Intermediates for synthesizing FAPI, their preparation method, and their use.

Abstract

The present invention provides an intermediate for synthesizing Evans blue-modified FAPI, a preparation method thereof, and its use in synthesizing Evans blue-modified FAPI. The synthesis of Evans blue-modified FAPI using the intermediate according to the present invention can improve production efficiency and reduce production costs, making it suitable for industrial production.

Description

[Technical Field]

[0001] This invention relates to the field of drug synthesis, and more specifically to intermediates, preparation methods, and uses for synthesizing Evans blue-modified FAPI (LNC1004). [Background technology]

[0002] Molecular imaging radiotracers targeting fibroblast-activating protein (FAP) have shown promising preclinical and clinical outcomes in tumor diagnosis. LNC1004 is a newly developed Evans Blue-modified fibroblast-activating protein inhibitor (Evans Blue-modified FAPI), with the chemical name 2,2',2''-(10-(2-(((S)-1-((4'-((E)-(8-amino-1-hydroxy-5,7-disulfonaphthalene-2)-yl)diazenyl)-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-6 -(4-(4-(3-((4-((2-((S)-2-cyanopyrrolidine-1-yl)-2-oxyethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazine-1-yl)-4-oxobutyramide)-1-oxohexyl-2-yl)amino)-2-oxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)trifluoroacetate. In conventional technology, LNC1004 is used for tumor-targeted therapy. 177 It was found that labeling with Lu and adding an Evans Blue fragment extended the circulating half-life, improving pharmacokinetics (PK), increasing tumor uptake, and enhancing the effectiveness of radiotherapy. Therefore, radiolabeled LNC1004 can be used as a novel long-acting cancer treatment.

[0003] In the prior art (reference: Evans blue-modified radiolabeled fibroblast activation protein inhibitor as long-acting cancer therapeutics, Theranostics 2022; 12(1): 422-433), a synthesis scheme for LNC1004 has been reported. JPEG0007838187000001.jpg86170JPEG0007838187000002.jpg46170

[0004] The above scheme can obtain the target product, but it has the following drawbacks: 1) Using an Evans blue fragment containing a sulfonic acid group as a starting material to synthesize Evans blue-modified FAPI (LNC1004) increases the polarity of intermediates involved in the entire synthesis scheme, making separation and purification difficult using conventional workup methods (e.g., extraction, column chromatography). On the other hand, using preparative liquid-phase chromatography for purification increases the difficulty of separation, is inefficient, and is unsuitable for industrial production. 2) DOTA-NHS is used for coupling the final fragment, but DOTA-NHS itself is structurally unstable, easily decomposes at high temperatures, causing the ring to open, leading to many side reactions, low yield, and high overall cost due to DOTA-NHS itself being expensive.

[0005] Therefore, in order to facilitate the industrial scale-up production of LNC1004, its synthesis method needs to be improved. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Based on the above technical background, the technical problem that this invention aims to solve is to improve the yield of LNC1004 synthesis, reduce costs, and make it more suitable for industrial scale-up production.

[0007] The main objective of the present invention is to provide a novel compound used as a key intermediate for synthesizing Evans blue-modified FAPI. Synthesizing Evans blue-modified FAPI using this key intermediate not only reduces production costs but also significantly improves production efficiency and yield, making it suitable for large-scale industrial production.

[0008] Another object of the present invention is to provide a method for preparing the aforementioned important intermediates.

[0009] A further object of the present invention is to provide a method for synthesizing Evans blue-modified FAPI using the aforementioned key intermediates, that is, to provide the use of the aforementioned key intermediates in the synthesis of Evans blue-modified FAPI. [Means for solving the problem]

[0010] To achieve the above objectives, the present invention employs the following technical solutions.

[0011] In a first aspect, the present invention provides an intermediate for synthesizing Evans blue-modified FAPI (LNC1004) whose structure is represented by formula (I). [ka]

[0012] In a second aspect, the present invention is Step a involves reacting (S)-N-(2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)-6-(3-(piperazine-1-yl)propoxy)quinoline-4-carboxamide (i.e., compound II) with succinic anhydride to obtain compound III, Step b involves coupling compound III with tert-butyl(S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohexa-2-yl)carbamate (i.e., compound IV) to obtain compound V, Step c involves deprotecting compound V to obtain compound VI, followed by substitution reactions and treatments to obtain compound VII. The present invention provides a method for preparing the above-mentioned intermediate (i.e., the compound of formula I), comprising step d, which involves hydrolyzing compound VII to obtain the intermediate having the structure of formula I.

[0013] Specifically, the reaction scheme for the intermediate of the present invention (i.e., compound I of formula) is as follows. JPEG0007838187000004.jpg68170JPEG0007838187000005.jpg177170

[0014] In a preferred embodiment of the present invention, in step b, specifically, compound III obtained in step a and tert-butyl(S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohexa-2-yl)carbamate (i.e., compound IV) are added to an N,N-dimethylcarboxamide solvent, followed by the addition of a condensing agent and an organic base, and the mixture is reacted at 20-45°C with stirring for 1-8 hours to obtain compound V.

[0015] In a more preferred embodiment of the present invention, in step b), the condensing agent is one of HATU, HBTU, TBTU, TSTU, PyAOP, and PyBOP, most preferably HATU, and in step b), the organic base is one of N,N-diisopropylethylamine and triethylamine, most preferably N,N-diisopropylethylamine.

[0016] In a preferred embodiment of the present invention, step c specifically involves organic meltAfter adding the compound V obtained in step b to the medium, an organic acid is added, and the reaction is carried out for 1 to 8 hours while stirring at 20 to 45 °C to obtain compound VI. Then, an excess of organic base is added. Next, 2,2’,2’’-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyltriacetate (DOTA-TRIS-TBU-ESTER NHS) is added, and the reaction is carried out for 1 to 8 hours while stirring at 15 to 45 °C, followed by treatment to obtain compound VII.

[0017] In a more preferred embodiment of the present invention, in step c, the melt medium is any one of dichloromethane, N,N-dimethylcarboxamide or a mixture of both, most preferably N,N-dimethylcarboxamide. In step c, the organic acid is any one of trifluoroacetic acid and p-toluenesulfonic acid, most preferably trifluoroacetic acid. In step c, the organic base is any one of N,N-diisopropylethylamine and triethylamine, most preferably N,N-diisopropylethylamine.

[0018] In a preferred embodiment of the present invention, in step d), specifically, after adding the compound VII obtained in step c to the melt medium, an organic acid is added, and the reaction is carried out for 1 to 8 hours while stirring at 20 to 45 °C, followed by treatment to obtain an intermediate of formula I structure.

[0019] In a more preferred embodiment of the present invention, in step d), the melt medium is dichloromethane or acetonitrile.

[0020] In a more preferred embodiment of the present invention, in step d), the organic acid is any one of trifluoroacetic acid and p-toluenesulfonic acid, most preferably trifluoroacetic acid.

[0021] The present invention provides a method for preparing the above-mentioned intermediate (i.e., compound I), wherein compound (I) is prepared by hydrolysis of compound (VII), and the preparation scheme is as follows: JPEG0007838187000006.jpg101170

[0022] To prepare the compound of formula (I) from the compound of formula (VII) described above, a preferred preparation method is organic melt The process involves adding compound (VII) to the medium, then adding an organic acid, and reacting the mixture at 20-45°C with stirring for 1-8 hours to obtain an intermediate with the structure of formula I.

[0023] Preferably, in the above preparation method, the organic melt The fertilizer is preferably dichloromethane or acetonitrile.

[0024] Preferably, in the above preparation method, the organic acid is preferably one of trifluoroacetic acid and p-toluenesulfonic acid, and more preferably trifluoroacetic acid.

[0025] Furthermore, the compound of formula (VII) described above can be obtained by a substitution reaction with the compound of formula (VI), and the preparation scheme is as follows. JPEG0007838187000007.jpg94170

[0026] To prepare compound (VII) from compound (VI) of formula above, a preferred preparation method is organic meltCompound VI is added to the medium, followed by the addition of an excess organic base. Then, 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) is added, and the mixture is reacted at 15-45°C with stirring for 1-8 hours to obtain compound VII.

[0027] Preferably, in the above preparation method, the organic melt The medium is one of dichloromethane, N,N-dimethylcarboxamide, or a mixture of both, most preferably N,N-dimethylcarboxamide.

[0028] Preferably, in the above preparation method, the organic base is one of N,N-diisopropylethylamine and triethylamine, most preferably N,N-diisopropylethylamine.

[0029] Furthermore, the compound of formula (VI) described above can be obtained by deprotecting the compound of formula (V), and the preparation scheme is as follows. JPEG0007838187000008.jpg71170

[0030] To prepare compound (VI) from compound (V) above, a preferred preparation method is organic melt Compound V is added to the medium, followed by the addition of an organic acid. The reaction is carried out at 20-45°C with stirring for 1-8 hours to obtain compound VI.

[0031] Preferably, in the above preparation method, the organic melt The medium is one of dichloromethane, N,N-dimethylcarboxamide, or a mixture of both, most preferably N,N-dimethylcarboxamide.

[0032] Preferably, in the above preparation method, the organic acid is one of trifluoroacetic acid and p-toluenesulfonic acid, most preferably trifluoroacetic acid.

[0033] Furthermore, the compound of formula (V) described above can be obtained by coupling it with the compound of formula (IV) and the compound of formula (III), and the preparation scheme is as follows. JPEG0007838187000009.jpg73170

[0034] To prepare compound (V) from the above-mentioned compounds of formula (IV) and formula (III), a preferred preparation method is to add compounds III and IV to an N,N-dimethylcarboxamide solvent, then add a condensing agent and an organic base, and react the mixture with stirring at 20-45°C for 1-8 hours to obtain compound V.

[0035] Preferably, in the above preparation method, the condensing agent is one of HATU, HBTU, TBTU, TSTU, PyAOP, and PyBOP, most preferably HATU.

[0036] Preferably, in the above preparation method, the organic base is one of N,N-diisopropylethylamine and triethylamine, most preferably N,N-diisopropylethylamine.

[0037] Furthermore, the compound of formula (III) described above can be obtained by reacting the compound of formula (II) with succinic anhydride, and the preparation scheme is as follows. JPEG0007838187000010.jpg23170

[0038] In a third aspect, the present invention provides a method for synthesizing Evans blue-modified FAPI (LNC1004) using the aforementioned intermediate (i.e., compound I of formula I) of the present invention, and the present invention also provides the use of the aforementioned intermediate (i.e., compound I of formula I) in the synthesis of Evans blue-modified FAPI (LNC1004).

[0039] Specifically, the above methods or uses include the following reaction scheme. JPEG0007838187000011.jpg97170

[0040] In the reaction steps of the above method or use, the intermediate whose structure is represented by formula (I) is dissolved in water, and hydrochloric acid solution and sodium nitrite are sequentially added while stirring at -5 to 25°C to react and produce a diazonium salt solution. Then, 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate are mixed to carry out an acid-base neutralization reaction to produce an aqueous solution of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate. While stirring at -5 to 25°C, the diazonium salt solution is added dropwise to the aqueous solution of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate, and the mixture is reacted for 1 to 8 hours. After post-treatment, Evans blue-modified FAPI (i.e., LNC1004) is obtained.

[0041] In a preferred embodiment of the present invention, in the above reaction step, the molar concentration of the hydrochloric acid solution is 0.5 mol / L to 6 mol / L, more preferably 1 to 3 mol / L, the equivalent amount of hydrochloric acid used (molar ratio with the intermediate) is 1.0 to 3.0, more preferably 1.5 to 2.0, the equivalent amount of sodium nitrite used (molar ratio with the intermediate) is 1.0 to 3.0, more preferably 1.0 to 1.5, the equivalent amount of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt used (molar ratio with the intermediate) is 1.0 to 3.0, more preferably 1.0 to 1.5, and the equivalent amount of sodium bicarbonate used (molar ratio with the intermediate) is 5.0 to 10.0, more preferably 8.0 to 10.0. [Effects of the Invention]

[0042] The method for synthesizing Evans Blue-modified FAPI (LNC1004) of the present invention is based on the premise of synthesizing the intermediate (compound of formula I) of the present invention, and ultimately obtains LNC1004 from the coupling of this intermediate with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt. Compared with conventional synthesis methods, the synthesis method of the present invention differs overall mainly in the following respects: The reaction with the given 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt is carried out in the final step. When introducing the DOTA group, a means of first coupling a carboxyl-protected DOTA group and then deprotecting it is used. Based on the above differences, the advantages of the present invention are as follows. 1) By performing the coupling of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and the intermediate in the final step of the synthesis, the polarity of the reaction intermediate is reduced compared to conventional synthesis methods. This allows all compounds prepared up to the intermediate in the synthesis scheme to be separated and purified by post-treatment means such as extraction or column chromatography, thereby improving the overall production efficiency and yield of the synthesis process and making it suitable for large-scale industrial production. 2) When introducing the DOTA group, a method is employed in which a carboxyl-protected DOTA group is coupled and then deprotected. Specifically, instead of DOTA-NHS, 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) is involved in the reaction, and the tert-butyl ester is later removed. This makes the synthesis process more efficient, further increases the yield, and reduces costs. First, 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) has more stable chemical properties than DOTA-NHS and its structure is less likely to be destroyed during the reaction. Therefore, in this invention, there are fewer side reactions and a higher yield in the one-step reaction. Furthermore, 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) is less polar than DOTA-NHS, so even after obtaining the corresponding compound (VII) by reaction, it can be separated and purified by column chromatography, making it suitable for industrial production.Furthermore, since 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) (approximately 1500 yuan / g) has a lower market price than DOTA-NHS (approximately 10000 yuan / g), in this invention, production costs are significantly reduced by using 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) instead of DOTA-NHS. [Brief explanation of the drawing]

[0043] [Figure 1] This is the mass spectrum of compound III from Example 1. [Figure 2] This is the mass spectrum of compound V in Example 1. [Figure 3] This is the mass spectrum of compound VII from Example 1. [Figure 4] This is the mass spectrum of the intermediate of formula I in Example 1. [Figure 5] This is the mass spectrum of the Evans Blue modified FAPI (LNC1004) from Example 2. [Modes for carrying out the invention]

[0044] The present invention can be better understood by referring to the following examples. However, it will be readily apparent to those skilled in the art that the specific material ratios, process conditions, and results described in the examples are for illustrative purposes only and do not limit the present invention as described in detail in the claims.

[0045] Example 1: Preparation of compound (I) Preparation of Compound III 10.06 g of (S)-N-(2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)-6-(3-(piperazin-1-yl)propoxy)quinoline-4-carboxamide (i.e., compound II) was dissolved in 70 ml of dichloromethane. Then, 16.9 ml of DIPEA and 2.81 g of succinic anhydride were sequentially added, and the mixture was reacted at room temperature with stirring for 2 hours. The mixture was purified by column chromatography to obtain 7.32 g of compound III. Yield 59.54%, theoretical [MH] - = 549.25, measured [MH] - = 549.24826. The spectrum representing its structure is shown in Figure 1.

[0046] Preparation of compound V Add 7.20g of compound III to 70ml of N,N-dimethylcarboxamide. tert-butyl (S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohexa-2-yl)carbamate (compound IV) 5.83 g, HATU 6.12 g, and DIPEA 2.66 g were added sequentially and reacted at room temperature with stirring for 2 hours. The mixture was purified by column chromatography to obtain compound V 11.66 g. Yield 91.30%, theoretical [M+H] + =973.53, measured [M+H] + =973.52750. The spectrum representing its structure is shown in Figure 2.

[0047] Preparation of Compound VII 11.50 g of Compound V was added to 200 ml of N,N-dimethylcarboxamide, and then 77.86 ml of trifluoroacetic acid was added. The mixture was reacted for 1 hour with stirring at 40 - 45 °C to obtain Compound VI. Then, 234 ml of N,N-diisopropylethylamine was added, and further 7.5 g of 2,2’,2’’-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyltriacetate (DOTA-TRIS-TBU-ESTER NHS) was added. The reaction was carried out for 6 hours with stirring at room temperature. After the reaction solution was rotary evaporated, it was added to 200 ml of dichloromethane and dissolved. The organic phase was washed twice with water, dried over anhydrous sodium sulfate, and then rotary evaporated. It was purified by column chromatography to obtain 20.85 g of Compound VII. Yield 123.51% (containing inorganic salts), theoretical [M + H] + = 1427.85, measured [M + H] + = 1427.84403. The spectrum representing its structure is shown in Figure 3.

[0048] Preparation of the compound of formula (I) To 20.5 g of Compound VII, 350 ml of dichloromethane and 350 ml of trifluoroacetic acid were added. After reacting for 1 hour with stirring at 40 - 45 °C, the reaction solution was dropped into 3.5 L of methyl tert-butyl ether, stirred to precipitate a solid, filtered, and then purified by preparative liquid chromatography to obtain 10.36 g of the compound of formula I. Yield 56.30%, theoretical [M + H] + = 1259.66, ([M + 2H] / 2) + = 630.33, measured ([M + 2H] / 2) + = 630.33554. The spectrum representing its structure is shown in Figure 4.

[0049] The reaction scheme of this example is as follows. JPEG0007838187000012.jpg143170JPEG0007838187000013.jpg104170

[0050] Example 2: Preparation of Evans Blue-modified FAPI (LNC1004) 10.3 g of compound (I) prepared in Example 1 was dissolved in 135 ml of water, and while stirring, 8.7 ml of 2 mol / L hydrochloric acid solution and 0.618 g of sodium nitrite were sequentially added and reacted to produce a diazonium salt solution. Then, 6.9 g of sodium bicarbonate was dissolved in 135 ml of water, and next, 3.1 g of monosodium 1-amino-8-naphthol-2,4-disulfonic acid salt was added to cause an acid-base neutralization reaction to produce 1-amino-8-naphthol-2,4-disulfonic acid. Aqueous solutions of monosodium hydroxynitrate and sodium bicarbonate were prepared. While stirring at 0-10°C, the diazonium salt solution was added dropwise to the aqueous solution of monosodium 1-amino-8-naphthol-2,4-disulfonic acid and sodium bicarbonate. After reacting for 1 hour, the reaction mixture was evaporated by rotation and purified by preparative liquid chromatography to obtain 7.01 g of radiolabelable fibroblast-activating protein inhibitor (LNC1004) modified with Evans blue. Yield 53.91%, theoretical ([M+2H] / 2) + = 795.31, measured ([M+2H] / 2) + =795.30666. The spectrum representing its structure is shown in Figure 5.

[0051] The reaction scheme for this embodiment is as follows: JPEG0007838187000014.jpg110170

[0052] Comparative Example 1: Synthesis of Compound 2 0.31 g, 1.0 mmol of tert-butyl (4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl) carbamate (compound 1) and 4 ml of acetonitrile were placed in 50 ml flasks. Under an ice bath, 1.5 ml of 2 M hydrochloric acid was added dropwise to the reaction flask and the mixture was allowed to react for 15 minutes. Next, sodium nitrite (0.068 g, 1.0 mmol) was dissolved in 2 ml of water, and this was added dropwise to the reaction flask and allowed to react for half an hour to form solution A. Separately, a 50 ml reaction flask was prepared, and 0.33 g, 1.0 mmol of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt, sodium carbonate (0.105 g, 1.0 mmol), and 5 ml of water were added to form solution B. Under an ice bath, solution A was slowly added dropwise to solution B and the mixture was allowed to react for 2 hours with stirring under an ice bath. The compound was purified by preparative liquid chromatography and freeze-dried to obtain pure compound 2 (yield 47%).

[0053] Synthesis of Compound 3 Under ice bath conditions, compound 2 (0.52 g, 1.0 mmol) was dissolved in trifluoroacetic acid, and the system was heated to room temperature and reacted for 2 hours. After the reaction was complete, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by preparative liquid chromatography and freeze-dried to obtain pure compound 3 (yield 73%).

[0054] Synthesis of Compound 4 Compound 3 (0.54 g, 1.0 mmol), Boc-Lys(Fmoc)-OH (0.47 g, 1.0 mmol), HATU (0.38 g, 1.0 mmol), N,N-diisopropylethylamine (0.26 g, 2.0 mmol), and 10 ml of N,N-dimethylcarboxamide were added to a 100 ml flask. The reaction mixture was stirred until the reaction was complete, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by preparative liquid chromatography and lyophilized to obtain pure compound 4 (yield 47%).

[0055] Preparation of compound 5 For compound 4, Fmoc was deprotected using piperidine (20% [v / v]) at room temperature. The reaction mixture was stirred for 1 hour, and then DMF was removed under high vacuum. The residue was purified by preparative liquid chromatography. The separation yield was 67%. Succinic anhydride (500 mg, 5 mmol) was dissolved in 5 ml of DMF and added to compound 4 intermediate (0.5 mmol, 375 mg) from which Fmoc protection had been removed, followed by the addition of 10 mmol of DIPEA. The mixture was stirred at room temperature for 12 hours until it was completely converted to compound 5. Then, DMF was removed under high vacuum. The residue was purified by preparative liquid chromatography (278 mg, 64% yield).

[0056] Preparation of compound 8 (S)-4-(3-((4-((2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)aminoformyl)quinoline-6-yl)oxy)propyl)piperazine-1-tert-butylcarboxylate (compound 6) (110 mg, 0.2 mmol) and 4-methylbenzenesulfonic acid monohydrate (380 mg, 2 mmol) were dissolved in 10 ml of acetonitrile. The reaction mixture was shaken overnight at 45°C to obtain compound 7. Acetonitrile was removed by rotary evaporation, and the residue was dissolved in DMF. Then, compound 5 (174 mg, 0.2 mmol), N,N-diisopropylethylamine (129 mg, 1 mmol), and HATU (76 mg, 0.2 mmol) were added. The reaction mixture was stirred at room temperature for 6 hours to obtain compound 8. DMF was removed under high vacuum, and the residue was purified by preparative liquid chromatography (210 mg, 81% yield).

[0057] Preparation of LNC1004 At room temperature, the BOC group of compound 8 (26 mg, 0.02 mmol) was removed over 1 hour in DCM (v / v) using 10% TFA. Subsequently, the TFA and DCM were removed under a nitrogen stream, and the residue was reacted with 1.2 equivalents of DOTA-NHS ester and 8 equivalents of DIPEA in DMF. After stirring at room temperature for 2-3 hours, the reaction mixture was purified by preparative liquid chromatography to obtain the desired product (23 mg, 73% yield).

[0058] The reaction scheme is as follows: JPEG0007838187000015.jpg76170JPEG0007838187000016.jpg86170

[0059] A comparison of Examples 1 and 2 of the present invention with Comparative Example 1 revealed that in Comparative Example 1, LNC1004 was synthesized through a 7-step reaction, requiring purification by preparative liquid chromatography after each step. The overall yield was 4.09%. In contrast, in Examples 1 and 2 of the present invention, the synthesis scheme involved a total of 6 steps to synthesize LNC1004, requiring purification by preparative liquid chromatography only in the last two steps. The overall yield was 20.38%. The synthesis method of the present invention was found to significantly improve the yield of LNC1004.

Claims

1. An intermediate for synthesizing Evans Blue modified FAPI, An intermediate characterized by having a structure represented by formula (I).

2. A method for preparing an intermediate according to claim 1, Step a involves reacting (S)-N-(2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)-6-(3-(piperazine-1-yl)propoxy)quinoline-4-carboxamide (compound II) with succinic anhydride to obtain compound III, Step b involves coupling compound III with compound IV, which is tert-butyl(S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohexa-2-yl)carbamate, to obtain compound V. Step c involves deprotecting compound V to obtain compound VI, followed by a substitution reaction, and then purification by extraction and column chromatography to obtain compound VII. The step includes: treating compound VII by hydrolysis to obtain the intermediate having the structure of formula I, The method is characterized by having the following specific reaction equation.

3. The method according to 2, characterized in that, in step b, specifically, compound III obtained in step a and tert-butyl(S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohexa-2-yl)carbamate are added to an N,N-dimethylcarboxamide solvent, a condensing agent and an organic base are added, and the reaction is carried out at 20 to 45°C with stirring for 1 to 8 hours to obtain compound V.

4. The condensing agent is HATU (2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, CAS: 148893-10-1), HBTU(O-(1H-BENZOTRIAZOL-1-YL)-N,N,N',N'-TETRAMETHYLURONIUM HEXAFLUOROPHOSPHATE, CAS: 94790-37-1), TBTU (benzotriazol-1-yl-tetramethyluroniumtetrafluoroborate, CAS: 125700-67-6), TSTU(O-(N-SUCCINIMIDYL)-1,1,3,3-TETRAMETHYLURONIUM TETRAFLUOROBORATE, CAS: 105832-38-0), PyAOP((3-Hydroxy-3H-1,2,3-triazolo[4,5-b]pyridinato-O)tri-1-pyrrolidinylphosphonium hexafluorophosphate, CAS: The method according to claim 3, characterized in that it is one of the following: 156311-83-0), PyBOP (BENZOTRIAZOLYL-N-OXYTRIS-(DIMETHYLAMINO-PHOSPHONIUM HEXAFLUOROPHOSPHATE), CAS: 128625-52-5).

5. The method according to claim 3, characterized in that the organic base is one of N,N-diisopropylethylamine and triethylamine.

6. The method according to 2, wherein in step c, specifically, compound V obtained in step b is added to an organic solvent, then an organic acid is added, and the mixture is reacted at 20 to 45°C with stirring for 1 to 8 hours to obtain compound VI, then an excess organic base is added, and then 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate is added, and the mixture is reacted at 15 to 45°C with stirring for 1 to 8 hours, followed by extraction and purification by column chromatography to obtain compound VII.

7. The method according to 6, characterized in that the organic solvent is one of dichloromethane, N,N-dimethylcarboxamide, or a mixture of both.

8. The method according to 6, characterized in that the organic acid is one of trifluoroacetic acid and p-toluenesulfonic acid.

9. The method according to 6, characterized in that the organic base is one of N,N-diisopropylethylamine and triethylamine.

10. The method according to 2, wherein, specifically in step d, compound VII obtained in step c is added to an organic solvent, an organic acid is added, and the mixture is reacted at 20 to 45°C with stirring for 1 to 8 hours, followed by precipitation and purification by preparative liquid chromatography to obtain an intermediate with structure I.

11. The method according to 10, characterized in that the organic acid is one of trifluoroacetic acid and p-toluenesulfonic acid.

12. The use of the intermediate according to claim 1 in the synthesis of Evans Blue modified FAPI (LNC1004), The method of use is characterized by the following steps: dissolving the intermediate whose structure is represented by formula I in water, sequentially adding hydrochloric acid solution and sodium nitrite while stirring at -5 to 25°C to react and produce a diazonium salt solution; mixing 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate to cause an acid-base neutralization reaction to produce an aqueous solution of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate; adding the diazonium salt solution dropwise to the aqueous solution of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate while stirring at -5 to 25°C to react for 1 to 8 hours; and purifying by preparative liquid chromatography to obtain Evans blue modified FAPI (LNC1004), the specific reaction formula being as follows.

13. The use according to claim 12, characterized in that the molar concentration of the hydrochloric acid solution is 0.5 mol / L to 6 mol / L.

14. The use according to claim 12, characterized in that the molar ratio of hydrochloric acid to the intermediate after adding the hydrochloric acid solution is 1.0 to 3.

0.

15. The use according to claim 12, characterized in that the molar ratio of sodium nitrite to the intermediate is 1.0 to 3.

0.

16. The use according to claim 12, characterized in that the molar ratio of the 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt to the intermediate is 1.0 to 3.

0.

17. The use according to claim 12, characterized in that the molar ratio of sodium bicarbonate to the intermediate is 5.0 to 10.0.

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