Intermediates for the synthesis of FAPI and their preparation and 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] The present invention relates to the field of drug synthesis, and in particular to intermediates for synthesizing Evans Blue-modified FAPI (LNC1004), preparation methods and uses thereof. [Background technology]

[0002] Molecular imaging radiotracers targeting fibroblast activation protein (FAP) have shown promising preclinical and clinical results in tumor diagnosis. LNC1004 is a newly developed Evans Blue-modified fibroblast activation protein inhibitor (Evans Blue-modified FAPI), whose chemical name is 2,2',2''-(10-(2-(((S)-1-((4'-((E)-(8-amino-1-hydroxy-5,7-disulfonaphthalen-2)-yl)diazenyl)-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-6 -(4-(4-(3-((4-((2-((S)-2-cyanopyrrolidin-1-yl)-2-oxyethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-4-oxobutyramido)-1-oxohexyl-2-yl)amino)-2-oxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid trifluoroacetate. Prior art studies have reported the use of LNC1004 for targeted tumor therapy. 177 We found that the addition of Evans Blue fragments to the radiolabeled LNC1004 extended its circulating half-life, improved its pharmacokinetics (PK), and increased its tumor uptake, potentially enhancing the efficacy of radiotherapy. Therefore, radiolabeled LNC1004 can be used as a novel, long-acting cancer therapeutic agent.

[0003] A synthetic scheme for LNC1004 has been reported in the prior art (reference: Evans blue-modified radiolabeled fibroblast activation protein inhibitor as long-acting cancer therapeutics, Theranostics 2022; 12(1): 422-433). JPEG2025534068000002.jpg86170JPEG2025534068000003.jpg46170

[0004] Although the above scheme can produce the desired product, it has the following drawbacks. 1) The use of sulfonic acid-containing Evans Blue fragments as starting materials for synthesizing Evans Blue-modified FAPI (LNC1004) increases the polarity of the intermediates involved in the overall synthesis scheme, making separation and purification difficult using conventional workup methods (e.g., extraction, column chromatography, etc.). Furthermore, the use of preparative liquid-phase chromatography for purification increases the difficulty of separation, resulting in low efficiency and making it unsuitable for industrial production. 2) DOTA-NHS is used for coupling the final fragment, but DOTA-NHS itself is structurally unstable and prone to decomposition and ring opening at high temperatures, resulting in numerous side reactions and low yields. Furthermore, the high cost of DOTA-NHS increases the overall cost.

[0005] Therefore, there is a need to improve the synthesis of LNC1004 to facilitate its industrial scale-up production. Summary of the Invention [Problem to be solved by the invention]

[0006] In light of the above technical background, the technical problem to be solved by the present invention 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 that can be used as a key intermediate in the synthesis of Evans Blue-modified FAPI. The synthesis of 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] Yet another object of the present invention is to provide a process for the preparation of said key intermediate.

[0009] It is a further object of the present invention to provide a method for synthesizing Evans Blue-modified FAPI utilizing said key intermediate, i.e., the use of said key intermediate in the synthesis of Evans Blue-modified FAPI. [Means for solving the problem]

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

[0011] In a first aspect, the present invention provides an intermediate for synthesizing an Evans Blue-modified FAPI (LNC1004) having the structure of formula (I). [ka]

[0012] In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Step a) reacting (S)—N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazin-1-yl)propoxy)quinoline-4-carboxamide (i.e., compound II) with succinic anhydride to obtain compound III; Step b: treating compound III with tert-butyl (S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohex-2-yl)carbamate (i.e., compound IV) by coupling reaction to obtain compound V; Step c) of deprotecting compound V to obtain compound VI, which is then subjected to a substitution reaction and treatment to obtain compound VII; and step d. treating said compound VII by hydrolysis to obtain said intermediate of formula I structure.

[0013] Specifically, the reaction scheme for the intermediates of the present invention (ie, compounds of formula I) is as follows: JPEG2025534068000005.jpg68170JPEG2025534068000006.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-oxohex-2-yl)carbamate (i.e., compound IV) are added to N,N-dimethylcarboxamide solvent, followed by adding a condensing agent and an organic base, and reacting and treating for 1 to 8 hours with stirring at 20 to 45°C to obtain compound V.

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

[0016] In a preferred embodiment of the present invention, in step c, specifically, compound V obtained in step b is added to an organic base solvent, followed by addition of an organic acid, and the mixture is stirred at 20 to 45°C for 1 to 8 hours to obtain compound VI. After that, an excess organic base is added, and then 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-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 stirred at 15 to 45°C for 1 to 8 hours to obtain compound VII.

[0017] In a more preferred embodiment of the present invention, in step c, the organic base solvent 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; and 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, compound VII obtained in step c is added to an organic base solvent, and then an organic acid is added, and the mixture is reacted and treated at 20 to 45°C for 1 to 8 hours with stirring to obtain an intermediate having a structure of formula I.

[0019] In a more preferred embodiment of the invention, in step d), the organic base solvent is dichloromethane or acetonitrile.

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

[0021] The present invention provides a method for preparing the above intermediate (i.e., the compound of formula I), and the compound of formula (I) is prepared by hydrolyzing the compound of formula (VII), and the preparation scheme is as follows: JPEG2025534068000007.jpg101170

[0022] A preferred method for preparing the compound of formula (I) from the compound of formula (VII) is to add the compound (VII) to an organic base solvent, then add an organic acid, and react and treat for 1 to 8 hours with stirring at 20 to 45°C to obtain an intermediate of formula I.

[0023] Preferably, in the above preparation method, said organic base solvent is preferably dichloromethane or acetonitrile.

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

[0025] Furthermore, the compound of formula (VII) can be obtained by subjecting the compound of formula (VI) to a substitution reaction, and the preparation scheme is as follows: JPEG2025534068000008.jpg94170

[0026] A preferred method for preparing compound (VII) from compound (VI) is to add compound VI to an organic base solvent, then add an excess amount of organic base, and then add 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS), and react and treat at 15 to 45°C for 1 to 8 hours with stirring to obtain compound VII.

[0027] Preferably, in the above preparation process, the organic base solvent is any 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 any one of N,N-diisopropylethylamine and triethylamine, most preferably N,N-diisopropylethylamine.

[0029] Furthermore, the compound of formula (VI) can be obtained by deprotecting the compound of formula (V), and the preparation scheme is as follows. JPEG2025534068000009.jpg71170

[0030] A preferred method for preparing the compound of formula (VI) from the compound of formula (V) is to add the compound V to an organic base solvent, then add an organic acid, and react at 20 to 45°C for 1 to 8 hours with stirring to obtain the compound VI.

[0031] Preferably, in the above preparation process, the organic base solvent is any 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 any one of trifluoroacetic acid and p-toluenesulfonic acid, most preferably trifluoroacetic acid.

[0033] Furthermore, the compound of formula (V) can be obtained by coupling the compound of formula (IV) and the compound of formula (III), and the preparation scheme is as follows: JPEG2025534068000010.jpg73170

[0034] A preferred method for preparing compound (V) from compound (IV) and compound (III) is to add compound III and compound IV to N,N-dimethylcarboxamide solvent, then add a condensing agent and an organic base, and react and treat at 20 to 45°C for 1 to 8 hours with stirring to obtain compound V.

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

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

[0037] Furthermore, the compound of formula (III) can be obtained by reacting the compound of formula (II) with succinic anhydride, and the preparation scheme is as follows. JPEG2025534068000011.jpg23170

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

[0039] Specifically, the above method or use includes the following reaction scheme: JPEG2025534068000012.jpg97170

[0040] In the reaction step of the above method or use, the intermediate represented by Formula (I) is dissolved in water, and hydrochloric acid and sodium nitrite are added successively at −5 to 25°C with stirring to form a diazonium salt solution. Subsequently, 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate are added to the solution to carry out an acid-base neutralization reaction to form an aqueous solution of 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate. 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 with stirring at −5 to 25°C, and the reaction is carried out 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 of the hydrochloric acid used (molar ratio to the intermediate) is 1.0 to 3.0, more preferably 1.5 to 2.0, the equivalent of the sodium nitrite used (molar ratio to the intermediate) is 1.0 to 3.0, more preferably 1.0 to 1.5, the equivalent of the 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt used (molar ratio to the intermediate) is 1.0 to 3.0, more preferably 1.0 to 1.5, and the equivalent of the sodium bicarbonate used (molar ratio to 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 premised on synthesizing the intermediate (compound of formula I) of the present invention, and ultimately obtains LNC1004 by coupling this intermediate with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt. Compared with conventional synthetic methods, the synthetic method of the present invention differs overall mainly in the following respects: the reaction with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt is carried out in the final step; and when introducing the DOTA group, a means is used in which a carboxyl-protected DOTA group is first coupled and then deprotected. Based on the above differences, the advantages of the present invention are as follows: 1) By coupling 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt with the intermediate in the final step of the synthesis, the polarity of the reaction intermediate is reduced compared to conventional synthesis methods, and all of the compounds prepared up to the point of obtaining the intermediate in the synthesis scheme can be separated and purified by post-treatment means such as extraction or column chromatography, thereby improving the production efficiency and yield of the entire synthesis process and making it suitable for large-scale industrial production. 2) When introducing the DOTA group, a method is adopted in which the carboxy-protected DOTA group is first coupled and then deprotected. That is, 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) is used instead of DOTA-NHS, and the tert-butyl ester is subsequently removed. This makes the synthesis process more efficient, further increasing yields and further reducing costs. First, 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) has chemical properties that are more stable than DOTA-NHS, and its structure is less likely to be destroyed during the reaction. Therefore, in the present invention, side reactions are reduced and the one-step reaction yield is higher. Furthermore, 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-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, and therefore can be separated and purified by column chromatography even after the corresponding compound (VII) is obtained by the reaction, making it suitable for industrial production.Furthermore, the commercial price 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-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) (approximately 1,500 RMB / g) is lower than that of DOTA-NHS (approximately 10,000 RMB / g). Therefore, in the present invention, 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri-tert-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS) is used instead of DOTA-NHS, thereby significantly reducing production costs. [Brief explanation of the drawings]

[0043] [Figure 1] 1 is a mass spectrum of the compound III of Example 1. [Figure 2] 1 is a mass spectrum of Compound V of Example 1. [Figure 3] 1 is a mass spectrum of Compound VII of Example 1. [Figure 4] 1 is a mass spectrum of the intermediate of Formula I in Example 1. [Figure 5] 1 is a mass spectrum of the Evans Blue-modified FAPI (LNC1004) of Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention may be better understood by reference to the following examples, however, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are used only to illustrate the present invention and are not intended to limit the invention as detailed in the claims.

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

[0046] Preparation of Compound V To 70 ml of N,N-dimethylcarboxamide, 7.20 g of compound III, 5.83 g of (S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohex-2-yl)carbamate (compound IV), 6.12 g of HATU, and 2.66 g of DIPEA were added in that order, and the mixture was stirred at room temperature for 2 hours. The mixture was then purified by column chromatography to give 11.66 g of compound V. Yield: 91.30%, theoretical [M+H] + =973.53, measured [M+H] + =973.52750. The spectrum representing the structure is shown in Figure 2.

[0047] Preparation of Compound VII To 200 ml of N,N-dimethylcarboxamide, 11.50 g of compound V was added, followed by 77.86 ml of trifluoroacetic acid. The mixture was stirred at 40 to 45 ° C. for 1 hour to give compound VI. Then, 234 ml of N,N-diisopropylethylamine was added, followed by 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-butyl triacetate (DOTA-TRIS-TBU-ESTER NHS). The mixture was stirred at room temperature for 6 hours, and the reaction solution was evaporated by rotary evaporation. The mixture was then dissolved in 200 ml of dichloromethane. The organic phase was washed twice with water and dried over anhydrous sodium sulfate. The organic phase was then evaporated by rotary evaporation and purified by column chromatography to give 20.85 g of compound VII. Yield 123.51% (including inorganic salts), theoretical [M+H] + =1427.85, measured [M+H] + =1427.84403. The spectrum representing the structure is shown in Figure 3.

[0048] Preparation of Compounds of Formula (I) 20.5 g of compound VII was added to 350 ml of dichloromethane and 350 ml of trifluoroacetic acid, and the mixture was stirred at 40-45°C for 1 hour. The reaction mixture was then added dropwise to 3.5 L of methyl tert-butyl ether, stirred, and the solid precipitated. After filtration, the solid was purified by preparative liquid chromatography to obtain 10.36 g of compound I. Yield: 56.30%, theoretical [M+H] + =1259.66, ([M+2H] / 2) + =630.33, measured ([M+2H] / 2) + =630.33554. The spectrum representing the structure is shown in Figure 4.

[0049] The reaction scheme of this example is as follows: JPEG2025534068000013.jpg143170JPEG2025534068000014.jpg104170

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

[0051] The reaction scheme of this example is as follows: JPEG2025534068000015.jpg110170

[0052] Comparative Example 1: Synthesis of compound 2 A 50 mL flask was charged with tert-butyl (4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)carbamate (Compound 1) (0.31 g, 1.0 mmol) and 4 mL of acetonitrile. In an ice bath, 1.5 mL of 2 M hydrochloric acid was added dropwise to the reaction flask and allowed to react for 15 minutes. Sodium nitrite (0.068 g, 1.0 mmol) was dissolved in 2 mL of water and added dropwise to the reaction flask. The resulting mixture was reacted for half an hour, resulting in Solution A. A separate 50 mL reaction flask was charged with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt (0.33 g, 1.0 mmol), sodium carbonate (0.105 g, 1.0 mmol), and 5 mL of acetonitrile to form Solution B. Solution A was slowly added dropwise to Solution B in an ice bath, and the mixture was allowed to react for 2 hours with stirring in the ice bath. Purification by preparative liquid chromatography and lyophilization gave pure compound 2 (47% yield).

[0053] Synthesis of compound 3 Compound 2 (0.52 g, 1.0 mmol) was dissolved in trifluoroacetic acid in an ice bath, and the mixture was allowed to warm to room temperature for 2 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain the crude product. The crude product was purified by preparative liquid chromatography and lyophilized to obtain pure compound 3 (73% yield).

[0054] Synthesis of compound 4 A 100 ml flask was charged with 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. 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 (47% yield).

[0055] Preparation of Compound 5 Compound 4 was deprotected using piperidine (20% [v / v]) at room temperature. The reaction mixture was stirred for 1 hour, and then the DMF was removed under high vacuum. The residue was purified by preparative liquid chromatography. The isolated yield was 67%. Succinic anhydride (500 mg, 5 mmol) was dissolved in 5 mL of DMF and added to the compound 4 intermediate (0.5 mmol, 375 mg) from which the Fmoc protection had been removed. Then, 10 mmol of DIPEA was added. The mixture was stirred at room temperature for 12 hours until complete conversion to compound 5 was achieved. The DMF was then 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-cyanopyrrolidin-1-yl)-2-oxoethyl)aminoformyl)quinolin-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 give compound 7. The 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 give compound 8. The DMF was removed under high vacuum, and the residue was purified by preparative liquid chromatography (210 mg, 81% yield).

[0057] Preparation of LNC1004 The BOC group of compound 8 (26 mg, 0.02 mmol) was removed in DCM (v / v) with 10% TFA at room temperature for 1 h. After removing the TFA and DCM under a stream of nitrogen, 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 h, the reaction mixture was purified by preparative liquid chromatography to give the desired product (23 mg, 73% yield).

[0058] The reaction scheme is as follows: JPEG2025534068000016.jpg76170JPEG2025534068000017.jpg86170

[0059] Comparing Examples 1 and 2 of the present invention with Comparative Example 1, it was found that in Comparative Example 1, LNC1004 was synthesized through a seven-step reaction, with post-reaction purification by preparative liquid chromatography required for each step. The overall yield was 4.09%. In contrast, in Examples 1 and 2 of the present invention, the synthesis scheme synthesizes LNC1004 through a total of six reaction steps, with only the last two steps requiring purification by preparative liquid chromatography. The overall yield was 20.38%. It was found that the synthesis method of the present invention can significantly improve the yield of LNC1004.

Claims

1. An intermediate for synthesizing Evans Blue-modified FAPI, An intermediate having the structure of formula (I):

2. 10. A process for preparing the intermediate of claim 1, comprising: Step a) reacting (S)—N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazin-1-yl)propoxy)quinoline-4-carboxamide (compound II) with succinic anhydride to obtain compound III; Step b: treating compound III with compound IV, tert-butyl (S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohex-2-yl)carbamate, via a coupling reaction to obtain compound V; Step c: deprotecting compound V to obtain compound VI, followed by substitution, extraction, and purification by column chromatography to obtain compound VII; and step d) treating said compound VII by hydrolysis to obtain said intermediate of formula I structure; The specific reaction scheme is as follows:

3. The method of claim 2, wherein in step b, the 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-oxohex-2-yl)carbamate are added to an N,N-dimethylcarboxamide solvent, followed by adding a condensing agent and an organic base, and reacting and treating at 20 to 45°C for 1 to 8 hours with stirring 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: 156311-83-0), PyBOP (BENZOTRIAZOLYL-N-OXYTRIS-(DIMETHYLAMINO-PHOSPHONIUM HEXAFLUOROPHOSPHATE), CAS: 128625-52-5), or PyBOP (BENZOTRIAZOLYL-N-OXYTRIS-(DIMETHYLAMINO-PHOSPHONIUM HEXAFLUOROPHOSPHATE), CAS: 128625-52-5).

5. 4. The method according to claim 3, wherein the organic base is any one of N,N-diisopropylethylamine and triethylamine.

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

7. 7. The method of claim 6, wherein the organic solvent is any one of dichloromethane, N,N-dimethylcarboxamide, or a mixture of both.

8. 7. The method according to claim 6, wherein the organic acid is one of trifluoroacetic acid and p-toluenesulfonic acid.

9. 7. The method according to claim 6, wherein the organic base is any one of N,N-diisopropylethylamine and triethylamine.

10. The method according to claim 2, characterized in that in step d, the compound VII obtained in step c is added to an organic solvent, and then an organic acid is added, and the mixture is reacted at 20 to 45°C for 1 to 8 hours with stirring, and then precipitated and purified by preparative liquid chromatography to obtain an intermediate having a structure of formula I.

11. 11. The method according to claim 10, wherein the organic acid is one of trifluoroacetic acid and p-toluenesulfonic acid.

12. 10. Use of the intermediate of claim 1 in the synthesis of Evans Blue modified FAPI (LNC1004), The method comprises the steps of: dissolving the intermediate represented by Formula I in water; sequentially adding hydrochloric acid solution and sodium nitrite at −5 to 25° C. with stirring to form a diazonium salt solution; then mixing 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt with sodium bicarbonate to form an acid-base neutralization reaction to form 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 with stirring at −5 to 25° C. for 1 to 8 hours; and purifying by preparative liquid chromatography to obtain Evans blue-modified FAPI (LNC1004), the specific reaction scheme of which is as follows:

13. 13. The use according to claim 12, wherein the molarity of the hydrochloric acid solution is between 0.5 mol / L and 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. 13. The use according to claim 12, wherein the molar ratio of sodium nitrite to the intermediate is from 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. 13. The use according to claim 12, wherein the molar ratio of sodium bicarbonate to the intermediate is from 5.0 to 10.0.

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