Use of oligopeptide containing L-selenomethionine in preparation of anti-tumor drugs

Abstract

The application relates to application of L-selenomethionine oligopeptide in preparation of an antitumor drug, and L-selenomethionine oligopeptide (III) is further prepared by using synthesized L-selenomethionine (II). The L-selenomethionine oligopeptide has good antitumor activity, and (II-1) to (II-10) in the application have good inhibitory activity on cervical cancer cells. The results obtained by the application show that the L-selenomethionine oligopeptide has wide application prospects in a drug development system, a new and wider idea is provided for synthesizing and screening the L-selenomethionine oligopeptide, and a more effective way is provided for treating related diseases.

Description

(I) Technical Field

[0001] This invention relates to the application of L-selenomethionine-containing oligopeptides in the preparation of antitumor drugs. (II) Background Technology

[0002] Methionine, also known as methionine (I), is one of the essential amino acids for the human body and participates in protein synthesis. Because it cannot be produced by the body itself, it must be obtained from external sources. A deficiency in methionine can lead to impaired protein synthesis and cause damage to the body. Lipid peroxides can damage primary and secondary lysosomal membranes, causing the release of acid phosphatases contained within the lysosomes, which are used for hydrolysis. These phosphatases can damage important organelles such as cell and mitochondrial membranes. Methionine combats this damage through multiple pathways.

[0003]

[0004] Selenium is an essential trace element for humans and animals. In recent decades, its application in chemistry and biology has developed rapidly. Over the past 50 years, hundreds of organoselenium compounds have been produced. Many of these compounds possess unique biochemical activities and have been found to act as antitumor agents, antioxidants, neuroprotective agents, and exhibit antiparasitic, antibacterial, and antiviral activities. The bioavailability, bioactivity, and toxicity of selenium compounds depend on their chemical structure rather than their total content. Compared to inorganic selenium, organoselenium has better bioavailability, higher nutritional value, and lower toxicity. As a representative substance of organoselenium, SeMet can be considered an atomic mutant of natural methionine. In many organisms, it "infiltrates" proteins and enzymes, replacing methionine and thus endowing these proteins with additional redox activity and participating extensively in various physiological and biochemical activities. In protein chemistry, SeMet plays a unique role in elucidating protein structure and function, becoming a primary target substrate for developing chemically active protein substances.

[0005] This invention synthesizes a series of chiral oligopeptides with different sequences using optically pure L-SeMet as a raw material and tests their toxic effects on HeLa (cervical cancer) cells. The results of this invention indicate that L-selenomethionine-containing oligopeptides possess good antitumor activity and can be developed into related drugs.

[0006] (III) Summary of the Invention

[0007] The purpose of this invention is to provide an L-selenomethionine-containing oligopeptide and its preparation method, as well as the application of this type of oligopeptide in the preparation of antitumor active drugs. This type of oligopeptide has a simple synthesis process, good antitumor activity, and good application value.

[0008] The technical solution of the present invention is as follows:

[0009] Application of the L-selenomethionine-containing oligopeptide of formula (III) in the preparation of antitumor drugs:

[0010]

[0011] In formula (III), R1 is an amino acid residue, dipeptide residue, or tripeptide residue that forms a peptide bond with the amino group in the formula via a carbonyl group, and R2 is a hydroxyl group or an amino acid residue that forms a peptide bond with the carbonyl group in the formula via an amino group.

[0012] Furthermore, R1 is one of the following groups:

[0013] Furthermore, R2 is a hydroxyl group or one of the following groups:

[0014]

[0015] Using a technical solution, 2-3 different amino acids are combined with selenomethionine to form selenium-containing tripeptides and selenium-containing tetrapeptides with anti-tumor activity.

[0016] Furthermore, the L-selenomethionine-containing oligopeptide of the present invention is preferably one of the following:

[0017]

[0018]

[0019] Preferably, the L-selenomethionine-containing oligopeptide is a compound of formula III-1 to III-10. More preferably, it is a compound of formula III-1, III-6, or III-4. Particularly preferred is a compound of formula III-6.

[0020] Furthermore, the tumor is cervical cancer, preferably HeLa cells.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] (1) In this invention, L-selenomethionine oligopeptide (III) was further prepared by using synthesized L-selenomethionine (II).

[0023] (2) This type of L-selenomethionine oligopeptide has good anti-tumor activity. In this invention, (II-1) to (II-10) have good inhibitory activity against HeLa (cervical cancer) cells.

[0024] (3) The results obtained by this invention show that L-selenomethionine oligopeptide has broad application prospects in drug development system, and provides new and broader ideas for the synthesis and screening of L-selenomethionine oligopeptide, in order to provide a more effective way for the treatment of related diseases. (IV) Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0026] Example 1: Preparation of L-selenomethionine (II)

[0027] The reaction formula is as follows:

[0028]

[0029] a. Preparation of L-aminobutyrolactone hydrochloride II-2

[0030] Weigh 0.595 g (5 mmol) of L-homoserine into a flask and add 6 M·L -1 7.5 mL of HCl aqueous solution was added, and the mixture was heated under reflux for 4 h until the substrate reacted completely. After cooling, the mixture was filtered to obtain a white solid. After washing, the solid was dried under vacuum at room temperature to obtain product II-2. The yield was 0.648 g, with a yield of 94.3%. The measured melting point of the product was 218 °C.

[0031] b. Preparation of L-bromohomoserine hydrobromide II-3

[0032] Weigh 0.548 g (4 mmol) of the above product II-2 into a pressure-resistant bottle, add acetic acid solution (acetic acid mass fraction of 33%), stir and heat at 110 °C for 5.5 h until the substrate reacts completely. After cooling to room temperature, a white precipitate is formed. Filter to obtain product II-3, dry under vacuum at room temperature to obtain 0.994 g of product, yield 94.5%. 1 H NMR (500MHz, CD3OD) δ4.17(s,1H),3.66(td,J=6.9,2.4Hz,2H),2.63–2.47(m,1H),2.39(d,J=6.9Hz,1H). 13 C NMR (126MHz, CD3OD) δ174.33,55.91,38.18,31.80.

[0033] c. Preparation of L-bromohomoserine methyl ester hydrochloride II-4

[0034] Weigh 0.973 g (3.5 mmol) of product II-3 into a flask, add 3 mL of SOCl2, reflux under nitrogen for 1 h, evaporate the solvent, cool to 0 °C, add 3 mL of methanol, and then slowly heat to 50 °C. After the reaction is complete, evaporate the solvent to obtain a pale yellow syrupy substance II-4, which is dried under vacuum at room temperature to obtain 0.758 g of product, with a yield of 93.2%.

[0035] d. Preparation of L-Boc-bromohomoserine methyl ester II-5

[0036] Weigh 0.693 g (3 mmol) of product II-4 into a flask, add 3 mL of dichloromethane and stir to dissolve. Then add 0.334 g of Et3N (3.3 mmol) and 0.719 g of (Boc)2O (3.3 mmol) and react overnight at room temperature. After the substrate reaction is complete, evaporate the solvent to obtain a solid, dry under vacuum at room temperature, and purify by column chromatography [V(Pet):V(EtoAc) = 4:1] to obtain the initial product. Collect the solvent, evaporate the solvent to dryness, and dry under vacuum at room temperature to obtain the target product II-5, yielding 0.832 g of product, with a yield of 93.7%. 1 H NMR (500MHz, CDCl3) δ5.19(s,1H),4.41(s,1H),3.73(s,3H),3.42(s,2H),2.37(s,1H),2.19(s,1H),1.41(s,9H).

[0037] e. Preparation of L-Boc-selenomethionine methyl ester II-6

[0038] First, 0.564 g (3 mmol) of dimethyl diselenide was weighed into a flask and dissolved in 3 mL of anhydrous methanol. Then, 0.158 g (6.6 mmol) of dry NaH was rapidly added. This process was carried out under strictly anhydrous and oxygen-free conditions. A vigorous initial reaction was observed, and the reaction proceeded at room temperature for 40 min. 0.590 g (2 mmol) of the completely dried product II-5 was weighed and dissolved in 3 mL of anhydrous methanol, then added to the reaction solution. Substrate consumption and product formation were monitored by GC-MS. After 5 h of reaction, the substrate reaction was complete, and the crude product was a pale yellow syrup. The solvent was evaporated to dryness, and the product was purified by column chromatography. The solvent was collected, evaporated to dryness, and dried under vacuum at room temperature to obtain the target product II-6. The yield was 0.849 g, or 91.2%.

[0039] f. Preparation of L-selenomethionine methyl ester II-7

[0040] Weigh 0.467 g (1.5 mmol) of the obtained substance II-6 into a flask and add 5 mL of 4 M·L⁻¹ solution. -1The product was dissolved in a solution of HCl and stirred at room temperature for 2 hours, resulting in the precipitation of a white precipitate. The precipitate was filtered, washed with diethyl ether, and then dried under vacuum at room temperature to give 0.293 g of the product, with a yield of 93.1%.

[0041] Preparation of g, L-selenomethionine II

[0042] Weigh 2.11 g (10 mmol) of the above substance II-7 into a flask, add 10 mL of methanol and 5 mL of water. Stir to dissolve, add 48 mg (20 mmol LiOH), and monitor the reaction by TLC. After the reaction is complete and cooled, a white precipitate forms. After filtration, washing with ether, and drying under vacuum at room temperature, L-selenomethionine is obtained, yielding 1.494 g of the product, with a yield of 76.2%. ESI-MS, m / z: M + 197. 1 H NMR (500MHz, D2O) δ3.87–3.77(m,1H),2.62(td,J=7.9,1.4Hz,2H),2.30–2.12(m,2H),2.02(d,J=1.5Hz,3H). 13 C NMR(125MHz,D2O)δ174.32,55.91,38.18,31.80.[α] D +21.7°(C=0.5, 2N HCl), mp=275℃.

[0043] The overall reaction of this invention adopts a strategy of synthesis from the C-terminus to the N-terminus of the amino acid in one step. The synthesis of other tripeptides is consistent with the above-described steps, with the difference being that the types of amino acids condensed and coupled are different or the amino acid coupling order is different. The synthesis of tetrapeptides is based on tripeptides with amino acids protected by Boc and no protecting group at the carboxyl terminus, and then condensed and coupled with a fourth amino acid. The other steps are consistent with the synthesis of tripeptides.

[0044] Example 2: Preparation of L-selenomethionine-containing oligopeptide Ala-Gly-SeMet-OH(III-1)

[0045] The reaction formula is as follows:

[0046]

[0047] a. Synthesis of Boc-Ala-Gly-OMe

[0048] Take a flask, weigh out Boc-Ala-OH (1 mmol, 0.198 g) and dissolve it in DCM (5 mL). Add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g). Stir and activate for 5 min, then add L-NH2-Gly-OMe·HCl (1.3 mmol, 0.163 g) and react for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.249 g of product, with a yield of 95.9%.

[0049] b. Synthesis of Boc-Ala-Gly-OH

[0050] The product Boc-Ala-Gly-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Boc-Ala-Gly-OH, yielding 0.227 g (96.2% yield).

[0051] c. Synthesis of Boc-Ala-Gly-SeMet-OMe

[0052] The Boc-Ala-Gly-OH (0.95 mmol, 0.234 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.39 g of product, with a yield of 96.5%.

[0053] d. Synthesis of Boc-Ala-Gly-SeMet-OH

[0054] The product Boc-Ala-Gly-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Boc-Ala-Gly-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.356 g of product was obtained, with a yield of 94.3%.

[0055] e. Synthesis of NH2-Ala-Gly-SeMet-OH

[0056] The above-obtained Boc-Ala-Gly-SeMet-OH was dissolved in 4 M·L under ice bath conditions. -1 The reaction was monitored by TLC using HCl 1,4-dioxane solution. After the substrate had reacted completely and new spots formed, 50 mL of diethyl ether was added, resulting in the precipitation of a white precipitate. The mixture was then stirred thoroughly for 2 hours, at which point the substance became hydrophilic. The mixture was filtered and washed three times with 150 mL of diethyl ether. The solvent was removed by vacuum evaporation, yielding 0.269 g of the product with a purity of 98.8% and a yield of 97.4%. Its structure is characterized as follows: 1 H NMR (500MHz, (CD3)2SO) δ12.67(s,1H),8.77(s,1H),8.30(dd,J=13.5,6.7Hz,4H),4.28(d,J=5. 2Hz,1H),3.90–3.72(m,3H),2.45(d,J=8.0Hz,2H),1.99–1.82(m,4H),1.32(d,J=6.9Hz,3H).13C NMR (126MHz, (CD3)2SO) δ173.44,170.30,168.83,66.81,52.32,48.62,32.16,21.21,17.53,3.98.

[0057] Example 3: Preparation of L-selenomethionine-containing oligopeptide Pro-Ile-SeMet-OH(III-2)

[0058]

[0059] a. Synthesis of Pro-Ile-OMe

[0060] Take a flask, weigh out Pro (1 mmol, 0.115 g), dissolve it in DCM (5 mL), add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g), and stir to activate for 5 min. Then add L-NH2-Ile-OMe·HCl (1.3 mmol, 0.236 g) and react for 5 h. After the reaction is complete as monitored by TLC, add DCM (60 mL) and stir well. Then wash successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). Then dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to obtain 0.232 g of product, with a product yield of 95.9%.

[0061] b. Synthesis of Pro-Ile-OH

[0062] The product Pro-Ile-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Pro-Ile-OH, yielding 0.211 g of product, with a yield of 96.2%.

[0063] c. Synthesis of Pro-Ile-SeMet-OMe

[0064] The Pro-Ile-OH (0.95 mmol, 0.217 g) obtained in the previous step was dissolved in DCM (5 mL), and N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g) were added and stirred for 5 min to activate the mixture. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the mixture was reacted for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.374 g of product, with a product yield of 96.5%.

[0065] d. Synthesis of Pro-Ile-SeMet-OH

[0066] The product Pro-Ile-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.341 g of the product was obtained, with a yield of 94.3%. Its structure is characterized as follows: 1 H NMR(500MHz, (CD3)2SO) δ12.88(s,1H),10.50(dd,J=11.7,6.1Hz,1H),8.96(d,J=8.6Hz,1H),8.69(p,J=6.2,5. 7Hz, 1H), 8.61 (d, J = 7.7Hz, 1H), 4.54 (ddd, J = 9.5, 7.7, 4.5Hz, 1H), 4.42 (t, J = 8.2Hz, 2H), 3.39 (ddp, J = 27.0, 17. 3,5.7Hz,2H),2.78(ddd,J=12.4,8.6,5.1Hz,1H),2.72(d,J=6.9Hz,1H),2.58–2.43(m,1H),2.14(s,4H),2.11–1 .91(m,4H),1.69(dtd,J=15.0,7.5,3.3Hz,1H),1.41–1.25(m,1H),1.10(d,J=6.7Hz,3H),1.04(t,J=7.5Hz,3H). 13 C NMR (125MHz, (CD3)2SO) δ173.43,171.17,168.54,66.82,58.91,57.97,52.15,46.05,36.87,30.37,24.80,23.90,21.23,15.71,11.37,3.99.

[0067] Example 4: Preparation of L-selenomethionine-containing oligopeptide Phe-Pro-SeMet-OH(Ⅲ-3)

[0068]

[0069] a. Synthesis of Boc-Phe-Pro-OMe

[0070] Take a flask, weigh out Boc-Phe-OH (1 mmol, 0.265 g) and dissolve it in DCM (5 mL). Add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g). Stir and activate for 5 min, then add L-NH2-Pro-OMe·HCl (1.3 mmol, 0.215 g) and react for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.361 g of product, with a yield of 95.9%.

[0071] b. Synthesis of Boc-Phe-Pro-OH

[0072] The product Boc-Phe-Pro-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Boc-Phe-Pro-OH, yielding 0.348 g of product (96.2% yield).

[0073] c. Synthesis of Boc-Phe-Pro-SeMet-OMe

[0074] The Boc-Phe-Pro-OH (0.95 mmol, 0.344 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.494 g of product, with a yield of 96.5%.

[0075] d. Synthesis of Boc-Phe-Pro-SeMet-OH

[0076] The product Boc-Phe-Pro-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Boc-Phe-Pro-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.455 g of the product was obtained, with a yield of 94.3%.

[0077] e. Synthesis of NH2-Phe-Pro-SeMet-OH

[0078] The obtained Boc-Phe-Pro-SeMet-OH was dissolved in 4 M·L⁻¹ HCl 1,4-dioxane solution under ice bath conditions. The reaction was monitored by TLC. After the substrate had reacted completely and a new spot was formed, 50 mL of diethyl ether was added, at which point a white precipitate formed. The mixture was then stirred thoroughly for 2 h, at which point the substance became hydrophilic. The mixture was filtered to obtain the above substance and washed three times with 150 mL of diethyl ether. The solvent was removed by evaporation under reduced pressure to obtain the product. The final product with a purity of 98.8% was obtained, yielding 0.356 g of product, with a yield of 97.4%. Its structural characterization is as follows: 1H NMR (500MHz, (CD3)2SO) δ12.48 (s, 1H), 8.74–8.59 (m, 2H), 8.39 (d, J = 7.8Hz, 1H), 7.35–7.27 (m, 3H), 7. 21(d,J=6.8Hz,2H),4.28–4.21(m,1H),4.19–4.07(m,2H),3.55(s,2H),3.21(s,1H),2.92(s,1H),2.63 –2.51(m,1H),2.49–2.43(m,1H),2.10(dtd,J=14.3,9.4,4.9Hz,1H),2.04–1.96(m,1H),1.91(d,J=8.9 Hz,3H),1.74(dhept,J=14.1,5.1,4.5Hz,1H),1.63(d,J=7.5Hz,2H),1.37(dp,J=14.4,5.3Hz,1H).13C NMR(126MHz,(CD3)2SO)δ173.20,171.32,167.40,135.16,129.91,128.98,1 27.81,66.82,60.14,52.56,47.08,37.09,31.53,29.67,23.89,21.61,3.95.

[0079] Example 5: Preparation of L-selenomethionine-containing oligopeptide Pro-Ile-SeMet-Gly-OH(Ⅲ-4)

[0080]

[0081] a. Synthesis of Pro-Ile-OMe

[0082] Take a flask, weigh out Pro (1 mmol, 0.115 g), dissolve it in DCM (5 mL), add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g), and stir to activate for 5 min. Then add L-NH2-Ile-OMe·HCl (1.3 mmol, 0.218 g) and react for 5 h. After the reaction is complete as monitored by TLC, add DCM (60 mL) and stir well. Then wash successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). Then dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to obtain 0.232 g of product, with a product yield of 95.9%.

[0083] b. Synthesis of Pro-Ile-OH

[0084] The product Pro-Ile-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Pro-Ile-OH, yielding 0.21 g of product (96.2% yield).

[0085] c. Synthesis of Pro-Ile-SeMet-OMe

[0086] The Pro-Ile-OH (0.95 mmol, 0.217 g) obtained in the previous step was dissolved in DCM (5 mL), and N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g) were added and stirred for 5 min to activate the mixture. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the mixture was reacted for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.373 g of product, with a yield of 96.5%.

[0087] d. Synthesis of Pro-Ile-SeMet-OH

[0088] The product Pro-Ile-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.341 g of product was obtained, with a yield of 94.3%.

[0089] e. Synthesis of Pro-Ile-SeMet-Gly-OMe

[0090] The Pro-Ile-SeMet-OH (0.95 mmol, 0.372 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-Gly-OMe·HCl (1.24 mmol, 0.138 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 386 g of product, with a product yield of 96.5%.

[0091] f、Pro-Ile-SeMet-Gly-OH

[0092] The product Pro-Ile-SeMet-Gly-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-Gly-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.354 g of the product was obtained, with a yield of 94.3%. Its structure is characterized as follows: 1H NMR(500MHz, (CD3)2SO) δ12.50(s,1H),10.29(dt,J=12.8,6.1Hz,1H),8.76(d,J=8.6Hz,1H),8.46(q,J=17.1,12. 5Hz,1H),8.29(s,1H),8.20(d,J=8.0Hz,1H),4.38(td,J=8.2,4.9Hz,1H),4.32–4.23(m,1H),4.21(d,J=8.1Hz,1H) ,3.79(s,1H),3.69(s,1H),3.18(qt,J=14.0,8.5,7.3Hz,2H),2.51(d,J=4.3Hz,2H),2.29(dt,J=15.6,6.2Hz,1H), 2.04–1.68(m,8H),1.45(dtt,J=15.3,7.8,3.8Hz,1H),1.10(ddt,J=21.1,17.2,8.9Hz,1H),0.93–0.75(m,6H).13C NMR(126MHz,(CD3)2SO)δ171.62,171.41,170.85,168.66,66.82,58.92,58.14,53 .00,46.06,41.11,36.69,33.49,30.37,24.85,23.93,20.71,15.81,11.41,3.93.

[0093] Example 6: Preparation of L-selenomethionine-containing oligopeptide Pro-Ile-SeMet-Val-OH(Ⅲ-5)

[0094]

[0095] a. Synthesis of Pro-Ile-OMe

[0096] Take a flask, weigh out Pro (1 mmol, 0.115 g), dissolve it in DCM (5 mL), add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g), and stir to activate for 5 min. Then add L-NH2-Ile-OMe·HCl (1.3 mmol, 0.236 g) and react for 5 h. After the reaction is complete as monitored by TLC, add DCM (60 mL) and stir well. Then wash successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). Then dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to obtain 0.232 g of product, with a product yield of 95.9%.

[0097] b. Synthesis of Pro-Ile-OH

[0098] The product Pro-Ile-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Pro-Ile-OH, yielding 0.21 g of product (96.2% yield).

[0099] c. Synthesis of Pro-Ile-SeMet-OMe

[0100] The Pro-Ile-OH (0.95 mmol, 0.217 g) obtained in the previous step was dissolved in DCM (5 mL), and N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g) were added and stirred for 5 min to activate the mixture. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the mixture was reacted for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.374 g of product, with a yield of 96.5%.

[0101] d. Synthesis of Pro-Ile-SeMet-OH

[0102] The product Pro-Ile-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.340 g of product was obtained, with a yield of 94.3%.

[0103] e. Synthesis of Pro-Ile-SeMet-Val-OMe

[0104] The Pro-Ile-SeMet-OH (0.95 mmol, 0.386 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-Val-OMe·HCl (1.24 mmol, 0.208 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.42 g of product, with a product yield of 96.5%.

[0105] f、Pro-Ile-SeMet-Val-OH

[0106] The product Pro-Ile-SeMet-Val-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-Val-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product, yielding 0.381 g, with a yield of 94.3%. Its structure is characterized as follows:

[0107] 1 H NMR (500MHz, (CD3)2SO) δ12.65(s,1H),10.29(s,1H),8.75(d,J=8.6Hz,1H),8.48(t,J=9.9Hz,1H),8.28(d,J=8 .0Hz,1H),7.94(d,J=8.3Hz,1H),4.44(td,J=8.1,5.1Hz,1H),4.23(t,J=8.1Hz,2H),4.13(dd,J=8.4,5.6Hz,1H) ,3.53–3.31(m,1H),3.17(tq,J=20.7,6.3Hz,2H),2.51(d,J=3.0Hz,2H),2.38–2.22(m,1H),2.07(tq,J=13.4,6 .8Hz,1H),1.92(m,4H),1.89–1.72(m,3H),1.45(dqd,J=15.1,7.5,3.3Hz,1H),1.08(s,1H),0.91–0.76(m,12H). 13 C NMR(125MHz,(CD3)2SO)δ172.60,170.83,170.13,166.36,66.82,58.92,58.14,52.54 ,52.00,46.06,41.11,36.69,33.49,30.37,24.85,23.93,20.71,15.81,11.41,3.93.

[0108] Example 7: Preparation of L-selenomethionine-containing oligopeptide Pro-Ile-Phe-SeMet-OH(Ⅲ-6)

[0109]

[0110] a. Synthesis of Pro-Ile-OMe

[0111] Take a flask, weigh out Pro (1 mmol, 0.115 g), dissolve it in DCM (5 mL), add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g), and stir to activate for 5 min. Then add L-NH2-Ile-OMe·HCl (1.3 mmol, 0.236 g) and react for 5 h. After the reaction is complete as monitored by TLC, add DCM (60 mL) and stir well. Then wash successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). Then dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to obtain 0.232 g of product, with a product yield of 95.9%.

[0112] b. Synthesis of Pro-Ile-OH

[0113] The product Pro-Ile-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Pro-Ile-OH, yielding 0.210 g of product, with a yield of 96.2%.

[0114] c. Synthesis of Pro-Ile-Phe-OMe

[0115] The Pro-Ile-OH (0.95 mmol, 0.217 g) obtained in the previous step was dissolved in DCM (5 mL), and N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g) were added and stirred for 5 min to activate the mixture. Then, NH2-Phe-OMe·HCl (1.24 mmol, 0.267 g) was added and the mixture was reacted for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.343 g of product, with a product yield of 95.6%.

[0116] d. Synthesis of Pro-Ile-Phe-OH

[0117] The product Pro-Ile-Phe-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, and then extracted with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-Phe-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product, yielding 0.311 g, with a yield of 94.3%.

[0118] e. Synthesis of Pro-Ile-Phe-SeMet-OMe

[0119] The Pro-Ile-Phe-OH (0.95 mmol, 0.357 g) obtained in the previous step was dissolved in DCM (5 mL). DIPEA (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until it was complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to give 0.454 g of the product, with a yield of 96.5%.

[0120] f、Pro-Ile-Phe-SeMet-OH

[0121] The product Pro-Ile-SeMet-Leu-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-Phe-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product, yielding 0.402 g, with a yield of 94.3%. The structure of the selenomethionine oligopeptide is characterized as follows:

[0122] 1 H NMR(500MHz, (CD3)2SO) δ10.24(s,1H),8.64(d,J=8.9Hz,1H),8.46(td,J=10.7,6.8Hz,1H),8.34(dd,J=14.5,8.1Hz,2H),7.28(d,J=7.0 Hz,2H),7.22(t,J=7.5Hz,2H),7.16(d,J=7.4Hz,1H),4.65–4.53(m,1H),4.34(ddd,J=9.2,7.8,4.7Hz,1H),4.28–4.15(m,2H),3.18(dtd . 87–1.74(m,2H),1.73–1.59(m,2H),1.36(dqd,J=15.0,7.4,4.2Hz,1H),1.10–0.92(m,1H),0.78(t,J=7.4Hz,3H),0.73(d,J=6.8Hz,3H). 13 C NMR(125MHz,(CD3)2SO)δ178.12,176.37,175.36,173.10,142.96,134.44,133.13,131.35,71.60,63.69, 62.75,58.91,57.11,50.84,45.24,44.24,41.88,37.08,35.09,29.47,28.70,25.91,20.41,16.16,8.74.

[0123] Example 8: Preparation of L-selenomethionine-containing oligopeptide Leu-Gly-SeMet-Ile-OH (Ⅲ-7)

[0124]

[0125] a. Synthesis of Boc-Leu-Gly-OMe

[0126] Take a flask, weigh out Boc-Leu-OH (1 mmol, 0.231 g) and dissolve it in DCM (5 mL). Add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g). Stir and activate for 5 min, then add L-NH2-Gly-OMe·HCl (1.3 mmol, 0.163 g) and react for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.29 g of product, with a yield of 95.9%.

[0127] b. Synthesis of Boc-Leu-Gly-OH

[0128] The product Boc-Phe-Pro-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Boc-Leu-Gly-OH, yielding 0.266 g of product (96.2% yield).

[0129] c. Synthesis of Boc-Leu-Gly-SeMet-OMe

[0130] The Boc-Leu-Gly-OH (0.95 mmol, 0.274 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.440 g of product, with a product yield of 96.5%.

[0131] d. Synthesis of Boc-Leu-Gly-SeMet-OH

[0132] The product Boc-Leu-Gly-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Boc-Leu-Gly-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.403 g of product was obtained, with a yield of 94.3%.

[0133] e. Synthesis of Boc-Leu-Gly-SeMet-Ile-OMe

[0134] The Boc-Leu-Gly-SeMet-OH (0.95 mmol, 0.456 g) obtained in the previous step was dissolved in DCM (5 mL), and N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C. Immediately afterwards, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g) were added and stirred for 5 min to activate the mixture. Then, NH2-Ile-OMe·HCl (1.24 mmol, 0.225 g) was added and the mixture was reacted for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.48 g of product, with a yield of 96.5%.

[0135] f. Synthesis of Boc-Leu-Gly-SeMet-Ile-OH

[0136] The product from the previous step, Boc-Leu-Gly-SeMet-Ile-OMe, was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product, Boc-Leu-Gly-SeMet-Ile-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.4 g of product was obtained, with a yield of 94.3%.

[0137] Synthesis of g, NH2-Leu-Gly-SeMet-Ile-OH

[0138] The obtained Boc-Leu-Gly-SeMet-Ile-OH was dissolved in 4 M·L⁻¹ HCl 1,4-dioxane solution under ice bath conditions. The reaction was monitored by TLC. After the substrate had reacted completely and a new spot was formed, 50 mL of diethyl ether was added, at which point a white precipitate formed. The mixture was then stirred thoroughly for 2 h, at which point the substance became hydrophilic. The mixture was filtered to obtain the above substance and washed three times with 150 mL of diethyl ether to obtain a final product with a purity of 98.8%, yielding 0.345 g of product, with a yield of 97.4%. Its structural characterization is as follows:

[0139] 1H NMR(500MHz, (CD3)2SO) δ12.56(s,1H),8.95(s,1H),8.41(d,J=5.6Hz,3H),8.22(d,J=8.2Hz,1H),8.09(d,J =8.1Hz,1H),4.45(td,J=8.0,5.9Hz,1H),4.13(d,J=2.3Hz,1H),3.93–3.67(m,3H),2.48(dddt,J=19.0,12. 1,9.6,6.5Hz,2H),2.00–1.86(m,4H),1.86–1.75(m,1H),1.70(dq,J=13.2,6.6Hz,1H),1.58(d,J=7.2Hz,2H ),1.40(dqd,J=15.1,7.6,4.2Hz,1H),1.20(ddddd,J=17.7,13.8,10.7,7.1,2.8Hz,1H),0.92–0.78(m,12H). 13 C NMR(125MHz,(CD3)2SO)δ173.17,171.56,169.76,168.48,66.82,65.37,56.91,53.19 ,51.36,42.45,36.54,33.76,25.18,24.01,22.93,22.77,20.73,16.02,11.79,3.94.

[0140] Example 9: Preparation of L-selenomethionine-containing oligopeptide Lys-SeMet-Pro-OH(Ⅲ-8)

[0141] The reaction formula is as follows:

[0142]

[0143] a. Synthesis of Boc-Lys-SeMet-OMe

[0144] Take a flask, weigh 3.1 (1 mmol, 0.693 g) and dissolve it in DCM (5 mL). Add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g). Stir and activate for 5 min, then add L-NH2-SeMet-OMe·HCl (1.3 mmol, 0.321 g) and react for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.516 g of product, with a yield of 95.9%.

[0145] b. Synthesis of Boc-Lys-Se-Met-OH

[0146] The product Boc-Lys-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Boc-Lys-SeMet-OH, yielding 0.484 g of product (96.2% yield).

[0147] c. Synthesis of Boc-Lys-SeMet-Pro-OMe

[0148] The Boc-Lys-SeMet-OH (0.95 mmol, 0.512 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-Pro-OMe·HCl (1.24 mmol, 0.204 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.57 g of product, with a yield of 97.2%.

[0149] d. Synthesis of Boc-Lys-SeMet-Pro-OH

[0150] The product Boc-Lys-SeMet-Pro-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Boc-Lys-SeMet-Pro-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.540 g of product was obtained, with a yield of 96.8%.

[0151] e. Synthesis of NH2-Lys-SeMet-Pro-OH

[0152] The obtained Boc-Lys-SeMet-Pro-OH was dissolved in 4M·L⁻¹ HCl 1,4-dioxane solution under ice bath conditions. The reaction was monitored by TLC. After the substrate had reacted completely and new spots were formed, 50 mL of diethyl ether was added, at which point a white precipitate formed. The mixture was then stirred thoroughly for 2 h, at which point the substance became hydrophilic. The mixture was filtered to obtain the above substance and washed three times with 150 mL of diethyl ether. 0.368 g of the product was obtained, with a yield of 98.2%. Its structural characterization is as follows: 1 H NMR (500MHz, (CD3)2SO) δ12.75 (s, 1H), 8.46–8.33 (m, 3H), 8.19 (t, J = 6.4Hz, 3H), 4. 61(tt,J=12.1,5.8Hz,1H),4.29–4.21(m,1H),3.85(h,J=6.5Hz,1H),3.72–3.66(m,1 H),3.60(dt,J=9.9,6.6Hz,1H),2.77–2.58(m,4H),2.15(dq,J=12.5,7.7Hz,1H),2.0 0–1.87(m,5H),1.86–1.66(m,4H),1.59(t,J=4.9Hz,2H),1.36(q,J=8.4,7.9Hz,2H). 13 C NMR (125MHz, (CD3)2SO) δ178.26,174.41,173.70,63.75,56.73,56.00,51.89,43.44,36.96,35.40,33.83,31.26,29.82,26.14,25.54,8.82.

[0153] Example 10: Preparation of L-selenomethionine-containing oligopeptide Lys-SeMet-Ala-OH (Ⅲ-9)

[0154] The reaction formula is as follows:

[0155]

[0156] a. Synthesis of Boc-Lys-SeMet-OMe

[0157] Take a flask, weigh 3.1 (1 mmol, 0.693 g) and dissolve it in DCM (5 mL). Add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g). Stir and activate for 5 min, then add L-NH2-SeMet-OMe·HCl (1.3 mmol, 0.321 g) and react for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.516 g of product, with a yield of 95.9%.

[0158] b. Synthesis of Boc-Lys-Se-Met-OH

[0159] The product Boc-Lys-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and then extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Boc-Lys-SeMet-OH, yielding 0.484 g of product (96.2% yield).

[0160] c. Synthesis of Boc-Lys-SeMet-Ala-OMe

[0161] The Boc-Lys-SeMet-OH (0.95 mmol, 0.512 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-Ala-OMe·HCl (1.24 mmol, 0.173 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until it was complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. 0.547 g of product was obtained, with a yield of 97.3%.

[0162] d. Synthesis of Boc-Lys-SeMet-Ala-OH

[0163] The product Boc-Lys-SeMet-Ala-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Boc-Lys-SeMet-Ala-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.523 g of product was obtained, with a yield of 97.8%.

[0164] e. Synthesis of NH2-Lys-SeMet-Ala-OH

[0165] The obtained Boc-Lys-SeMet-Ala-OH was dissolved in 4M·L⁻¹ HCl 1,4-dioxane solution under ice bath conditions. The reaction was monitored by TLC. After the substrate had reacted completely and new spots were formed, 50 mL of diethyl ether was added, at which point a white precipitate formed. The mixture was then stirred thoroughly for 2 h, at which point the substance became hydrophilic. The mixture was filtered to obtain the above substance and washed three times with 150 mL of diethyl ether. 0.342 g of the product was obtained, with a yield of 98.2%. Its structural characterization is as follows:

[0166] 1H NMR(500MHz,(CD3)2SO)δ12.56(s,1H),8.85(d,J=7.8Hz,1H),8.41(dd,J=10.8, 6.2Hz,3H),8.18(d,J=5.9Hz,3H),4.36(d,J=5.2Hz,1H),4.16(t,J=7.2Hz,1H), 3.85(d,J=5.6Hz,1H),2.71(q,J=6.8Hz,2H),2.65–2.44(m,2H),1.93(s,4H),1. 80–1.65(m,2H),1.65–1.50(m,2H),1.38(t,J=7.9Hz,2H),1.26(d,J=7.2Hz,3H). 13 C NMR (125MHz, (CD3)2SO) δ179.07,175.63,173.63,58.19,56.90,52.81,43.44,38.32,35.42,31.35,26.17,25.44,22.18,8.70.

[0167] Example 11: Preparation of L-selenomethionine-containing oligopeptide Pro-Ile-SeMet-Phe-OH(Ⅲ-10)

[0168]

[0169] a. Synthesis of Pro-Ile-OMe

[0170] Take a flask, weigh out Pro (1 mmol, 0.115 g), dissolve it in DCM (5 mL), add N,N-diisopropylethylamine (2.4 mmol, 0.310 g) at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.2 mmol, 0.230 g) and 1-hydroxybenzotriazole (HOBT) (1.2 mmol, 0.162 g), and stir to activate for 5 min. Then add L-NH2-Ile-OMe·HCl (1.3 mmol, 0.236 g) and react for 5 h. After the reaction is complete as monitored by TLC, add DCM (60 mL) and stir well. Then wash successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). Then dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate under vacuum to obtain 0.232 g of product, with a product yield of 95.9%.

[0171] b. Synthesis of Pro-Ile-OH

[0172] The product Pro-Ile-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and the mixture was stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with 0.5 M·L⁻¹ HCl, and the solution was extracted with ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the pure product Pro-Ile-OH, yielding 0.210 g of product, with a yield of 96.2%.

[0173] c. Synthesis of Pro-Ile-SeMet-OMe

[0174] The Pro-Ile-OH (0.95 mmol, 0.217 g) obtained in the previous step was dissolved in DCM (5 mL), and N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g) were added and stirred for 5 min to activate the mixture. Then, NH2-SeMet-OMe·HCl (1.24 mmol, 0.305 g) was added and the mixture was reacted for 5 h. After the reaction was monitored by TLC until it was complete, DCM (60 mL) was added. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to give 0.374 g of product, with a yield of 96.5%.

[0175] d. Synthesis of Pro-Ile-SeMet-OH

[0176] The product Pro-Ile-SeMet-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. The eluent was collected and concentrated under reduced pressure to obtain the product. 0.349 g of the product was obtained, with a yield of 94.3%.

[0177] e. Synthesis of Pro-Ile-SeMet-Phe-OMe

[0178] The Pro-Ile-SeMet-OH (0.95 mmol, 0.512 g) obtained in the previous step was dissolved in DCM (5 mL). N,N-diisopropylethylamine (2.28 mmol, 0.295 g) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.14 mmol, 0.219 g) and 1-hydroxybenzotriazole (HOBT) (1.14 mmol, 0.154 g). The mixture was stirred and activated for 5 min. Then, NH2-Phe-OMe·HCl (1.24 mmol, 0.386 g) was added and the reaction was carried out for 5 h. After the reaction was monitored by TLC until complete, DCM (60 mL) was added and stirred until homogeneous. The mixture was then washed successively with 5% phosphoric acid aqueous solution (120 mL), saturated sodium bicarbonate solution (120 mL), and saturated sodium chloride solution (120 mL). The organic phase was then dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to obtain 0.47 g of product, with a product yield of 96.5%.

[0179] f、Pro-Ile-SeMet-Phe-OH

[0180] The product Pro-Ile-SeMet-Leu-OMe from the previous step was dissolved in 5 mL of methanol. Under ice bath conditions, 5 mL of 1 M·L⁻¹ lithium hydroxide aqueous solution was added and stirred thoroughly. The reaction progress was monitored by TLC. After the reaction was complete, the pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product Pro-Ile-SeMet-Phe-OH. The crude product was then purified by column chromatography with an eluent ratio of V(Pet):V(EtoAc) of 4:1. The process was monitored by TLC combined with ninhydrin and iodine staining. 0.433 g of the product was obtained, with a yield of 94.3%. Its structure is characterized as follows: 1 H NMR(500MHz,(CD3)2SO)δ12.77(s,1H),10.28(s,1H),8.90–8.63(m,1H),8.44( d,J=31.9Hz,1H),8.36–8.02(m,2H),7.21(d,J=27.2Hz,5H),4.40(d,J=39.1Hz ,2H),4.33–4.06(m,2H),3.32–2.98(m,3H),2.93(d,J=10.6Hz,1H),2.39(dd,J =47.3,33.2Hz,3H),2.12–1.57(m,8H),1.43(s,1H),1.09(s,1H),0.80(s,6H). 13C NMR (125MHz, (CD3)2SO) δ173.08,171.18,170.72,168.59,137.88,129.54,128.58,126.85,66.83,58. 93,58.06,53.83,53.03,46.05,36.98,36.77,33.71,30.38,24.84,23.93,20.62,15.79,11.41,3.91.

[0181] Example 12: Preparation of L-selenomethionine-containing oligopeptide Phe-Val-SeMet-OH(Ⅲ-12)

[0182]

[0183] a. Synthesis of N-tert-butoxycarbonylphenylalanylvaline methyl ester Boc-Phe-Val-OMe

[0184] The procedure was the same as step a in Example 2, except that 1-1 (1 mmol, 0.231 g) was replaced with 3-1 (1 mmol, 0.265 g) and glycine methyl ester hydrochloride (1.3 mmol, 0.163 g) was replaced with valine methyl ester hydrochloride (1.3 mmol, 0.218 g), resulting in a final product of 0.362 g and a yield of 95.7%.

[0185] b. Synthesis of N-tert-butoxycarbonylphenylalanylvaline Boc-Phe-Val-OH

[0186] The procedure was the same as step b in Example 2, except that 1-2 was replaced with 3-2, ultimately yielding 0.356 g of product, with a yield of 96.3%. c. Synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionine methyl ester Boc-Phe-Val-Se-Met-OMe

[0187] The procedure was the same as step c in Example 2, except that 1-3 (0.9 mmol, 0.259 g) was replaced with 2-3 (0.9 mmol, 0.328 g), and the final product was 0.488 g, with a yield of 97.5%.

[0188] The synthesis of N-tert-butoxycarbonylphenylalanylvaline selenomethionine Boc-Phe-Val-SeMet-OH was performed in the same manner as step d in Example 2, except that 1-4 was replaced with 2-4. The final product was 0.447 g, with a yield of 93.8%.

[0189] e. Synthesis of phenylalanylvaline selenomethionine NH2-Phe-Val-Se-Met-OH

[0190] The operation is the same as step e in Example 2, except that 1-5 is replaced with 2-5, and the final product is 0.352g, with a yield of 96.6%. Its structural characterization is as follows: ¹H NMR (500MHz, (CD₃)₂SO) δ 12.65 (s, ¹H), 8.78 (d, J = 8.8 Hz, ¹H), 8.44 (d, J = 7.6 Hz, ¹H), 8.42–8.25 (m, 2H), 7.31–7.17 (m, 5H), 4.32 (ddd, J = 9.6, 7.6, 4.4 Hz, 1H), 4.27–4.10 (m, 2H), 3.16 (dd, J = 14.0, 5.6 Hz, 1H), 3.01 (dd, J = 14.1, 6.8 Hz, 1H), 2.56 (s, 2H), 2.11–1.92 (m, 6H), 0.91 (t, J = 6.9 Hz, 6H). ¹³C NMR(125MHz,(CD3)2SO)δ173.38,171.01,168.14,135.36,130.14,128.80,127. 42,66.82,58.38,53.47,52.34,37.17,32.08,31.36,21.24,19.58,18.89,3.95.

[0191] Example 13: Determination of the inhibitory effect of L-selenomethionine-containing oligopeptides (III-1) to (III-10), (III-12) on cell growth

[0192] Cervical cancer (HeLa) cells were purchased from the National University of Singapore.

[0193] (1) Cervical cancer (HeLa) cell culture

[0194] Culture conditions: DMEM high glucose culture medium (purchased from Hangzhou Zeheng Biotechnology Co., Ltd.) + 10% / high-quality fetal bovine serum, 37℃, gas phase: air (95%), carbon dioxide (5%).

[0195] Frozen storage conditions: DMEM high glucose culture medium + 10% fetal bovine serum + 10% DMSO.

[0196] Passaging method: HeLa cells were placed in T25 cell culture dishes for adherent culture under the conditions of 5% CO2 and 37°C, using DMEM solution containing 10% FBS. When the cells reached the logarithmic growth phase, the T25 cell culture dishes were removed from the incubator, and 2 mL of trypsin was added for digestion. After complete digestion, 1 mL of trypsin was pipetted into a 1.5 mL EP tube, centrifuged at 1500 rpm for 5 min, and the trypsin was discarded. The cells were resuspended in culture medium and diluted to a cell density of 1 × 10⁵ / mL. The cell suspension was then pipetted into 96-well plates (100 μL per well). A blank control was added to each well with 100 μL of cell-free culture medium. The 96-well plates were incubated overnight in a cell culture incubator.

[0197] (2) Antitumor activity assay (MTT method)

[0198] HeLa cells were cultured adherently in T25 cell culture dishes under 5% CO2 and 37°C conditions in DMEM solution containing 10% FBS. When the cells reached the logarithmic growth phase, the T25 cell culture dishes were removed from the incubator, and 2 mL of trypsin was added for digestion. After complete digestion, 1 mL of trypsin was pipetted into a 1.5 mL EP tube, centrifuged at 1500 rpm for 5 min, the trypsin was discarded, and the cells were resuspended in culture medium and diluted to a cell density of 1 × 10⁻⁶ cells / mL. 5 / mL. Add 1mL of cell suspension to each well of a 96-well plate using a pipette, with a volume of 100μL per well. Add 100μL of cell-free culture medium to the blank control. Incubate the 96-well plate overnight in a cell culture incubator. When the cells in the 96-well plate reach the logarithmic growth phase, add 1μL of different concentrations of the test sample to each experimental group, with three replicates for each concentration. Incubate for 24 hours. After incubation, add 10μL of CCK8 reagent to each well and incubate for another 2 hours at 37℃ and 5% CO2. Add 1μL of DMSO at the corresponding concentration to the control group. After 2 hours of incubation, measure the absorbance at 450nm using a microplate reader. Calculate cell viability using the following formula:

[0199]

[0200] By replacing compound III-1 in the above steps with Cisplatin, and then with compounds III-2, III-3, III-4, III-5, III-6, III-7, III-8, III-9, and III-10 respectively, while keeping III-12 unchanged, the antitumor effects of compounds III-2 to III-10 and III-12 can be determined. The sample test results are shown in Table 1.

[0201] Table 1. Inhibitory activity of L-selenomethionine (II) and L-selenomethionine-containing oligopeptides on HeLa cells.

[0202]

[0203] Note: The compounds corresponding to Ⅲ-1—Ⅲ-10 and Ⅲ-12 are Ala-Gly-SeMet-OH, Ile-Pro-SeMet-OH, Phe-Pro-SeMet-OH, Pro-Ile-SeMet-Gly-OH, Pro-Ile-SeMet-Val-OH, Phe-Val-Se-Met-Pro-OH, Leu-Gly-SeMet-Ala-OH, Lys-SeMet-Pro-OH, Lys-SeMet-Ala-OH, Pro-Ile-SeMet-Phe-OH, and Phe-Val-SeMet-OH, respectively.

[0204] As shown in the table above, compound III-12 had little inhibitory effect on HeLa cells regardless of the concentration gradient, while compounds III-1 to III-10 all had significant inhibitory effects on HeLa cells. At a sample concentration of 10 μg / mL, Cisplatin inhibited HeLa cells by 77.9%, and the inhibition rates of compounds III-1 to III-10 were generally between 51% and 76%. Compounds III-6, III-1, and III-4 showed the best inhibitory effects, with inhibition concentrations reaching 76%, 62%, and 61% respectively.

[0205] At a concentration of 20 μg / mL, Cisplatin inhibited HeLa cells by 87%. The inhibition rates of compounds III-1 to III-10 on HeLa cells were generally between 56% and 86%. The compounds III-6, III-1, and III-5 showed the best inhibitory effects, with inhibition concentrations of 86% and 64% on HeLa cells, respectively.

[0206] When the drug concentration was further increased to 40 ug / mL, Cisplatin inhibited HeLa cells by 92%, slightly higher than compound III-6's 89%.

[0207] When the drug concentration was 80 ug / mL, compounds III-1 to III-10 showed good inhibition rates against HeLa cells, ranging from 63% to 92%, with compound III-6 showing an inhibition rate of 90%, close to that of Cisplatin (92%).

[0208] In summary, compounds III-1 to III-10 all exhibited significant inhibitory effects on HeLa cells. At low concentrations, their inhibitory activity against HeLa cells was close to that of Cisplatin, while at high concentrations, their inhibitory activity against HeLa cells was lower than that of Cisplatin.

[0209] Overall, when the concentrations of the 10 selenopeptides (compounds III-1 to III-10) ranged from 10 μg / mL to 80 μg / mL, their inhibitory rates on HeLa cells generally remained stable and increased slowly with increasing concentration. Within this concentration range, the inhibition rate fluctuated relatively smoothly, and all compounds showed an inhibition rate of over 51% against this tumor cell, demonstrating a good ability to inhibit this type of cervical cancer cell.

Claims

1. Application of the L-selenomethionine-containing oligopeptide shown in formula (III) in the preparation of antitumor drugs: the tumor is cervical cancer, In formula (III), R1 is an amino acid residue, dipeptide residue, or tripeptide residue that forms a peptide bond with the amino group in the formula via a carbonyl group; R2 is a hydroxyl group or an amino acid residue that forms a peptide bond with the carbonyl group in the formula via an amino group; the L-selenomethionine-containing oligopeptide is one of the following:

2. The application as described in claim 1, characterized in that: The L-selenomethionine-containing oligopeptide is a compound represented by formula III-1, III-6, or III-4.

3. The application as described in claim 2, characterized in that: The L-selenomethionine-containing oligopeptide is the compound shown in Formula III-6.

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