OLIGOPEPTIDE LINKER INTERMEDIATE AND PREPARATION METHOD THEREOF

Abstract

The invention provides a new oligopeptide linker intermediate and a preparation method thereof. The preparation method of the oligopeptide intermediate is easily carried out under mild reaction conditions, and since almost no side reactions occur in the reaction, the method produces a high-purity product with fewer impurities and easy to be purified, achieving unexpected technical effects.

Claims

1. An oligopeptide linker intermediate having the structure represented by formulas (1)-(4): ##STR00042## wherein, the AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4 are any amino acid.

2. The oligopeptide linker intermediate according to claim 1, wherein the AA.sub.1, AA.sub.2, AA.sub.3, and AA.sub.4 are each independently selected from the group consisting of -valine- (-Val-), -citrulline -(-Cit-), -alanine- (-Ala-), -lysine- (-Lys-), -lysine(trityl)- (-Lys(Trt)-), -lysine(monomethoxytrityl)- (-Lys(Mmt)-), -lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-), -arginine- (-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-), -leucine- (-Len-) and -isoleucine- (-Ile-).

3. The oligopeptide linker intermediate according to claim 2, wherein the -AA.sub.1-AA.sub.2- is selected from the group consisting of -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -valine-lysine- (-Val-Lys-), -valine-lysine(trityl)- (-Val-Lys(Trt)-), -valine-lysine(monomethoxytrityl)- (-Val-Lys(Mmt)-), -valine-lysine(fluorenylmethyloxycarbonyl)- (-Val-Lys(Fmoc)-), -valine-arginine- (-Val-Arg-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylpropyl-lysine- (-Phe-Lys-), -phenylalanine-lysine(trityl)- (-Phe-Lys(Trt)-), -phenylalanine-lysine(monomethoxytrityl)- (-Phe-Lys (Mmt)-), -phenylalanine-lysine (fluorenylmethyloxycarbonyl)- (-Phe-Lys(Fmoc)-), leucine-citrulline- (-Leu-Cit-), isoleucine-citrulline- (-Ile-Cit-) and -phenylalanine-arginine- (-Phe-Arg-); the -AA.sub.1-AA.sub.2-AA.sub.3- is -phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); and the -AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the group consisting of -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leucine-glycine- (-Gly-Phe-Leu-Gly-) and -alanine-leucine-alanine-leucine (-Ala-Leu-Ala-Leu-).

4. The oligopeptide linker intermediate according to claim 3, wherein the oligopeptide linker intermediate includes the following structure: ##STR00043## ##STR00044## ##STR00045## ##STR00046##

5. An antibody-drug conjugate having a linker, wherein the precursor of the linker used in the antibody-drug, conjugate is the oligopeptide linker intermediate according to any one of claims 1 to 4.

6. A method for preparing the oligopeptide linker intermediate of formulas (1) to (3) according to claim 1, comprising: 1) performing a condensation reaction between a carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid AA.sub.1, and then amino acid AA.sub.2, or amino acid AA.sub.1, amino acid AA.sub.2 and amino acid AA.sub.3 in sequence, or amino acid AA.sub.1, amino acid AA.sub.2, amino acid AA.sub.3 and amino acid AA.sub.4 in sequence, to obtain a 2-(trimethylsilyl)ethoxycarbonyl-oligopeptide condensate; 2) reacting the resulting 2-(trimethylsilyl)ethoxycarbonyl-oligopeptide condensate and p-aminobenzyl alcohol to obtain a 2-(trimethylsilyl)ethoxycarbonyl-oligopeptide-p-aminobenzyl alcohol condensate, wherein the carbonylation reagent is any compound containing a carbonyl group.

7. The method according to claim 6, wherein the oligopeptide linker intermediate of formula (1) is obtained via the following reaction path: ##STR00047## wherein the method comprises the following steps: 1) performing a condensation reaction between a carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; 2) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino acid AA.sub.2 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; and 3) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and p-aminobenzyl alcohol to obtain a 2-(trimethylsilyl)ethoxycarbonyl-dipeptide-p-aminobenzyl alcohol condensate.

8. The method according to claim 6, wherein the oligopeptide linker intermediate of formula (2) is obtained via the following reaction path: ##STR00048## wherein the method comprises the following steps: 1) performing a condensation reaction between a carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; 2) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino acid AA.sub.2 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; 3) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino acid AA.sub.3 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; and 4) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and p-aminobenzyl alcohol to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tripeptide-p-aminobenzyl alcohol condensate.

9. The method according to claim 6, wherein the oligopeptide linker intermediate of formula (3) is obtained via the following reaction path: ##STR00049## wherein the method comprises the following steps: 1) performing a condensation reaction between a carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; 2) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino acid AA.sub.2 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; 3) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino acid AA.sub.3 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; 4) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and amino acid AA.sub.4 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate; and 5) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate and p-aminobenzyl alcohol to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide-p-aminobenzyl alcohol condensate.

10. A method for preparing the oligopeptide linker intermediate of formula (4) according to claim 1, comprising: performing a condensation reaction between a carbonylation reagent, 2-(trimethylsilyl)ethanol and amino acid AA.sub.1, amino acid AA.sub.2, amino acid AA.sub.3 and amino acid AA.sub.4 in sequence, to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate, wherein the carbonylation reagent is any compound containing a carbonyl group.

11. The method according to claim 10, wherein the oligopeptide linker intermediate of formula (4) is obtained via the following reaction path: ##STR00050## 1) performing a condensation reaction between a carbonylation reagent, 2-(trimethylsilyl)ethanol, and amino acid AA.sub.1 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate; 2) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-amino acid condensate and amino acid AA.sub.2 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate; 3) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-dipeptide condensate and amino acid AA.sub.3 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate; and 4) performing a condensation reaction between the 2-(trimethylsilyl)ethoxycarbonyl-tripeptide condensate and amino acid AA.sub.4 to obtain a 2-(trimethylsilyl)ethoxycarbonyl-tetrapeptide condensate.

12. The method according to any one of claims 6-11, wherein the carbonylation reagent has a structure of formula (5): ##STR00051## wherein: the R.sub.1 and R.sub.2 are each independently selected from the group consisting of: ##STR00052##

13. The method according to claim 12, wherein the carbonylation reagent is selected from the group consisting of: ##STR00053##

14. The method according to any one of claims 6 to 11, wherein the AA.sub.1, AA.sub.2, AA.sub.3, and AA.sub.4 are each independently selected from the group consisting of: -valine- (-Val-), -citrulline -(-Cit-), -alanine- (-Ala-), -lysine- (-Lys-), -lysine(trityl)- (-Lys(Trt)-), -lysine(monomethoxytrityl)- (-Lys(Mmt)-), -lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-), -arginine- (-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-), -leucine- (-Leu-) and -isoleucine- (-Ile-).

15. The method according to claim 14, wherein the -AA.sub.1-AA.sub.2- is selected from the group consisting of -valine-citrulline- (-Val-Cit-), -valine-alanine- (-Val-Ala-), -valine-lysine- (-Val-Lys-), -valine-lysine(trityl)- (-Val-Lys(Trt)-), -valine-lysine(monomethoxytrityl)- (-Val-Lys(Mmt)-), -valine-lysine(fluorenylmethyloxycarbonyl)- (-Val-Lys(Fmoc)-), -valine-arginine- (-Val-Arg-), -phenylalanine-citrulline- (-Phe-Cit-), -phenylpropyl-lysine- (-Phe-Lys-), -phenylalanine-lysine(trityl)- (-Phe-Lys(Trt)-), -phenylalanine-lysine(monomethoxytrityl)- (-Phe-Lys (Mmt)-), -phenylalanine-lysine (fluorenylmethyloxycarbonyl)- (-Phe-Lys(Fmoc)-), leucine-citrulline- (-Leu-Cit-), isoleucine-citrulline- (-Ile-Cit-) and -phenylalanine-arginine- (-Phe-Arg-); the -AA.sub.1-AA.sub.2-AA.sub.3- is -phenylalanine-arginine-arginine-(-Ala-Arg-Arg-); and the -AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the group consisting of -glycine-glycine-phenylalanine-glycine- (-Gly-Gly-Phe-Gly-), -glycine-phenylalanine-leucine-glycine- (-Gly-Phe-Leu-Gly-), and -alanine-leucine-alanine-leucine (-Ala-Leu-Ala-Leu-).

16. The method of claim 15, wherein the -AA.sub.1-AA.sub.2- is selected from the following structures: ##STR00054## ##STR00055## ##STR00056##

17. The method according to claim 15, wherein the -AA.sub.1-AA.sub.2-AA.sub.3- is ##STR00057##

18. The method according to claim 15, wherein the -AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4- is selected from the following structures: ##STR00058##

19. The method according to any one of claims 6 to 18, wherein solvent used in the condensation reaction is a polar solvent or non-polar solvent; preferably, the solvent is one or more selected from the group consisting of tetrahydrofuran, dioxane, acetonitrile, DMF, DMSO, DMAc, DMPU, HMPA, ethylene glycol dimethyl ether, diethyl ether, tert-butyl methyl ether, tert-butanol, water, ethyl acetate, methanol, ethanol, isopropanol, dichloromethane, chloroform, and carbon tetrachloride; more preferably, the solvent is one or more selected from the group consisting of tetrahydrofuran, dioxane, acetonitrile, DMF, DMSO, water, methanol, dichloromethane, chloroform, carbon tetrachloride and ethanol.

20. Use of the intermediate according to any one of claims 1-4 in the preparation of an antibody-drug conjugate.

21. Use of the method according to any one of claims 6-19 in the preparation of an antibody-drug conjugate.

Description

BRIEF DESCRIPTION OF THE FIGURE

[0067] FIGS. 1-A to 1-F show the relevant LC-MS diagrams of the Val-Cit-PAB prepared by Teoc protecting group method, in which FIG. 1-A is a mass spectrum of the blank control, F1-B is a liquid phase spectrum of the blank control, FIG. 1-C is a mass spectrum of the Val-Cit-PAB prepared by Teoc protecting group method, FIG. 1-D is a liquid phase spectrum of the product, FIG. 1-E is a positive ion mass spectrum of the product, and FIG. 1-F is a negative ion mass spectrum of the product.

[0068] FIG. 2-A to FIG. 2-H show relevant LC-MS diagrams of the Val-Cit-PAB prepared by Cbz protecting group method, in which FIG. 2-A is a mass spectrum of the blank control, FIG. 2-B is a liquid phase spectrum of the blank control, FIG. 2-C is a mass spectrum of the Val-Cit-PAB prepared by Cbz protecting group method, FIG. 2-D is a liquid phase spectrum of the product, FIG. 2-E is a positive ion mass spectrum of the product, FIG. 2-F is a negative ion mass spectrum of the product, FIG. 2-G is a ion mass spectrum of the impurity, and FIG. 2-H is a negative ion mass spectrum of the impurity.

[0069] FIG. 3-A to FIG. 3-D show relevant LC-MS diagrams of the Val-Cit-BAB prepared by Boc protecting group method (hydrochloric acid deprotection method), in which FIG. 3-A is a mass spectrum of the Val-Cit-PAB prepared by Boc protecting group method, FIG. 3-B is a liquid phase spectrum of the product, FIG. 3-C is a positive ion spectrum, and FIG. 3-D is a negative ion spectrum.

[0070] FIG. 4-A to FIG. 4-D show relevant LC-MS diagrams of the Val-Cit-PAB prepared by Boc protecting group method (trifluoroacetic acid deprotection method), in which FIG. 4-A is a mass spectrum of the Val-Cit-PAB prepared by Boc protecting group method, FIG. 4-B is a liquid phase spectrum of the product, FIG. 4-C is a positive ion spectrum, and FIG. 4-D is a negative ion spectrum.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviation

[0071] Unless otherwise stated, all abbreviations used in the present invention have the same meaning as understood by those of ordinary skill in the art. As used in the present invention, the common abbreviations and their definitions are as follows:

TABLE-US-00002 Abbreviation Definition Fmoc fluorenylmethyloxycarbonyl Cbz carbobenzyloxy Boc tert-butoxycarbonyl Teoc 2-(trimethylsilyl)ethoxycarbonyl Val valine Cit citrulline Ala alanine Lys lysine Arg arginine Phe phenylalanine

Definition

[0072] Various terms related to various aspects of the specification are used throughout the specification and claims. Unless otherwise indicated, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in a manner consistent with the definitions provided herein.

[0073] As used herein, the terms “a” and “an” and “the” are used in accordance with standard practice and mean one or more, unless the context indicates otherwise. Thus, for example, reference to “an antibody drug conjugate” includes a combination of two or more antibody drug conjugates and the like.

[0074] It should be understood that wherever an aspect is described herein with the word, “comprising” it also provides similar aspects described with “consisting of” and/or “substantially consisting of”.

[0075] Although the numerical ranges and parameter approximations shown in the broad scope of the present invention, the numerical values shown in the specific examples are described as accurately as possible. However, any numerical value inherently must contain a certain amount of error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein are understood to cover any and all subranges contained therein. For example, a recorded range of “1 to 10” should be considered to include any and all subranges between a minimum of 1 and a maximum of 10 (inclusive); that is, all subranges beginning with a minimum of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as “incorporated herein” is to be understood as being incorporated in its entirety.

[0076] As used in the present invention, means that the group containing connected to another group through a chemical bond here.

[0077] The term “linking group” used in the present invention refers to a bifunctional or multifunctional molecule, which can react with a protein/antibody molecule and a dipeptide linker, respectively, and thus function as a “bridge” to link the protein/antibody with the dipeptide linker.

[0078] The term “oligopeptide linker” used in the present invention generally refers to a linking structure containing two or more amino acid residues, which further comprises a p-benzyl alcohol residue. The two amino acids are linked together by means of dehydration condensation. The amino acids referred to herein generally refer to those organic compounds containing both amino and carboxyl groups, including all essential amino acids and non-essential amino acids. Preferably, the amino acid includes, but is not limited to, -valine- (-Val-), -citrulline -(-Cit-), -alanine- (-Ala-), -lysine- (-Lys-), -lysine(trityl)- (-Lys(Trt)-), -lysine(monomethoxytrityl)- (-Lys(Mmt)-), -lysine(fluorenylmethyloxycarbonyl)- (-Lys(Fmoc)-), -arginine- (-Arg-), -phenylalanine- (-Phe-), -glycine- (-Gly-), -leucine- (-Leu-), and -isoleucine- (-Ile-).

SPECIFIC EXAMPLES

[0079] The following further describes the present invention in combination with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally performed under conventional conditions or conditions recommended by the manufacturer. The reagents without specific sources are conventional reagents purchased on the market. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0080] The units in weight-volume percentage in the present invention are well known to those skilled in the art, and for example, refer to the weight of a solute in 100 ml of a solution.

[0081] Unless otherwise defined, all specialties and sciences used herein are used in the same sense as those familiar to those skilled in the art. In addition, any method or material similar or equal to the content described can be used in the method of the present invention. The preferred embodiments and materials described herein are for illustration purposes only.

Example 1 Preparation of Val-Cit-PAB by Teoc Protecting Group Method

(1) Preparation of Teoc-Val-Cit

[0082] ##STR00033##

[0083] 14.16 g of 2-(trimethylsilyl)ethanol (120 mmol), 38.7 g of N,N-diisopropylethylamine (300 mmol), 30.4 g of bis(4-nitrophenyl)carbonate (100 mmol), and 400 mL of acetonitrile were sequentially added into a 500 mL single-neck flask. After the addition, the mixture was stirred at room temperature for 16 h to form reaction solution 1. 14.08 g of L-valine (120 mmol) and 25.8 g of N,N-diisopropylethylamine (200 mmol) were added into a 1 L single-neck flask, and dissolved in 400 mL of water to form reaction solution 2. The reaction solution 1 was added to the reaction solution 2 with stirring, and after stirring at room temperature for 16 h, the completion of the reaction was detected by LC-MS method. After passing the detection, the reaction solution was rotary evaporated to remove the solvent. After evaporation, 500 mL of water was added, and 1 mol/L hydrochloric acid was added dropwise with stirring to adjust the pH to 1. Then, the aqueous phase was extracted twice, each with 300 mL of ethyl acetate. The extracts were combined and washed twice, each with 300 mL of water. After washing with water, the extract was dried by adding anhydrous magnesium sulfate, and the solvent was rotary evaporated to obtain 54 g of a pale yellow viscous solid product (i.e., 2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture containing 4-nitrophenol (simply referred to as 2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture)). LC-MS (M-H)-: 260.1. The 2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture was used in the next reaction without purification.

[0084] 10.5 g of the 2-(trimethylsilyl)ethoxycarbonyl-L-valyl mixture (40 mmol) as prepared above, 24.3 g of N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (80 mmol), 25.8 g N,N-diisopropylethylamine (200 mmol), and 200 mL N,N-dimethylformamide were sequentially added into a 500 mL single-necked flask. After the addition, the mixture was stirred at room temperature for 6 h to form reaction solution 3. 21 g of L-citrulline (120 mmol) was added into a 1 L single-neck flask, and completely dissolved under stirring with the addition of 400 mL of water, to form a reaction solution 4. 15,5 g of N,N-diisopropylethylamine (120 mmol) and 200 mL of N,N-dimethylformamide were added into a 500 mL beaker, to form a reaction solution 5. The reaction solution 5 was added to the reaction solution 4, then transferred to a low-temperature bath, and stirred at −10° C. for 0.5 h. Then, the reaction solution 3 was slowly added dropwise to the above reaction system under stirring at −10° C., which was controlled to be completed in about 2 h. After the completion of the addition, the mixture was kept at −10° C. for 16 h under stirring. Then, the completion of the reaction was detected by LC-MS method. After passing the detection, the system was rotary evaporated to remove the solvent, and after rotary evaporation, 500 mL of 0.1 mol/L hydrochloric acid was added and stirred well. The aqueous phase was extracted twice, each with 300 mL of ethyl acetate. The extracts were combined and washed twice, each with 300 mL 0.1 mol/L hydrochloric acid, and then washed twice with 300 mL of water. After washing with water, the extract was dried by adding anhydrous magnesium sulfate, and the solvent was rotary evaporated before 100 mL of ethyl acetate and 1000 mL of toluene were added with stirring for 16 h and then filtered. The filter cake was dissolved in 50 mL of THF, and 1 L of methyl tert-butyl ether:n-hexane=1:1 was added to prepare a solution. The mixture was stirred at room temperature for 16 hours and then filtered. The filter cake was dissolved in 50 mL of THF before 1 L of toluene was added. After stirring at room temperature for 6 h, the mixture was filtered and the filter cake was dried to obtain 11.1 g of an off-white powdery solid product with a yield of 68%. LC-MS(M+H).sup.+: 418.7, LC-MS(M-H).sup.−: 416.7.

(2) Preparation of Teoc-Val-Cit-PAB

[0085] ##STR00034##

[0086] 7.5 g of 2-(trimethylsilyl)ethoxycarbonyl-L-valyl-L-citrulline (18 mmol), 9 g of N-ethoxyacyl-2-ethoxy-1,2-dihydroquinoline (36 mmol), 4.5 g of p-aminobenzyl alcohol (36 mmol), 150 mL of dichloromethane and 75 mL of methanol were sequentially added into a 500 mL single-neck flask. After the addition, the mixture was stirred at room temperature for 16 h. Then, the completion of the reaction was detected by LC-MS method. After passing the detection, the reaction solution was dried by rotary evaporation, and after evaporation, 200 mL of dichloromethane and 200 mL of ethyl acetate were added and stirred for 16 h, and then filtered. To the filter cake, 100 mL of dichloromethane, 100 mL of ethyl acetate, and 200 mL of n-hexane were added with stirring for 16 h, and then filtered. The filter cake was dried to obtain 7.5 g of an off-white powdery solid product (the product was 2-(trimethylsilyl)ethoxycarbonyl-L-valyl-L-citrull amido-p-benzyl alcohol, namely Teoc-Val-Cit- PAB) with a yield of 80%. LC-MS(M+H).sup.+: 523.5, LC-MS(M-H).sup.−: 522.

(3) Preparation of Val-Cit-PAB (Relative Molecular Weight: 379.46)

[0087] ##STR00035##

[0088] 3.15 g of 2-(trimethylsilyl)ethoxycarbonyl-L-valyl-L-citrull amido-p-benzyl alcohol (6.2 mmol), 2.16 g of potassium fluoride (37.2 mmol), 12.4 mL of a 1.0 mol/L tetrabutylammonium fluoride solution in tetrahydrofuran, and 100 mL of N,N-dimethylformamide were sequentially added into a 500 mL single-necked flask. After the addition, the mixture was stirred at room temperature for 24 h and then the completion of the reaction was detected by LC-MS method. After the completion of the reaction, the reaction solution was filtered. After rotatory evaporation of the solvent of the filtrate, 100 mL of anhydrous ethanol was added to fully dissolve the residue. 18 mL of a 1 mol/L dilute hydrochloric acid solution was added and stirred well before the mixture was dried by adding anhydrous magnesium sulfate and then filtered. The filtrate was dried by rotary evaporation, and then 30 mL of ethanol was added to completely dissolve the residue. Under stirring at room temperature, a mixed solution of 200 mL of methyl tert-butyl ether and 200 mL of dichloromethane was added and stirred for 1 h before filtered. The filter cake was dried to obtain 1.83 g of an off-white powdery solid product (the product was L-valyl-L-citrull amido-p-benzyl alcohol, that is, Val-Cit-PAB) with a yield of 80%.

[0089] In the LC-MS detection, FIG. 1-A is a mass spectrum of the blank control, F1-B is a liquid phase spectrum of the blank control, FIG. 1-C is a mass spectrum of the Val-Cit-PAB prepared by Teoc protecting group method (the product in this example), FIG. 1-D is a liquid phase spectrum of the product, FIG. 1-E is a positive ion mass spectrum of the product, and FIG. 1-F is a negative ion mass spectrum of the product. By comparing FIG. 1-A with FIG. 1-C, it can be seen that the peak at the time point of 2.87 min is the product ion peak. By comparing FIG. 1-B with FIG. 1-D, it can be seen that the product has a strong HPLC signal, and by combining with FIG. 1-A and FIG. 1-C, it can be seen that the Val-Cit-PAB prepared by the Teoc protecting group method has fewer impurities. By analyzing the positive ion detection spectrum (2.849 min) and negative ion detection spectrum (2.866 min) at 2.87 min, it can be known that: LC-MS(M+H).sup.+: 379.6, LC-MS(M-H).sup.−: 378.0.

Example 2 Preparation of Val-Cit-PAB by Cbz Protecting Group Method

(1) Preparation of Cbz-Val-Cit

[0090] ##STR00036##

[0091] 10 g of N-benzyloxycarbonyl-L-valine (i.e., Cbz-Val) (40 mmol), 18.2 g of N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (60 mmol), 15.5 g of N,N-dlisopropylethylamine (120 mmol), and 300 mL of acetonitrile were sequentially added into a 1 L single-neck flask. After the addition, the mixture was stirred at room temperature for 4 h. After dissolving 7.7 g of L-citrulline (44 mmol) in 300 ml of water, the mixture was added to the above reaction system and stirred at room temperature for 16 h. Samples were taken for detection by LC-MS, and then the reaction was completed. Under stirring, 0.1 mol/L hydrochloric acid was added dropwise to adjust the pH to 1. After the solvent was dried by rotary evaporation, 800 mL of water was added, stirred at room temperature for 16 h, and filtered. After the filter cake was dried under vacuum, 400 mL of ethyl acetate and 400 mL of dichloromethane were added and stirred for 16 h before filtered. After the filter cake was dried under vacuum, 200 mL of ethyl acetate, 200 mL of dichloromethane, and 400 mL of n-hexane were added, and the mixture was stirred for 16 hours and then filtered. The filter cake was dried to obtain 12.65 g of an off-white powdery solid product (i.e., N-benzyloxycarbonyl-L-valyl-L-citrulline) with a yield of 77%. LC-MS(M+H).sup.+: 408.7, LC-MS(M-H).sup.−: 406.9.

(2) Preparation of Cbz-Val-Cit-PAB

[0092] ##STR00037##

[0093] 12.65 g of N-benzyloxycarbonyl-L-valyl-L-citrulline (31 mmol), 15.74 g of N-ethoxyacyl-2-ethoxy-1,2-dihydroquinoline (62 mmol), 7.53 g of p-aminobenzyl alcohol (62 mmol), 200 ml, of dichloromethane, and 100 mL of methanol were sequentially added into a 500 mL single-neck flask. After stirring at room temperature for 16 h, samples were taken for detection by LC-MS, and then the reaction was completed. After the reaction solution was dried by rotary evaporation, 200 mL of dichloromethane and 200 mL of ethyl acetate were added, stirred for 16 h, and filtered, 100 mL of dichloromethane, 100 mL of ethyl acetate, and 200 mL of n-hexane were added to the filter cake, stirred for 16 h and filtered. The filter cake was dried to obtain 9.1 g of an off-white powdery solid product with a vied of 60%. LC-MS(M+H).sup.+: 513.6.

(3) Preparation of Val-Cit-PA13 (Relative Molecular Weight: 379.46)

[0094] ##STR00038##

[0095] 1.12 g of N-benzyloxycarbonyl-L-valyl-L-citrull amido-p-benzyl alcohol (2 mmol), 0.3 g of palladium on carbon, and 100 mL of methanol were sequentially added into a 250 mL single-neck flask and stirred at 10° C. The reaction system was covered with a balloon filled with hydrogen gas, and the mixture was stirred at 10° C. for 16 h. Samples were taken for detection by LC-MS, and then the reaction was completed. The reaction solution was filtered, and the filtrate was dried by rotary evaporation. 400 mL of n-hexane was added, stirred for 16 h., and then filtered. The filter cake was dried to obtain 0.69 g of an off-white powdery solid product with a yield of 91%.

[0096] In the LC-MS detection, FIG. 2-A is a mass spectrum of the blank control, FIG. 2-B is a liquid phase spectrum of the blank control, FIG. 2-C is a mass spectrum of the Val-Cit-PAB prepared by Cbz protecting group method (the product in this example), FIG. 2-D is a liquid phase spectrum of the product, FIG. 2-E is a positive ion mass spectrum of the product, FIG. 2-F is a negative ion mass spectrum of the product, FIG. 2-G is a ion mass spectrum of the impurity, and FIG. 2-H is a negative ion mass spectrum of the impurity. By Comparing FIG. 2-A with FIG. 2-C, it can be seen that there are two stronger ion peaks: at 4.33 min and at 6.54 min. By comparing FIG. 2-B and 2-D, it can be seen that there are two peaks with strong HPLC signals at 4.12 min and at 5.39 min, and combining with FIG. 2-A and FIG. 2-C, it can be inferred that one of the two peaks is a product peak and one is an impurity peak, and the content of impurities is higher. By analyzing the positive ion mass spectrum and negative ion mass spectrum at 4.33 min and at 5.54 min, it can be found that the peak at 4.33 min is the product peak (LC-MS(M+H).sup.+: 379.6, LC-MS(M-H).sup.−: 378.0), and the peak at 5.54 min is the impurity peak (LC-MS(M+H).sup.+: 363.6), which is speculated to be a by-product of Val-Cit-PAB dehydroxylation (with a molecular weight difference of only 16), and its content is higher. Since the chemical properties of the by-product are very similar to the product, the by-product is very hard to be removed, and has a great effect on the subsequent reactions and product quality. in addition, hydrogen gas is used as a reactant in the deprotection process, which poses a safety risk.

Example 3 Preparation of Phe-Cit-PAB by Boc Protecting Group Method

(1) Preparation of Boc-Phe-Cit

[0097] ##STR00039##

[0098] 2.65 g of N-(tert-butoxycarbonyl)-L-phenylalanine (10 mmol) (i.e., Boc-Phe), 3.61 g of N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (12 mmol), 3.87 g of N,N-diisopropylethylamine (30 mmol), and 50 mL of N,N-dimethylformamide were sequentially added into a 250 mL single-neck flask, and stirred at room temperature for 4 h.

[0099] 1.75 g of L-citrulline (10 mmol) was dissolved in 50 ml of water, which is added to the above reaction system and stirred at room temperature for 16 h. Samples were taken for detection by LC-MS, and then the reaction was completed. Under stirring, 0.1 mol/L hydrochloric acid was added dropwise to adjust the pH to 1. After the system was dried by rotary evaporation, 200 mL of water was added, stirred at room temperature for 16 h, and filtered. After the filter cake was dried, 100 mL of ethyl acetate and 100 mL of dichloromethane were added, stirred for 16 h and filtered. The filter cake was dried to obtain 3 g of a yellow powdery solid product (that is, N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrulline, Boc-Phe-Cit) with a yield of 71%. LC-MS(M-H).sup.−: 421.4.

(2) Preparation of Boc-Phe-Cit-PAB

[0100] ##STR00040##

[0101] 3 g of N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrulline (7.1 mmol) (i.e., Boc-Phe-Cit), 3.5 g of N-ethoxyacyl-2-ethoxy-1,2-dihydroquinoline (14.2 mmol), 1.75 g of p-aminobenzyl alcohol (14.2 mmol), 60 mL of dichloromethane, and 30 mL of methanol were sequentially added into a 250 mL single-neck flask, and stirred at room temperature for 16 h. Then samples were taken for detection by LC-MS, and then the reaction was completed. After the reaction solution was dried by rotary evaporation, 100 mL of dichloromethane and 100 mL of ethyl acetate were added, stirred for 16 h, and centrifuged. 100 mL of n-hexane was added to the white solid in the lower layer, stirred for 2 h and centrifuged. The white solid in the lower layer was dried to obtain 2.3 g of a off-white powdery solid product (that is, N-(cert-butoxycarbonyl)-L-phenylalanyl-L-citrull amido-p-benzyl alcohol, Boc-Phe-Cit-PAB) with a yield of 62%. LC-MS(M-H).sup.−: 526.2.

(3) Preparation of Phe-Cit-PAB (Relative Molecular Weight: 427.51)

[0102] The Boc protecting group is removed by hydrochloric acid deprotection and trifluoroacetic acid deprotection, respectively, shown as follows.

##STR00041##

(a) Hydrochloric Acid Deprotection Method

[0103] 0.5 g of N-(tert-butoxycarbonyl)-L-phenylal amido-p-benzyl alcohol (1 mmol) (i.e., Boc-Phe-Cit-PAB), 3 mL of 4 mol/L hydrogen chloride solution in dioxane and 3 mL of dioxane were sequentially added into a 10 mL single-neck flask. After the addition, the mixture was stirred at room temperature for 3 h. Samples were taken for detection by LC-MS, and then the reaction was completed.

[0104] In the LC-MS detection, FIG. 3-A is a mass spectrum of the Phe-Cit-PAB prepared by Boc protecting group method (the product in this example), FIG. 3-B is a liquid phase spectrum of the product, FIG. 3-C is a positive ion spectrum, and FIG. 3-D is a negative ion spectrum. It can be seen from FIG. 3-A that there is a strong ion peak at 5.39 min. By analyzing the positive ion mass spectrum (5.390 min) and negative ion mass spectrum (5.403 min) at this position, the value of LC-MS(M+H).sup.+: 446.2, LC-MS(M-H).sup.−: 444.1 was obtained. It is speculated that most of the products prepared by this method are by-products in which the hydroxyl group at the benzyl position is replaced by chlorine.

(b) Trifluoroacetic Acid Deprotection Method

[0105] 0.5 g of N-(tert-butoxycarbonyl)-L-phenylalanyl-L-citrull amido-p-benzyl alcohol (1 mmol) and 3 mL of trifluoroacetic acid were sequentially added into a 10 mL single-neck flask. After the addition, the mixture was stirred at room temperature for 2 h and the completion of the reaction was detected by LC-MS.

[0106] In the LC-MS detection, FIG. 4-A is a mass spectrum of the Phe-Cit-PAB prepared by Boc protecting group method (the product in this example), FIG. 4-B is a liquid phase spectrum of the product, FIG. 4-C is a positive ion spectrum, and FIG. 4-D is a negative ion spectrum. It can be seen from FIG. 4-A that there is a strong ion peak at 5.77 min. By analyzing the positive ion mass spectrum (5.768 min) and negative ion mass spectrum (5.781 min) at this position, the value of LC-MS(+H:).sup.+: 524.4, LC-MS(M-H).sup.−: 522.1 was obtained. It is speculated that most of the products prepared by this method are by-products in which the hydroxyl group at the benzyl position forms trifluoroacetate.

[0107] In summary, the method for preparing Val-Cit-PAB using Teoc as a protecting group provided by the present invention is easily carried out under mild reaction conditions, and since almost no side reactions occur in the reaction, the method produces a high-purity product with fewer impurities and easy to be purified. The method for preparing Val-Cit-PAB using Cbz as a protecting group produces large amounts of by-products from dehydroxylation. Because the chemical properties of the by-products are very similar to the products, they are very difficult to be removed, greatly affecting the subsequent reactions and product quality. In addition, hydrogen gas is used as a reactant in the deprotection process, which poses a safety risk. The method of preparing Val-Cit-PAB by using Boc as a protecting group has the problems that the hydroxyl group at the benzyl position is replaced by chlorine, and forms trifluoroacetate in a high conversion rate. Therefore, compared with the other two methods, the Teoc method provided by the present invention can be easily carried out and produces few impurities, thus achieving unexpected. technical effects.

TABLE-US-00003 Easy or Protecting difficult to group By-products purify Reaction conditions Teoc — very few easy mild Cbz By-products from more difficult Use of hydrogen poses safety dehydroxylation of risks Val-Cit-PAB Boc By-products in which most difficult Use of large amounts of acid the hydroxyl group at presents safety risks and is not the benzyl position of environmentally friendly. Val-Cit-PAB is replaced by chlorine By-products in which most difficult the hydroxyl group at the benzyl position of Val-Cit-PAB forms trifluoroacetate

[0108] The invention has been exemplified by various specific embodiments. However, those of ordinary skill in the art can understand that the present invention is not limited to the specific embodiments. Those of ordinary skill in the art can make various changes or modifications within the scope of the present invention, and various technical features mentioned in various places in this specification can be combined with each other without departing from the spirit and scope of the present invention. Such modifications and variations are all within the scope of the present invention.