, J = 7.7 Hz, 1H), 7.35 (d,J = 4.4 Hz, 3H), 7.33 – 7.28 (m, 1H), 7.08 – 7.03 (m, 1H), 5.48 (d, J = 7.1 Hz, 1H), 5.19 – 5.12 (m, 2H), 4.62-4.55 (m, 1H), 4.46 (dd, J = 7.5, 4.9 Hz, 1H), 4.22 (dd, J = 17.2, 7.9 Hz, 1H), 4.15 – 4.10 (m, 1H), 4.07 (d, J = 7.1 Hz, 1H), 3.65 – 3.59 (m, 1H), 3.56 (dd, J = 17.1, 5.1 Hz, 1H), 2.20 – 2.15 (m, 2H), 2.09 – 2.03 (m, 1H), 2.00 (dd, J = 11.3, 5.3 Hz, 1H), 1.77 – 1.56 (m, 4H), 1.40 (s, 9H), 1.04 (s, 9H), 0.88 (dd, J = 12.8, 6.4 Hz, 6H). 13 13C NMR (150 MHz, CDCl3) δ 172.7, 172.2, 171.7, 169.1, 135.91, 128.5, 128.1, 128.0, 77.24, 77.0, 76.8, 66.4, 61.3, 59.7, 50.9, 48.3, 43.2, 40.7, 33.9, 29.4, 28.2, 26.5, 24.8, 24.7, 22.8, 21.8; ESI-MS calcd for [C 31 H 48 N4O7+ H + 588.7, found 588.7..

[0060] 5.3 Synthesis of Boc-D-Phg-Pro-Gly-D-Leu-OBn (9c)

[0061] HOBt (4.45 g, 33 mmol) and Boc-D-Phg-OH8c (7.53 g, 30 mmol) were added to a 150 mL round-bottom flask under argon protection. 60 mL of dry THF was added, and the mixture was stirred at 0 °C. When the temperature inside the flask dropped to 0 °C, DIC (5.1 mL, 33 mmol) was slowly added dropwise. After 0.5 h, the tripeptide Pro-Gly-D-Leu-OBn7a (11.28 g, 30 mmol), which had been previously dissolved in THF, was added. After stirring at room temperature for 48 h, the insoluble matter was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 15.52 g of the tetrapeptide Boc-D-Phg-Pro-Gly-D-Leu-OBn (9c), with a yield of 85%. White solid, Mp142–144 o C; [α] D 25 = -2.0 ( c 0.5, CHCl3); 1 H NMR (600 MHz, CDCl3) δ 7.45 – 7.39(m, 2H), 7.38 – 7.32 (m, 6H), 7.27 (s, 1H), 7.22 (d, J = 7.0 Hz, 1H), 5.72 (d, J =5.2 Hz, 2H), 5.38-5.30 (m, 1H), 5.17 (d, J = 3.3 Hz, 2H), 4.66 – 4.61 (m, 1H), 4.47 – 4.39 (m, 1H), 4.04 (dd, J = 16.8, 6.6 Hz, 1H), 3.98– 3.94 (m, 1H), 3.83(dd, J = 12.7, 6.3 Hz, 1H), 3.38 – 3.29 (m, 1H), 2.21 – 2.16 (m, 1H), 2.09–1.99 (m, 2H), 1.75 – 1.70 (m, 2H), 1.39 (d, J = 4.9 Hz, 9H), 1.13 (d, J = 6.5 Hz, 4H), 0.96 – 0.90 (m, 6H). 13C NMR (150 MHz, CDCl3) δ 172.5, 171.5, 169.0,135.7, 129.2, 129.1, 128.5, 128.4, 128.1 (dd, J = 12.3, 9.2 Hz), 80.5, 77.2,77.0, 76.8, 66.8, 61.2, 51.0, 47.3, 43.4, 42.1, 41.1, 28.2, 24.6, 23.4, 22.8,21.8; ESI-MS calcd for [C 33 H 44 N4O7+ H + 609.3, found 609.3.

[0062] 5.4 Synthesis of the tetrapeptide Boc-D-Chg-Phe-Pro-Gly-D-Leu-OBn (9d)

[0063] HOBt (4.45 g, 33 mmol) and Boc-D-Chg-OH8d (7.71 g, 30 mmol) were added to a 150 mL round-bottom flask under argon protection. 60 mL of dry THF was added, and the mixture was stirred at 0 °C. When the temperature inside the flask dropped to 0 °C, DIC (5.1 mL, 33 mmol) was slowly added dropwise. After 0.5 h, the tripeptide Pro-Gly-D-Leu-OBn7a (11.28 g, 30 mmol), which had been previously dissolved in THF, was added. After stirring at room temperature for 48 h, the insoluble matter was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. After drying over anhydrous sodium sulfate, the solution was concentrated under reduced pressure and purified by column chromatography with petroleum ether and ethyl acetate to give 15.5 g of the tetrapeptide Boc-D-Chg-Phe-Pro-Gly-D-Leu-OBn (9d), yield 84%. White solid, Mp: 160–161. o C; [α] D 20 = -10 ( c 0.62, CHCl3); 1 H NMR (400 MHz, CDCl) 3) δ 7.37 – 7.30 (m, 5H), 5.28 – 5.26 (d, J= 6.4 Hz, 1 H), 5.21 –5.13 (m, 2H), 4.66 – 4.60 (m, 1H), 4.50 – 4.47 (m, 1H), 4.20 – 4.07 (m, 2H),4.00 – 3.96 (m, 1H), 3.64 – 3.56 (m, 2H), 2.21 – 2.16 (m, 2H), 2.10 – 1.98(m, 2H), 1.87 – 1.84 (m, 1H), 1.79 – 1.56 (m, 8H), 1.39 (s, 9H), 1.28 – 1.17(m, 4H), 1.31 – 1.11 (d, J = 6.8 Hz, 2H), 1.06 – 0.99 (m, 2H), 0.92 – 0.89 (m,6H); 13 13C NMR (100 MHz, CDCl3) δ 172.5, 171.7, 169.0, 157.0, 135.7, 128.4,128.0, 127.8, 80.3, 66.3, 60.9, 57.6, 50.7, 47.6, 43.1, 41.8, 40.8, 39.5,29.4, 29.2, 28.1, 26.0, 25.7, 25.6, 24.6, 23.4, 22.8, 21.7; ESI-MS calcd for[C 33 H 50 N4O7+ H + 614.4, found: 614.4. .

[0064] 5.5 Synthesis of Boc-D-Phe-Pro-Gly-D-Leu-OBn (9e)

[0065] HOBt (4.45 g, 33 mmol) and Boc-D-Phe-OH8e (7.95 g, 30 mmol) were added to a 150 mL round-bottom flask under argon protection. 60 mL of dry THF was added, and the mixture was stirred at 0 °C. When the temperature inside the flask dropped to 0 °C, DIC (5.1 mL, 33 mmol) was slowly added dropwise. After 0.5 h, the tripeptide Pro-Gly-D-Leu-OBn7a (11.28 g, 30 mmol), which had been previously dissolved in THF, was added. After stirring at room temperature for 48 h, the insoluble matter was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. After drying over anhydrous sodium sulfate, the solution was concentrated under reduced pressure and purified by column chromatography with petroleum ether and ethyl acetate to give the tetrapeptide Boc-D-Phe-Pro-Gly-D-Leu-OBn (9e), 16.07 g, yield 86%. White solid, MP: 160-161 ℃; [α] D 20 = -10 ( c 0.62, CHCl3); 1 H NMR(400 MHz, CDCl3) δ 7.40 - 7.19 (m, 10H), 5.50 (br, 1H), 5.18 (s, 1H), 4.65 -4.64 (m, 1 H), 4.41 - 4.38 (m, 1H), 4.32 - 4.30 (m, 1H), 4.16 - 4.10 (m, 1H), 3.69 - 3.61 (m, 2H), 2.68 - 2.65 (m, 1H), 2.05 - 2.02 (m, 1H), 1.91 - 1.56(m, 8H), 1.39 (s, 9H), 1.14 - 1.12 (m, 2H), 0.96 - 0.91 (m, 6H); 13 C NMR (100MHz, CDCl3) δ 172.6, 172.0, 171.5, 169.0 156.3,135.9, 135.7, 129.2, 128.6,128.5, 128.1, 127.9, 127.2, 80.5, 66.5, 60.9, 54.4, 50.7, 47.1, 43.1, 42.0,40.9, 3 8.1, 28.8, 28.1, 24.6, 24.4, 23.4, 22.9, 21.7; ESI-MS calcd for[C 34 H46 N4O7+H + 623.3, found 623.3.

[0066] 5.6 Synthesis of Boc-D-Phe-Pro-Gly-D-Phe-OBn (9f)

[0067] HOBt (3.71 g, 27.5 mmol) and Boc-D-Phe-OH8e (6.63 g, 25 mmol) were added to a 150 mL round-bottom flask under argon protection. 50 mL of dry THF was added, and the mixture was stirred at 0 °C. When the temperature inside the flask dropped to 0 °C, DIC (4.25 mL, 27.5 mmol) was slowly added dropwise. After 0.5 h, the tripeptide Pro-Gly-D-Phe-OBn7b (10.23 g, 25 mmol), which had been previously dissolved in THF, was added. After stirring at room temperature for 48 h, the insoluble matter was filtered off, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved in ethyl acetate, then washed successively with 1 M NaOH solution, water, 1 M hydrochloric acid solution, and saturated brine. After drying over anhydrous sodium sulfate and concentration under reduced pressure, the residue was purified by column chromatography with petroleum ether and ethyl acetate to give the tetrapeptide Boc-D-Phe-Pro-Gly-D-Phe-OBn (9f), 13.45 g, yield 82%. White solid, MPa 138-139.6. o C; [α] D 25 = -49.0 ( c 0.5, CHCl3); 1 HNMR (600 MHz, CDCl3) δ 7.33 – 7.28 (m, 4H), 7.25 – 7.18 (m, 8H), 7.16 – 7.13(m, 3H), 5.45 (d, J = 6.5 Hz, 1H), 5.13 – 5.08 (m, 2H), 4.88 (dd, J = 14.4, 6.6Hz, 1H), 4.46 – 4.41 (m, 1H), 4.33 (dd, J = 7.8, 3.7 Hz, 1H), 3.98 (dd, J = 17.0, 6.9 Hz, 1H), 3.73 (dd, J = 17.0, 5.7 Hz, 1H), 3.66 – 3.585 (b, 1H), 3.15 (dd, J=6.7, 2.6 Hz, 2H), 2.95 (t, J = 7.7 Hz, 2H), 2.67 (dd, J = 16.8, 7.6 Hz, 1H), 2.08– 2.02 (m, 1H), 1.84 – 1.75 (b, 3H), 1.57 – 1.51 (b, 1H), 1.39 (s, 9H). 13 C NMR(150 MHz, CDCl3) δ 171.9, 171.5, 171.4, 168.8, 136.2, 135.4, 129.4, 129.3,128.6, 128.5, 128.4, 128.3, 128.2, 127.2, 126.8, 80.5, 77.2, 77.0, 76.8,66.9, 60.8, 53.6, 47.1, 43.2, 38.2, 28.7, 28.3, 24.4; ESI-MS calcd for[C 37 H 44 N4O7+ H + 657.3, found 657.3.

[0068] (vi) Synthesis of intermediate compound 10 6.1 Synthesis of H2N-D-Val-Pro-Gly-D-Leu-OBn (10a)

[0069] The previously synthesized tetrapeptide Boc-D-Val-Pro-Gly-D-Leu-OBn9a (12.65 g, 22 mmol) was dissolved in CH2Cl2 (20 mL), and then TFA (20 mL) was slowly added dropwise at 0 °C, with stirring at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 9.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 10.03 g of colorless oily liquid H2N-D-Val-Pro-Gly-D-Leu-OBn (10a), with a yield of 96%. No purification was required, and it was used directly in the next reaction.

[0070] 6.2 Synthesis of H2N-D-Tle-Pro-Gly-D-Leu-OBn (10b)

[0071] The previously synthesized tetrapeptide Boc-D-Tle-Pro-Gly-D-Leu-OBn9b (12.96 g, 22 mmol) was dissolved in CH2Cl2 (25 mL), and then TFA (25 mL) was slowly added dropwise at 0 °C, with stirring at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 9.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.22 g of colorless oily liquid H2N-D-Tle-Pro-Gly-D-Leu-OBn (10b), with a yield of 95%. No purification was required, and it was used directly in the next reaction.

[0072] 6.3 Synthesis of H2N-D-Phg-Pro-Gly-D-Leu-OBn (10c)

[0073] The previously synthesized tetrapeptide Boc-D-Phg-Pro-Gly-D-Leu-OBn9c (13.4 g, 22 mmol) was dissolved in CH2Cl2 (25 mL), and then TFA (25 mL) was slowly added dropwise at 0 °C, with stirring at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 9.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.3 g of colorless oily liquid H2N-D-Phg-Pro-Gly-D-Leu-OBn (10c), with a yield of 92%. No purification was required, and it was used directly in the next reaction.

[0074] 6.4 Synthesis of the tetrapeptide H2N-D-Chg-Pro-Gly-D-Leu-OBn (10d)

[0075] The previously synthesized tetrapeptide Boc-D-Chg-Pro-Gly-D-Leu-OBn9d (13.53 g, 22 mmol) was dissolved in CH2Cl2 (25 mL), and then TFA (25 mL) was slowly added dropwise at 0 °C, with stirring at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH = 11.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.6 g of a colorless oily liquid, H2N-D-Chg-Pro-Gly-D-Leu-OBn (10d), with a yield of 94%. No purification was required, and it was used directly in the next reaction.

[0076] 6.5 Synthesis of H2N-D-Phe-Pro-Gly-D-Leu-OBn (10e)

[0077] The previously synthesized tetrapeptide Boc-D-Phe-Pro-Gly-D-Leu-OBn9e (13.7 g, 22 mmol) was dissolved in CH2Cl2 (25 mL), and then TFA (25 mL) was slowly added dropwise at 0 °C, with stirring at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 9.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed successively with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.5 g of a colorless oily liquid, H2N-D-Phe-Pro-Gly-D-Leu-OBn (10e), with a yield of 91%. No purification was required, and it was used directly in the next reaction.

[0078] 6.6 Synthesis of H2N-D-Phe-Pro-Gly-D-Phe-OBn (10f)

[0079] The previously synthesized tetrapeptide Boc-D-Phe-Pro-Gly-D-Phe-OBn9f (13.12 g, 20 mmol) was dissolved in CH2Cl2 (25 mL), and then TFA (25 mL) was slowly added dropwise at 0 °C, with stirring at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to remove as much TFA as possible. The residue was dissolved in CH2Cl2 and then neutralized with 1 M NaOH solution at 0 °C to approximately pH 11.0. The organic phase was separated, and the aqueous phase was extracted with CH2Cl2 (20 mL × 3). The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 10.62 g of colorless oily liquid H2N-D-Phe-Pro-Gly-D-Phe-OBn (10f), with a yield of 94%. No purification was required, and it was used directly in the next reaction.

[0080] The following are methods for preparing the specific compounds TP-1 to TP-6 ​​of this invention. Example 1

[0081] Synthetic tetrapeptide TP-1: NH2-D-Val-Pro-Gly-D-Leu-OH

[0082] The previously synthesized NH2-D-Val-Pro-Gly-D-Leu-OBn10a (10.0 g, 21 mmol) was dissolved in methanol (20 mL) and transferred to a 100 mL high-pressure reactor. Then, 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the reactor to 8 MPa at room temperature, and the mixture was stirred for 6 hours. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the tetrapeptide NH2-D-Val-Pro-Gly-D-Leu-OH (TP-1), 8.0 g, 100% yield. White solid, MPa: 152–154 °C; [α] D 20 = – 58.0 (c 0.85, H2O); 1 H NMR (600 MHz, D2O) δ 4.55 – 4.50 (m, 1H), 4.31 – 4.25 (m, 1H), 4.04(d, J = 17.1 Hz, 1H), 3.90 (d, J = 17.1 Hz, 1H), 3.87-3.83 (m, 1H), 3.77-3.73 (m,1H), 2.41 – 2.33 (m,2H), 2.11-2.04 (m, 3H), 1.74 – 1.60 (m, 4H), 1.13 (d,J =7.2 Hz, 3H), 1.05 (d, J = 12 Hz, 3H), 0.95 (d, J = 6 Hz 3H), 0.91 (d, J = 6 Hz, 3H). 13 C NMR (150 MHz, D2O) δ 179.7, 174.3, 170.4, 168.9, 61.1, 57.1, 53.5, 48.1,42.4, 40.5, 29.2, 28.7, 24.5, 24.3, 22.4, 20.5, 17.9, 15.9; HRMS calcd for[C 18 H 32 N4O5+ H + ] 385.2445, found 385.2438. Alternatively, the previously synthesized NH2-D-Val-Pro-Gly-D-Leu-OBn (10.0 g) was dissolved in ethanol (20 mL), transferred to a 100 mL high-pressure reactor, and then 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the mixture to 8 MPa at room temperature, and the mixture was stirred for 6 hours. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the tetrapeptide NH2-D-Val-Pro-Gly-D-Leu-OH (TP-1), a white solid, 7.45 g, with a yield of 93%. Example 2

[0083] Synthesis of TP-2: H2N-D-Tle-Pro-Gly-D-Leu-OH

[0084] The previously synthesized NH2-D-Tle-Pro-Gly-D-Leu-OBn10b (10.0 g, 20 mmol) was dissolved in methanol (20 mL) and transferred to a 100 mL high-pressure reactor. Then, 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the reactor to 8 MPa at room temperature, and the mixture was stirred for 6 hours. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the tetrapeptide NH2-D-Tle-Pro-Gly-D-Leu-OH (TP-2), 7.8 g, yield 98%. White solid, MPa: 204–205 °C, [α] D 20 = – 28.0 ( c 1.0, DMF); 1H NMR (600 MHz, D2O) 4.38 – 4.34 (m, 1H), 4.12 – 4.02 (m, 2H), 3.81 (q, J = 17.1 Hz, 2H), 3.72 – 3.63 (m, 2H), 3.21 (d, J = 0.4 Hz, 1H), 2.24 – 2.18 (m,1H), 1.91 (s, 3H), 1.57 – 1.52 (m, 1H), 1.50 – 1.42 (m, 2H), 0.97 (s, 9H), 0.76 (dd, J = 22.8, 5.8 Hz, 6H); 13 C NMR (150 MHz, D2O) δ 179.8, 174.3, 170.4,168.2, 61.0, 59.1, 53.7, 49.0, 42.4, 40.6, 33.9, 29.3, 25.3, 24.5, 24.26,22.4, 20.5; HRMS calcd for [C 19 H 30 N4O5+ H + ] 399.2602, found 399.2599. Example 3

[0085] Synthesis of TP-3: H2N-D-Phg-Pro-Gly-D-Leu-OH

[0086] The previously synthesized NH2-D-Phg-Pro-Gly-D-Leu-OBn10c (10.0 g, 19.5 mmol) was dissolved in methanol (20 mL) and transferred to a 100 mL high-pressure reactor. Then, 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the reactor to 8 MPa at room temperature, and the mixture was stirred for 6 h. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the tetrapeptide NH2-D-Phg-Pro-Gly-D-Leu-OH (TP-3), 7.8 g, yield 96%. White solid, MPa: 150–153 ℃, [α] D 20 = – 94.0( c 1.0, H2O); 1 H NMR (600 MHz, D2O) δ 7.62 – 7.42 (m, 5H), 5.47 (s, 1H), 4.50(dd,J = 9.0, 3.8 Hz, 1H), 4.31 (dd, J = 10.6, 3.2 Hz, 1H), 4.09 (d, J = 17.2 Hz, 1H), 3.89 (d, J = 17.2 Hz, 1H), 3.80 – 3.71 (m, 1H), 3.07 – 3.00 (m, 1H), 2.25 – 2.15 (m, 1H), 2.05 – 1.94 (m, 2H), 1.86 – 1.72 (m, 2H), 1.69 – 1.53 (m,2H), 1.01 – 0.86 (m, 6H); 13 C NMR (150 MHz, D2O) δ 179.9, 174.3, 170.3, 167.4,130.6, 129.8, 129.6, 128.9, 128.5, 61.5, 56.4, 53.6, 47.5, 42.3, 40.6, 28.9,24.6, 24.2, 22.4, 20.6; HRMS calcd for [C 21 H 30 N4O5+ H + ] 419.2289, found: 419.2285. Example 4

[0087] Synthesis of TP-4: H2N-D-Chg-Pro-Gly-D-Leu-OH

[0088] The previously synthesized NH₂-D-Chg-Pro-Gly-D-Leu-OBn10d (10.0 g, 19.4 mmol) was dissolved in methanol (20 mL) and transferred to a 100 mL high-pressure reactor. Then, 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the reactor to 8 MPa at room temperature, and the mixture was stirred for 6 hours. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to give the tetrapeptide NH₂-D-Chg-Pro-Gly-D-Leu-OH (TP-4), 7.94 g, yield 97%. White solid, MPa: 167–169 °C, [α] D 20 = – 96.0 ( c 1.0, H2O); 1H NMR (600 MHz, D2O) δ 4.55 – 4.46 (m, 1H), 4.33 – 4.20 (m, 2H), 4.03(d, J = 17.1 Hz, 1H), 3.93 – 3.70 (m, 3H), 2.40 – 2.32. (m, 1H), 2.13 – 1.94(m, 3H), 1.86 – 1.57 (m, 7H), 1.37 – 1.07 (m, 5H), 0.98 – 0.86 (m, 6H); 13 C NMR (150 MHz, D2O) δ 179.6, 174.3, 170.4, 168.8, 61.1, 56.6, 53.9, 53.4, 48.1,42.4, 40.5, 38.4, 29.3, 28.5, 27.1, 25.1, 24.5, 24.2, 22.4, 20.5; HRMS calcdfor [C 21 H 36 N4O5+ H + ] 425.2758, found 425.2748. Example 5

[0089] Synthesis of TP-5: NH2-D-Phe-Pro-Gly-D-Leu-OH

[0090] The previously synthesized NH₂-D-Phe-Pro-Gly-D-Leu-OBn9e (10.0 g, 19.1 mmol) was dissolved in methanol (20 mL) and transferred to a 100 mL high-pressure reactor. Then, 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the reactor to 8 MPa at room temperature, and the mixture was stirred for 6 hours. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the tetrapeptide NH₂-D-Phe-Pro-Gly-D-Leu-OH (TP-5), a white solid, 7.7 g, yield 93%. White solid, MPa: 198-200 ℃; [α] D 20 = –98.0 ( c 1.0, H2O); 1 H NMR (600 MHz, D2O) 7.45 - 7.39 (m, 3H), 7.35 (d, J = 6.9 Hz,2H), 4.62 - 4.57 (m, 1H), 4.37 (dd,J = 8.8, 4.6 Hz, 1H), 4.26 (dd, J = 10.5, 3.5Hz, 1H), 4.01 (d, J = 17.1 Hz, 1H), 3.88 (d, J = 17.2 Hz, 1H), 3.64 – 3.57 (m,1H), 3.30 – 3.18 (m, 2H), 2.86 – 2.80(m, 1H), 2.16 – 2.08 (m, 1H), 1.97 –1.85 (m, 2H), 1.73 – 1.56 (m, 4H), 0.92 (dd, J = 28.0, 6.0 Hz, 6H); 13 C NMR (150MHz, D2O) δ 179.8, 174.1, 170.3, 168.5, 133.3, 129.4, 129.1, 128.1, 61.0,53.6, 53.0, 47.7, 42.4, 40.5, 36.3, 29.1, 24.5, 24.0, 22.4, 20.5; HRMS calcdfor [C 22 H 32 N4O5+ H + ] 433.2445, found 419.2439. Example 6

[0091] Synthesis of TP-6: NH2-D-Phe-Pro-Gly-D-Phe-OH

[0092] The previously synthesized NH₂-D-Phe-Pro-Gly-D-Phe-OBn10f (10.2 g, 18 mmol) was dissolved in methanol (20 mL) and transferred to a 100 mL high-pressure reactor. Then, 500 mg of Pd / C (5%) was added. Hydrogen gas was bubbled through the reactor to 8 MPa at room temperature, and the mixture was stirred for 6 hours. Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the tetrapeptide NH₂-D-Phe-Pro-Gly-D-Phe-OH (TP-6), a white solid, 7.8 g, yield 92%. Mp 182.5–184.4 ℃, [α] D 20 = – 50.0 (c 0.5,DMF); 1H NMR (600 MHz, DMSO) δ 8.50 – 8.16 (m, 2H), 7.41 – 7.03 (m, 10H), 4.38 (m, 3H), 3.76 – 3.64 (m, 1H), 3.50 – 3.32 (m, 2H), 3.20 – 2.79 (m, 4H), 1.87– 1.60 (m, 3H), 1.40 – 1.26 (m, 1H), 0.98 (d, J = 6.5 Hz, 1H); 13 C NMR (150 MHz, DMSO) δ 175.0, 171.4, 170.2, 168.3, 139.3, 136.0, 129.9, 129.7, 128.9, 128.3,127.5, 126.2, 61.2, 52.9, 47.1, 42.1, 40.4 , 40.2, 40.1, 39.9, 39.7, 39.6,38.8, 29.3, 24.4, 23.7; HRMS calcd for [C 25 H 30 N4O5+ H + ] 467.2288, found: 467.2289.

[0093] II. Examples of the application of compound TP Example 7

[0094] Add an accurately weighed amount of the tetrapeptide catalyst TP (0.025 mmol) of this invention to a clean 5 mL round-bottom flask. N 87 mg, 0.5 mmol, of phenylmaleimide and 1.0 mL of dried redistilled toluene were added, and redistilled isobutyraldehyde (92 µL, 1.0 mmol) was added under ice-water bath and magnetic stirring. The reaction mixture was brought back to room temperature and the stirring continued. The reaction was monitored by TLC until it was complete. 3-4 drops of saturated ammonium chloride solution were added, and the mixture was extracted with ethyl acetate (10 mL × 3). The extract was washed with a small amount of saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residual oil was purified by column chromatography with petroleum ether and ethyl acetate to obtain the pure product.

[0095] The six tetrapeptide catalysts TP-1 to TP-6 ​​of this invention catalyze the reaction of isobutyraldehyde with... N The evaluation of the asymmetric conjugate addition reaction of phenylmaleimide is shown in Table 1 below. The results indicate that TP-3 and TP-5 exhibit the highest asymmetric catalytic efficiency.

[0096] Example 8. The effect of solvent on the asymmetric conjugated addition reaction of isobutyraldehyde and N-phenylmaleimide catalyzed by tetrapeptide TP-5.

[0097] Add accurately weighed tetrapeptide catalyst TP-5 (0.025 mmol, 10.8 mg) to a clean 5 mL round-bottom flask. N 87 mg, 0.5 mmol, of phenylmaleimide and 1.0 mL of dried redistilled reaction solvent were added. Redistilled isobutyraldehyde (92 µL, 1.0 mmol) was added under ice-water bath and magnetic stirring. The reaction mixture was brought back to room temperature and stirred continuously. The reaction was monitored by TLC until it was complete. 3-4 drops of saturated ammonium chloride solution were added, and the mixture was extracted with ethyl acetate (10 mL × 3). The extract was washed with a small amount of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The remaining oily residue was purified by column chromatography with petroleum ether and ethyl acetate to obtain the pure product.

[0098] The effects of various solvents on the reaction yield and ee value are shown in Table 2 below. The results show that, compared with toluene, acetonitrile significantly improves both the yield and ee value when used as the solvent.

[0099] Example 9. The effect of catalyst dosage on the catalytic efficiency of asymmetric conjugate addition reactions was observed.

[0100] Add accurately weighed tetrapeptide catalyst TP-5 or TP-3 (0.025 mmol) to a clean 5 mL round-bottom flask. N 87 mg, 0.5 mmol, of phenylmaleimide and 1.0 mL of dried redistilled reaction solvent were added. Redistilled isobutyraldehyde (92 µL, 1.0 mmol) was added under ice-water bath and magnetic stirring. The reaction mixture was brought back to room temperature and stirred continuously. The reaction was monitored by TLC until it was complete. 3-4 drops of saturated ammonium chloride solution were added, and the mixture was extracted with ethyl acetate (10 mL × 3). The extract was washed with a small amount of saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residual oil was purified by column chromatography with petroleum ether and ethyl acetate to obtain the pure product.

[0101] The reaction yields and ee values ​​are shown in Table 3 below. The results show that using 2.5 mol% of TP-3 tetrapeptide as a catalyst and acetonitrile as a solvent yields the best asymmetric catalytic effect.

[0102] Example 10 To observe the substrate adaptability of the asymmetric conjugated addition reaction of aliphatic aldehydes and maleimides catalyzed by tetrapeptide TP-3.

[0103] Accurately weighed tetrapeptide catalyst 2.5% TP-3 (0.0125 mmol, 5.3 mg), maleimide (0.5 mmol), and 1.0 mL of dried redistilled acetonitrile were added to a clean 5 mL round-bottom flask. Redistilled aliphatic aldehyde (1.0 mmol) was added under ice-water bath and magnetic stirring. The reaction mixture was brought back to room temperature and the reaction was stirred continuously. The reaction was monitored by TLC until it was complete. 3-4 drops of saturated ammonium chloride solution were added, and the mixture was extracted with ethyl acetate (10 mL × 3). The extract was washed with a small amount of saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The remaining oily substance was purified by column chromatography with petroleum ether and ethyl acetate to obtain the pure product.

[0104] The results are shown in Table 4.

[0105] Table 4. Asymmetric conjugated addition reaction of aliphatic aldehydes with maleimide catalyzed by tetrapeptide TP-3.

[0106] a The separation yield, ee, was determined by chiral HPLC.

[0107] The products P-1 to P-17 are characterized as follows.

[0108] P-1, [α] D 25 = -6.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz, CDCl3) δ 9.51 (s, 1H), 7.48-7.44 (m, 2H), 7.40 – 7.36 (m, 1H), 7.29-7.25 (m, 2H), 3.14 (dd, J = 9.6, 5.6 Hz, 1H), 2.96 (dd, J = 18.6, 9.6 Hz, 1H), 2.61 (dd, J =18.4, 5.6 Hz, 1H), 1.32 (s, 3H), 1.28 (s, 3H).

[0109] P-2, [α] D 25 = +2.0 ( c = 1.0, CHCl3); 1H NMR (600 MHz,CDCl3) δ 9.49 (s, 1H), 7.28 – 7.24 (m, 2H), 7.17-7.12 (m, 2H), 3.11 (dd, J =9.6, 5.6 Hz, 1H), 2.97 (dd, J = 18.3, 9.6 Hz, 1H), 2.61 (dd, J = 18.4, 5.6 Hz,1H), 1.35 (s, 3H), 1.28 (s, 3H).。

[0110] P-3, [α] D 25 = +3.0 ( c = 1.0, CHCl3); 1 H NMR (600MHz, CDCl3) δ 9.49 (s, 1H), 7.49 – 7.40 (m, 2H), 7.28 – 7.22 (m, 2H), 3.11(dd, J = 9.6, 5.6 Hz, 1H), 2.97 (dd, J = 18.3, 9.6 Hz, 1H), 2.62 (dd, J = 18.3, 5.6Hz, 1H), 1.36 (s, 3H), 1.29 (s, 3H).。

[0111] P-4, [α] D 25 = +3.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.49 (s, 1H), 7.62 – 7.57 (m, 1H), 7.23 – 7.15 (m, 2H), 3.11 (dd, J =9.6, 5.4 Hz, 1H), 2.97 (dd, J = 18.4, 9.6 Hz, 1H), 2.62 (dd, J = 18.4, 5.6 Hz,1H), 1.36 (s, 3H), 1.29 (s, 3H).。

[0112] P-5, [α] D25 = +6.0 ( c = 1.0, CHCl3); 1 H NMR (600MHz, CDCl3) δ 9.46 (s, 1H), 8.35 – 8.29 (m, 2H), 7.61 – 7.54 (m, 2H), 3.10(dd, J = 9.6, 5.4 Hz, 1H), 3.00 (dd, J = 18.6, 9.6 Hz, 1H), 2.65 (dd, J = 18.6, 5.4Hz, 1H), 1.41 (s, 3H), 1.30 (s, 3H).。

[0113] P-6, [α] D 25 = +5.0 ( c = 1.0, CHCl3); 1 H NMR (600MHz, CDCl3) δ 9.52 (s, 1H), 7.21 – 7.15 (m, 2H), 6.99 – 6.95 (m, 2H), 3.82(s, 3H), 3.13 (dd, J = 9.6, 5.4 Hz, 1H), 2.96 (dd, J = 18.4, 9.6 Hz, 1H), 2.60(dd, J = 18.6, 5.4 Hz, 1H), 1.32 (s, 3H), 1.28 (s, 3H).。

[0114] P-7, [α] D 25 = +6.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.52 (s, 1H), 7.27 (d, J = 7.2 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 3.14(dd, J = 9.6, 5.4 Hz, 1H), 2.97 (dd, J = 18.4, 9.6 Hz, 1H), 2.61 (dd, J= 18.4, 5.5Hz, 1H), 2.37 (s, 3H), 1.32 (s, 3H), 1.28 (s, 3H).。

[0115] P-8, [α] D 25 = +20.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.63 (s, 1H), 7.48 (t, J = 7.8 Hz, 2H), 7.40 (t, J = 7.5 Hz, 1H), 7.34 –7.28 (m, 2H), 3.31-3.26 (m, 1H), 3.12 – 3.06 (m, 1H), 2.93 (dd, J = 18.0, 9.7Hz, 1H), 2.60 (dd, J = 18.0, 5.7 Hz, 1H), 1.40 (d, J = 7.8 Hz, 3H).。

[0116] P-9, [α] D 25 = +16.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.78 (s, 1H), 7.50 – 7.45 (m, 2H), 7.41 – 7.36 (m, 1H), 7.31-7.28(m, 2H), 3.17 (dd, J = 7.2, 3.7 Hz, 1H), 3.11 – 2.99 (m, 1H), 2.87 (dd, J = 18.0,9.6 Hz, 1H), 2.73 (dd, J = 18.0, 6.0 Hz, 1H), 2.36 – 2.28 (m, 1H), 1.26 (d, J =6.6 Hz, 3H), 1.10 (d, J = 6.6 Hz, 3H).。

[0117] P-10, [α] D25 = +41.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.75 (s, 1H), 7.47 (t, J = 6Hz, 2H), 7.41-7.37 (m, 1H), 7.32-7.26 (m,2H), 3.36-3.32 (m, 1H), 3.03 – 2.96 (m, 2H), 2.57 (dd, J = 18.0, 5.4 Hz, 1H),1.94-1.88 (m, 1H), 1.70-1.62 (m, 1H), 1.51 – 1.45 (m, 2H), 1.42-1.36 (m, 2H),0.93 (t, J = 6 Hz, 3H).。

[0118] P-11, [α] D 25 = -40.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.39 (s, 1H), 7.49-7.46 (m, 2H), 7.40-7.37 (m, 1H), 7.34 – 7.29 (m,2H), 3.05-2.94(m, 2H), 2.58 (dd, J = 18, 5.4 Hz, 1H), 2.37 – 2.27 (m, 1H), 2.14– 2.03 (m, 2H), 1.88 – 1.69 (m, 5H).。

[0119] P-12, [α] D 25 = -5.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.55 (s, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.40-7.38 (m, 1H), 7.31 – 7.27(m, 2H), 3.22 (dd, J = 9.0, 6.0 Hz, 1H), 2.87 (dd, J= 18.0, 9.6 Hz, 1H), 2.68(dd, J = 18.2, 5.9 Hz, 1H), 1.99 – 1.85 (m, 3H), 1.67 – 1.49 (m, 6H).。

[0120] P-13, [α] D 25 = +10.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.63 (s, 1H), 7.47 (t, J = 7.8 Hz, 2H), 7.39 (t, J = 7.5 Hz, 1H), 7.29 –7.25 (m, 2H), 3.25 (dd, J = 9.6, 6.0 Hz, 1H), 2.97 (dd, J = 18.4, 9.6 Hz, 1H),2.69 (dd, J = 18.4, 6.0 Hz, 1H), 2.02 – 1.83 (m, 3H), 1.74 (dq, J = 15.0, 7.5 Hz,1H), 1.03 – 0.97 (m, 6H).。

[0121] P-14, [α] D 25 = -9.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz, CDCl3)δ 9.51 (s, 1H), 3.04 (dd, J = 9.6, 5.4 Hz, 1H), 2.99 (s, 3H), 2.82 (dd, J = 18.3,9.4 Hz, 1H), 2.45 (dd, J = 18.3, 5.4 Hz, 1H), 1.22 (d, J = 6.4 Hz, 6H).。

[0122] P-15, [α] D 25 = -11.0 ( c= 1.0, CHCl3); 1 H NMR (600 MHz,cdcl3) δ 9.51 (s, 1H), 3.98 – 3.91 (m, 1H), 2.97 (dd, J = 9.5, 5.2 Hz, 1H),2.74 (dd, J = 18.3, 9.5 Hz, 1H), 2.37 (dd, J = 18.3, 5.2 Hz, 1H), 2.16-2.08 m,2H), 1.80 (d, J = 13.4 Hz, 2H), 1.66-1.53 (m, 3H), 1.32-1.23 (m, 2H), 1.23 –1.12 (m, 7H).。

[0123] P-16, [α] D 25 = -14.0 ( c = 1.0, CHCl3); 1 H NMR (600MHz, CDCl3) δ 9.48 (s, 1H), 7.37 – 7.34 (m, 2H), 7.31-7.28 (m, 2H), 4.65 (q, J = 14.4 Hz, 2H), 3.03 (dd, J = 9.4, 5.4 Hz, 1H), 2.81 (dd, J = 18.3, 9.4 Hz, 1H),2.45 (dd, J = 18.3, 5.4 Hz, 1H), 1.16 (d, J = 2.3 Hz, 6H).。

[0124] P-17, [α] D 25 = +5.0 ( c = 1.0, CHCl3); 1 H NMR (600 MHz,CDCl3) δ 9.46 (s, 1H), 8.81 (s, 1H), 3.07 (dd, J = 9.6, 5.4 Hz, 1H), 2.83 (dd, J = 18.4, 9.6 Hz, 1H), 2.48 (dd,J = 18.4, 5.4 Hz, 1H), 1.21 (d, J = 6.9 Hz, 6H).。

Claims

1. A tetrapeptide compound TP-3 as shown in Formula 1, Its features R1 = Ph, R2 = i-Bu, with an amino acid sequence of H2N-D-Phg-Pro-Gly-D-Leu-OH, named TP-3.

2. The method for preparing compound TP-3 according to claim 1, characterized in that: 1) Compound 3a is prepared by any of the reactions in formula 2, 3, 4, 5, or 6. ， 2) Compound 4a is then prepared by any of the methods in Formula 7, Formula 8, or Formula 9. ， 3) Compound 7a was prepared by following the reaction mechanism shown in Formulas 10 and 11. ， 4) Compound 9c was prepared by the reaction shown in Formula 12. ， 5) The target compound TP-3 was then prepared using Equations 13 and 14, illustrating the reaction mechanism. 。 3. The tetrapeptide compound TP-3 of claim 1 is used to catalyze the asymmetric conjugated addition reaction of aliphatic aldehydes and maleimides.

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