Trifluoroacetate of fibroblast activation protein-targeting ligand and preparation method for trifluoroacetate

Abstract

Provided are trifluoroacetate of a fibroblast activation protein-targeting ligand and a preparation method for trifluoroacetate. Specifically, provided is a compound represented by the following formula (X), and the compound is used as a labeled precursor molecule, increasing the water solubility of the molecule, and facilitating the quality control of the labeling process.

Description

A trifluoroacetate salt of a ligand targeting fibroblast activation protein and its preparation method Technical Field

[0001] A trifluoroacetate salt of a ligand targeting a fibroblast activation protein and its preparation method belong to the pharmaceutical field. Background Technology

[0002] Fibroblast activation protein (FAP) is a type II transmembrane serine proteolytic enzyme that plays a crucial role in the metabolism of various endogenous peptides and peptide drugs. PCT / CN2024 / 091688 describes a novel class of ligands targeting FAP, highlighting the urgent need to find suitable salt types for industrial production. Summary of the Invention

[0003] This disclosure provides, in one aspect, a compound represented by formula (X),

[0004] The mass fraction of trifluoroacetic acid is selected from 10-30%.

[0005] In some embodiments, the compound of formula (X) provided in this disclosure has a trifluoroacetic acid mass fraction selected from 15-25%.

[0006] In some embodiments, the compound represented by formula (X) provided in this disclosure has a trifluoroacetic acid mass fraction selected from 18-22%.

[0007] In specific embodiments, the compound of formula (X) provided in this disclosure, wherein the mass fraction of trifluoroacetic acid is selected from 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11.0%, 11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12.0%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13.0%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13 ... 0.6%, 13.7%, 13.8%, 13.9%, 14.0%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15.0%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 1 5.7%, 15.8%, 15.9%, 16.0%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17.0%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18.0%, 18.1%, 18.2%, 18.3%, 18.4%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19.0%, 19.1%, 19.2%, 19.3%, 19.4%, 19.5%, 19.6%, 19.7%, 19.8% 19.9%, 20.0%, 20.1%, 20.2%, 20.3%, 20.4%, 20.5%, 20.6%, 20.7%, 20.8%, 20.9%, 21.0%, 21.1%, 21.2%, 21.3%, 21.4%, 21.5%, 21.6%, 21.7%, 21.8%, 21.9% %, 22.0%, 22.1%, 22.2%, 22.3%, 22.4%, 22.5%, 22.6%, 22.7%, 22.8%, 22.9%, 23.0%, 23.1%, 23.2%, 23.3%, 23.4%, 23.5%, 23.6%, 23.7%, 23.8%, 23.9%, 24. 0%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25.0%, 25.1%, 25.2%, 25.3%, 25.4%, 25.5%, 25.6%, 25.7%, 25.8%, 25.9%, 26.0%, 26.1%, 26.2%, 26.3%, 26.4%, 26.5%, 26.6%, 26.7%, 26.8%, 26.9%, 27.0%, 27.1%, 27.2%, 27.3%, 27.4%, 27.5%, 27.6%, 27.7%, 27.8%, 27.9%, 28.0%, 28.1%, 28.2%, 28.3%, 28.4%, 28.5%, 28.6%, 28.7%, 28.8%, 28.9%, 29.0%, 29.1%, 29.2%, 29.3%, 29.4%, 29.5%, 29.6%, 29.7%, 29.8%, 29.9%, 30.0%, or any value between two points. .

[0008] In some embodiments, the compound represented by formula (X) provided in this disclosure is a solid.

[0009] In some embodiments, the compound represented by formula (X) provided in this disclosure is an amorphous compound.

[0010] In some embodiments, the compound represented by formula (X) provided in this disclosure is a lyophilized powder.

[0011] This disclosure further provides a method for preparing the compound shown in formula (X) above, comprising the step of reacting compound 2 with 1.5-2 equivalents (molar ratio) of trifluoroacetic acid.

[0012] The method for preparing the compound represented by formula (X) disclosed herein may be wherein the multiple of trifluoroacetic acid may be selected from 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 equivalents (molar ratio), or any value between two points.

[0013] In some embodiments, the method for preparing the compound represented by formula (X) provided in this disclosure uses a reaction solvent that is a mixture of a nitrile solvent and water.

[0014] In an optional embodiment, the method for preparing the compound represented by formula (X) provided in this disclosure uses acetonitrile as the nitrile solvent.

[0015] In an optional embodiment, the method for preparing the compound represented by formula (X) provided in this disclosure further includes a freeze-drying step.

[0016] This disclosure further provides a method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, comprising: according to the aforementioned compound of formula (X), fluorine [ 18 The steps of reacting F ions with aluminum salts or their hydrates.

[0017] In some embodiments, the method for preparing the compound of formula (I) provided in this disclosure or its pharmaceutically acceptable salt or pharmaceutical composition thereof, wherein the aluminum salt or its hydrate is aluminum chloride hexahydrate.

[0018] This disclosure further provides the use of the aforementioned compound of formula (X) in the preparation of the compound of formula (I) or a pharmaceutical composition thereof.

[0019] The compound shown in formula (X) provided in this disclosure has increased molecular water solubility compared to a free base. In addition, the addition of a calculated amount of trifluoroacetic acid to form a trifluoroacetate salt with a specific content range helps in the quality control of the labeling process.

[0020] Radiation-resistant degradative agents: Stabilizers that protect organic molecules from radioactive degradation. For example, when gamma rays emitted from a radioactive nuclide break the bonds formed between the atoms of an organic molecule and free radicals, those free radicals are then removed by the stabilizer. This prevents the free radicals from undergoing any other chemical reactions that could lead to unwanted, potentially ineffective, or even toxic molecules.

[0021] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically pharmaceutically acceptable salts or prodrugs, along with other chemical components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and its biological activity.

[0022] The definition of "mass fraction of trifluoroacetic acid" in this disclosure is as follows:

[0023] ω(trifluoroacetic acid) = m(trifluoroacetic acid) / ∑(trifluoroacetic acid) m(trifluoroacetic acid) = m(trifluoroacetic acid) / m

[0024] In the formula, m(trifluoroacetic acid) is the mass of trifluoroacetic acid, and m = ∑(trifluoroacetic acid)m(trifluoroacetic acid) refers to the sum of the masses of all components of the mixture. In this disclosure, it refers to the sum of the masses of trifluoroacetic acid and compound 2. Detailed Implementation

[0025] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present disclosure.

[0026] Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0027] The HPLC / MS analytical chromatographic conditions in this disclosure are as follows:

[0028] 10 μl of sample was automatically injected per sample. Mobile phase: A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution. Flow rate: 1.5 mL / min. Gradient: B increased from 10% to 95% within 0-6 min, from 95% to 100% within 6-8 min, from 100% to 10% within 8-8.10 min, and maintained at 10% within 8.10-11.0 min.

[0029] Equipment model: Thermo Fisher ULTIMATE3000 ISQEM.

[0030] Column: Eclipse Plus C18, 3.5nm, 4.6×100mm.

[0031] UV detection wavelength: 254nM.

[0032] Compound purity data were obtained through manual integration, and molecular weight [M+1] was collected. + .

[0033] The chromatographic conditions for preparing the liquid phase in this disclosure are as follows:

[0034] Mobile phase: A: 0.1% aqueous trifluoroacetic acid solution, B: 0.1% trifluoroacetic acid acetonitrile solution. Flow rate: 16 ml / min. Gradient: B increases from 25% to 35% within 0-25.0 min, B increases from 35% to 70% within 25.0-25.1 min, and B remains at 70% within 25.1-33.0 min.

[0035] Equipment model: Agilent AGILENT1260Ⅱ.

[0036] Chromatographic column: HPLCONE, 5.0μm, 30×250mm.

[0037] UV detection wavelength: 254 nm. The target compound was collected and then freeze-dried.

[0038] Example 1. Preparation of 6-(trans-4-(((2-(4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-yl)acetyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (1)

[0039] Step 1. Preparation of methyl 6-aminoquinoline-4-carboxylic acid hydrochloride (1-h)

[0040] Under ice bath conditions, 12.5 g of acetyl chloride was slowly added dropwise to 100 ml of methanol, stirred at room temperature for 0.5 hours, 5.0 g of 6-aminoquinoline-4-carboxylic acid was added, and the mixture was heated under reflux for 12 hours. The solvent was evaporated, and the mixture was slurried with isopropyl ether and filtered to give compound 1-h (6.23 g, yield: 98.2%).

[0041] MS m / z(ESI): 203.03 [M+1] + .

[0042] Step 2. Preparation of methyl 6-(trans-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-yl)formamidoquinoline-4-carboxylic acid (1-g)

[0043] Under ice bath conditions, 500 mg of compound 1-h, 636 mg of trans-4-(tert-butoxycarbonylaminomethyl)cyclohexanecarboxylic acid, 2832 mg of ethyl acetate solution of propyl phosphate tricyclic anhydride (T3P, 50% mass concentration), and 959 mg of N,N-diisopropylethylamine were added to 30 mL of tetrahydrofuran. The reaction was carried out at 35 °C for 8 hours. The reaction solution was poured into 0.5 N hydrochloric acid, extracted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 1-g (887 mg, yield: 95.9%).

[0044] MS m / z(ESI): 442.32 [M+1] + .

[0045] Step 3. Preparation of 6-(trans-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-yl)formamidoquinoline-4-carboxylic acid (1-f)

[0046] Compound 1-g 600mg was added to 20ml of a mixed solvent of tetrahydrofuran / water = 1:1, and 171mg of lithium hydroxide monohydrate was added. The reaction was carried out at room temperature for 3 hours. The tetrahydrofuran in the solvent was evaporated, and the pH was adjusted to 3-4 with 0.5N hydrochloric acid. A large amount of solid precipitated out. The solid was filtered to obtain compound 1-f (543mg, yield: 93.6%).

[0047] MS m / z(ESI): 428.11 [M+1] +

[0048] Step 4. Preparation of (S)-1-(2-((tert-butoxycarbonyl)amino)acetyl)-4,4-difluoropyrrolidine-2-carboxynitrile (1-e)

[0049] Under ice bath conditions, 5.0 g of (S)-4,4-difluoropyrrolidone-2-carboxynitrile hydrochloride, 5.0 g of Boc-glycine, 31.5 g of ethyl acetate solution of propyl phosphate tricyclic anhydride (T3P, 50% by mass), and 10.68 g of N,N-diisopropylethylamine were added to 100 mL of tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into 0.5 N hydrochloric acid, extracted with ethyl acetate, and the organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 1-e (7.8 g, yield: 90.4%).

[0050] MS m / z(ESI): 290.08 [M+1] + .

[0051] Step 5. Preparation of (S)-1-aminoacetyl-4,4-difluoropyrrolidine-2-carboxynitrile (1-d)

[0052] 7.8 g of compound 1-e was dissolved in 100 ml of 3.0 mol / L ethyl hydrochloride solution, stirred at room temperature for 5 hours, and the solvent was evaporated to give compound 1-d (6.07 g, yield: 98.7%).

[0053] MS m / z(ESI): 190.13 [M+1] + .

[0054] Step 6. Preparation of 6-(trans-4-(((tert-butoxycarbonyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (1-c)

[0055] Under ice bath conditions, 500 mg of compound 1-f, 221 mg of 1-d, 1340 mg of an ethyl acetate solution of 1-propylphosphonic tricyclic anhydride (T3P) (T3P, 50% mass concentration), and 453 mg of N,N-diisopropylethylamine were added to 20 mL of tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into 0.5 N hydrochloric acid, extracted with ethyl acetate, and the organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 1-c (572 mg, yield: 81.7%).

[0056] MS m / z(ESI): 599.10 [M+1] + .

[0057] Step 7. Preparation of 6-(trans-4-(aminomethyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)carbamoylquinoline (1-b)

[0058] 572 mg of compound 1-c was dissolved in 30 mL of 3.0 mol / L ethyl acetate hydrochloride solution, stirred at room temperature for 5 hours, and the solvent was evaporated to give compound 1-b (461 mg, yield: 96.7%).

[0059] MS m / z(ESI): 499.13 [M+1] + .

[0060] Step 8. Preparation of 6-(trans-4-(((2-(4,7,10-tri-tert-butoxycarbonylmethyl-1,4,7,10-tetraazacyclododecane-1-yl)acetyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (1-a)

[0061] Compound 1-b (300 mg), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester (DOTA-tris(t-Bu ester)) (413 mg), HATU (412 mg), and N,N-diisopropylethylamine (233 mg) were dissolved in tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using a dichloromethane / methanol = methanol (0%–20%) eluent system to give compound 1-a (464 mg, yield: 73.2%).

[0062] MS m / z (ESI): 1053.46 [M+1] + .

[0063] Step 9. Preparation of 6-(trans-4-(((2-(4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-yl)acetyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (1)

[0064] 464 mg of compound 1-a was dissolved in 20 mL of dichloromethane, and 20 mL of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, the solvent was evaporated, and the solution was purified by preparative liquid chromatography and lyophilized to obtain the target compound 1 (166 mg, yield: 42.5%).

[0065] MS m / z (ESI): 885.36 [M+1] + .

[0066] Example 2. Preparation of 6-(trans-4-(((2-(4,7-dicarboxymethyl-1,4,7-triazacyclopentan-1-yl)acetyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)carbamoylquinoline (2)

[0067] Compound 2 was prepared using the same method as compound 1.

[0068] Step 1. Preparation of 6-(trans-4-(((2-(4,7-di-tert-butoxycarbonylmethyl-1,4,7-triazacyclonon-1-yl)acetyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (2-a)

[0069] Compound 1-b 300 mg, 1,4,7,-triazacyclononane-1,4,7-triacetic acid di-tert-butyl ester (NOTA-bis(t-Bu ester)) 300 mg, HATU 412 mg, and N,N-diisopropylethylamine 233 mg were dissolved in tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using a dichloromethane / methanol = methanol (0%–20%) eluent system to give compound 2-a (368 mg, yield: 68.2%).

[0070] MS m / z(ESI): 896.33 [M+1] + .

[0071] Step 2. Preparation of 6-(trans-4-(((2-(4,7-dicarboxymethyl-1,4,7-triazacyclopentan-1-yl)acetyl)amino)methyl)cyclohexane-1-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)carbamoylquinoline (2)

[0072] 368 mg of compound 2-a was dissolved in 20 mL of dichloromethane, and 20 mL of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, the solvent was evaporated, and the solution was purified by preparative liquid phase separation and lyophilized to obtain target compound 2 (125 mg, yield: 38.7%).

[0073] 1H NMR(400MHz,DMSO-d6)δ10.30(s,1H),9.10(t,1H),8.90(d,1H),8.57(s,1H),8.25(t,1H),8.03(m,2H),7.59(d,1H),5.15(dd,1H),4.11 -4.34(m,4H),3.78(d,2H),3.62(s,4H),2.81-3.01(m,16H),2.34-2.40(m,1H),1.80-1.92(m,4H),1.40-1.48(m,3H),0.93-1.02(m,2H).

[0074] MS m / z(ESI): 784.10 [M+1] + .

[0075] Example 3. Preparation of 6-((6r,9r)-N-(2-(4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-yl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (3)

[0076] Compounds 1-h and 1-d were prepared using the same method as compound 1.

[0077] Step 1. Preparation of methyl 6-((6r,9r)-N-tert-butoxycarbonyl-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamidoquinoline-4-carboxylic acid (3-g)

[0078] Under ice bath conditions, 500 mg of compound 1-h, 528 mg of (6r,9r)-N-Boc-1-oxa-4-azaspiro[5.5]undecane-9-carboxylic acid, 2832 mg of an ethyl acetate solution of 1-propylphosphonic tricyclic anhydride (T3P, 50% mass concentration), and 959 mg of N,N-diisopropylethylamine were added to 30 mL of tetrahydrofuran. The reaction was carried out at 35 °C for 8 hours. The reaction solution was poured into 0.5 N hydrochloric acid, extracted with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 3-g (815 mg, yield: 93.2%).

[0079] MS m / z(ESI): 484.35 [M+1] + .

[0080] Step 2. Preparation of 6-((6r,9r)-N-tert-butoxycarbonyl-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamidoquinoline-4-carboxylic acid (3-f)

[0081] Compound 3-g (815 mg) was added to 20 mL of a 1:1 mixture of tetrahydrofuran and water, along with 214 mg of lithium hydroxide monohydrate. The reaction was carried out at room temperature for 3 hours. The tetrahydrofuran in the solvent was evaporated, and the pH was adjusted to 3-4 with 0.5 N hydrochloric acid. A large amount of solid precipitated out. The solid was filtered to give compound 3-f (543 mg, yield: 95.7%). MS m / z (ESI): 470.31 [M+1] + .

[0082] Step 3. Preparation of 6-((6r,9r)-N-tert-butoxycarbonyl-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (3-c)

[0083] Under ice bath conditions, 500 mg of compound 3-f, 240 mg of 1-d, 1340 mg of ethyl acetate solution of tricyclic propyl phosphate anhydride (T3P, 50% by mass), and 453 mg of N,N-diisopropylethylamine were added to 20 mL of tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into 0.5 N hydrochloric acid, extracted with ethyl acetate, and the organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and the solvent was evaporated to give compound 3-c (521 mg, yield: 76.3%). MS m / z (ESI): 641.29 [M+1] + .

[0084] Step 4. Preparation of 6-((6r,9r)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (3-b)

[0085] 521 mg of compound 3-c was dissolved in 30 mL of 3.0 mol / L ethyl acetate hydrochloride solution, stirred at room temperature for 5 hours, and the solvent was evaporated to give compound 3-b (619 mg, yield: 94.8%).

[0086] MS m / z(ESI): 541.24 [M+1] + .

[0087] Step 5. Preparation of 6-((6r,9r)-N-(2-(4,7,10-tri-tert-butoxycarbonylmethyl-1,4,7,10-tetraazacyclododecane-1-yl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (3-a)

[0088] Compound 3-b 300 mg, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester (DOTA-tris(t-Bu ester)) 357 mg, HATU 356 mg, and N,N-diisopropylethylamine 202 mg were dissolved in 20 mL of tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using a dichloromethane / methanol = methanol (0%–20%) eluent system to give compound 3-a (395 mg, yield: 69.4%).

[0089] MS m / z(ESI): 1095.58 [M+1] + .

[0090] Step 6. Preparation of 6-((6r,9r)-N-(2-(4,7,10-tricarboxymethyl-1,4,7,10-tetraazacyclododecane-1-yl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (3)

[0091] 395 mg of compound 3-a was dissolved in 20 mL of dichloromethane, and 20 mL of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, the solvent was evaporated, and the solution was purified by preparative liquid phase separation and lyophilized to obtain the target compound 3 (89 mg, yield: 26.6%).

[0092] MS m / z(ESI): 927.34 [M+1] + .

[0093] Example 4. Preparation of 6-((6r,9r)-N-(2-(4,7-dicarboxymethyl-1,4,7-triazacyclopentan-1-yl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (4)

[0094] Compound 4 was prepared using the same method as compound 3.

[0095] Step 1. Preparation of 6-((6r,9r)-4-(N-(2-(4,7-di-tert-butoxycarbonylmethyl-1,4,7-triazacyclopentane-1-yl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (4-a)

[0096] Compound 3-b 300 mg, 1,4,7,-triazacyclononane-1,4,7-triacetic acid di-tert-butyl ester (NOTA-bis(t-Bu ester)) 259 mg, HATU (1.8 equivalent) 356 mg, and N,N-diisopropylethylamine 202 mg were dissolved in 20 mL of tetrahydrofuran and reacted at 35 °C for 5 hours. The reaction solution was poured into water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography using a dichloromethane / methanol = methanol (0%–20%) eluent system to give compound 4-a (386 mg, yield: 79.1%) MS m / z (ESI): 938.42 [M+1] + .

[0097] Step 2. Preparation of 6-((6r,9r)-4-(N-(2-(4,7-dicarboxymethyl-1,4,7-triazacyclopentane-1-yl)acetyl)-1-oxa-4-azaspiro[5.5]undecane-9-yl)formamido-4-((S)-2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoylquinoline (4)

[0098] 386 mg of compound 4-a was dissolved in 20 ml of dichloromethane, and 20 ml of trifluoroacetic acid was added. The mixture was stirred overnight at room temperature, the solvent was evaporated, and the solution was purified by preparative liquid phase separation and lyophilized to obtain the target compound 4 (179 mg, yield: 52.7%).

[0099] 1 H NMR(400 MHz,DMSO-d6)δ10.29(d,1H),9.07(s,1H),8.87(d,1H),8.52(d,1H),8.03(d,2H),7.56(d,1H ),5.15(t,1H),4.19-4.45(m,7H),3.58-4.19(m,24H),1.94(m,2H),1.67(m,4H),1.27(m,2H).

[0100] MS m / z(ESI): 826.33 [M+1]+ .

[0101] Example 5. Preparation of Compound 5

[0102] Add 0.1 mL of sodium acetate buffer to the reaction flask, then add 0.9 mL of... 68 The reaction mixture consisted of GaCl3 solution (0.1 N hydrochloric acid solution) and 5 μL of aqueous solution containing 6 nmol of compound 2. The pH of the reaction solution was maintained at 3.0–5.0, and the mixture was heated at 95 °C for 10–15 min.

[0103] The labeled reaction solution was diluted with 2 mL of physiological saline. ITLC analysis showed that the impurity content was less than 10%, allowing it to be used directly in animal imaging experiments.

[0104] Example 6. Preparation of Compound 6

[0105] Add 0.1 mL of sodium acetate buffer to the reaction flask, then add 0.9 mL of... 68 The reaction mixture consisted of GaCl3 solution (0.1 N hydrochloric acid solution) and 5 μL of aqueous solution containing 6 nmol of compound 4. The pH of the reaction solution was maintained at 3.0–5.0, and the mixture was heated at 95 °C for 10–15 min.

[0106] The labeled reaction solution was diluted with 2 mL of physiological saline. ITLC analysis showed that the impurity content was less than 10%, allowing it to be used directly in animal imaging experiments.

[0107] Example 7. Preparation of Compound 7

[0108] Add 1 μl of a 10 mM compound 2 aqueous solution to 50-100 μl of a 0.5 M acetate / sodium acetate buffer solution at pH 4.0-4.4, then add 2 mM AlCl3 aqueous solution at a molar ratio of 2:1, followed by 1850 MBq of QMA-purified [reagent / solution]. 18 F - 50 μl of ions were reacted at 100 °C for 10–15 min. Purification was performed using an HLB solid-phase extraction column (HLB cartridge), followed by elution with ethanol / water (1:1) and dilution with physiological saline to below 10% ethanol content. Radiochemical purity was greater than 90% as determined by radio-HPLC, allowing for direct application in animal imaging experiments.

[0109] Example 8. Preparation of the compound shown in formula (X)

[0110] The compound obtained in Example 2 was purified by high-pressure preparative reverse cyclic chromatography (HPLC) column (packing material: SP-100-8-ODS-P) to remove trifluoroacetic acid. The preparative solution of compound 2 was concentrated to a small volume by HPLC column (packing material: SP-100-8-ODS-P, mobile phase: acetonitrile / water). Based on the mass of free base in the column concentrate, a calculated amount (1.5-2 eq) of trifluoroacetic acid was added. After removing acetonitrile by vacuum concentration, the solution was lyophilized. The lyophilized powder was reconstituted and the trifluoroacetic acid content was determined. The standard was 10-30%.

[0111] Mass of free alkali in column concentrate (g) = Detection result of free alkali content in column concentrate (mg / ml) × Volume of column concentrate (ml) × 10 -3

[0112] Trifluoroacetic acid addition (g) = mass of free base ÷ molecular weight of free base × N × molecular weight of trifluoroacetic acid (N = 1.5~2.0)

[0113] Biological evaluation

[0114] The following test examples further describe and explain the present disclosure, but these test examples are not intended to limit the scope of the present disclosure.

[0115] Test Example 1: FAPα Enzyme Activity Assay

[0116] 1.1 Experimental Materials and Instruments

[0117] Table 1. Information on the source of experimental materials and instruments

[0118] 1.2 Experimental Procedure

[0119] Dilute the substrate (Z-Gly-Pro-AMC) to a 0.5 mM stock solution using DMSO. Dilute the 0.5 mM stock solution to a 50 μM solution with PBS before each experiment. Dilute the FAPα protein to a 0.5 ng / μl solution with PBS. Dilute the test compound and positive control to 100 nM or 200 nM concentrations with PBS for determining the single-point inhibition rate. For compounds with single-point inhibition rates comparable to the positive control, further IC50 measurements are performed. 50 The test compound and positive control were diluted with PBS to a maximum concentration of 10 μM and a minimum concentration of 0 nM, using a 5-fold serial dilution method, for a total of 8 gradients. Test method: Add 85 μl of FAPα dilution buffer and 10 μl of compound dilution buffer to the ELISA plate, mix well, and incubate at 37°C for 10 min. Add 5 μL of 50 μM substrate, mix well, and incubate at 37°C for 10 min. Read the values ​​using the ELISA reader: excitation light 380 nm, emission light 465 nm.

[0120] 1.3 Experimental Results

[0121] Table 2.1 Inhibition rate (100 nM) and IC50 of the disclosed compounds against FAPα enzyme. 50

[0122] The IC50 of compound 2 against FAPα enzyme was determined using the same method. 50 The value was 0.71 times that of the positive control drug FAPI-04, which was comparable to the positive control drug; the IC50 value of FAP-42 against FAPα enzyme was... 50 The value was 2.27 times that of the positive control drug FAPI-04. Note: The structure of FAPI-04 is shown below:

[0123] Reference: CN111699181A, p. 60

[0124] The structure of FAP-42 is shown below: CN111699181A, P.61

[0125] Test Example 2. 68 Ga、 18 PET imaging test of F-labeled compounds

[0126] 2.1 Experimental Materials

[0127] cell:

[0128] Cell information: U-87MG cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number: CL-0238, batch number: YBMIL8BQH0); Culture conditions: U-87MG cell-specific medium (MEM + 10% FBS + 1% P / S); Passages: 6-9 times;

[0129] Laboratory animals:

[0130] Strain: BALB / c nude mouse; Age: 4-5 weeks; Weight: 15-22g;

[0131] Reagents:

[0132] PBS (Solepro, P1020)

[0133] Matrigel (ABW, 0827045)

[0134] Trypsin-EDTA (Gibco, 25200-072)

[0135] U-87MG Special Culture Medium (Pronosai, CM-0238)

[0136] instrument:

[0137] Small animal PET / CT (ediso, nanoScan PET / CT 4heads)

[0138] Activity meter (Capintec)

[0139] Electronic balance (Changzhou Shuangjie, DT100)

[0140] 2.2 Experimental Procedure

[0141] Model building

[0142] Prepare a sufficient quantity of U-87MG cells and seed them into the posterior part of the right forelimb of B-NDG mice. The seeding volume is 100 μL, containing 50% Matrigel and 4 × 10⁴ cells. 6 Each cell.

[0143] Operating steps

[0144] Wipe the work surface with 75% medical alcohol and lay down a disposable sterile tablecloth. Prepare a 0.5mL insulin syringe, alcohol swabs, cotton swabs, and a marker in the injection room. Place the syringe in a mouse restraint and disinfect the mouse tail with an alcohol swab. Administer 0.2-0.5mL of the prepared test sample via the tail vein to each mouse, recording the injection time, syringe activity, and empty syringe activity for each injection. Anesthetize tumor-bearing mice with isoflurane and place them prone on a small animal PET bed, then fix them in place. Perform PET static image acquisition for 10 minutes at 0.5h, 1h, 2h, and 4h after administration; perform a whole-body CT scan before each static scan to obtain images of the labeled compound's distribution throughout the tumor-bearing mice. Obtain PET images of each experimental animal at different time points after administration. Select and delineate major organs, including tumors, muscles, bones, lungs, brain, liver, and kidneys. Observe the radioactive concentration and clearance of the labeled compound in tumor and non-target tissues within the tumor-bearing mice.

[0145] 3. Experimental Results

[0146] Table 3.1 Take-up of the disclosed compounds in tumors (tissues)

[0147] Table 3.2 Uptake of the disclosed compounds in tumors (tissues)

[0148] Table 3.3 Take-up of the disclosed compounds in tumors (tissues)

[0149] Table 3.4 Ratio of the disclosed compounds in tumor tissues to non-target organs (tissues)

[0150] Table 3.5 Ratio of the disclosed compounds in tumor tissues to non-target organs (tissues)

[0151] Conclusions: Compound 5 showed higher uptake in tumors than FAPI-04, but lower uptake in non-target organs (tissues); its tumor / non-target organ (tissue) ratio was superior to FAPI-04. Compound 7 showed higher uptake in tumors than FAPI-04, and its tumor / non-target organ (tissue) ratio was superior to FAPI-04.

[0152] Comparative Example 1. 18 PET Imaging Test of F-FAPI-42

[0153] Using the same experimental method as in Test Example 2, observe the markings. 18 The ratio of radioactive concentration of the F-FAPI-42 compound in tumor and non-target tissues in tumor-bearing mice.

[0154] Table 4.1 18 F-FAPI-42 tumor tissue to non-target organ (tissue) ratio

[0155] Experimental conclusion: In the U-87 mouse model, compound 7 compared to... 18 F-FAPI-42 exhibits lower uptake in non-target organs such as the gallbladder and intestine, implying that compound 7 has higher safety. Simultaneously, compound 7 has higher tumor / gallbladder ratios and tumor / intestinal uptake ratios, indicating that it can better distinguish tumor lesions from normal tissues and organs, demonstrating excellent targeting properties.

[0156] Comparative Example 2. Preparation of Compound HR-03

[0157] Step 1: Preparation of intermediate HR-03B

[0158] 1.00 g of intermediate HR-03A and 691 mg of 10% Pd / C were added to 100 mL of tetrahydrofuran, and the mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. The target product was detected by LCMS. The resulting mixture was filtered, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure to give 319 mg of intermediate HR-03B as a white solid.

[0159] LCMS(ESI,m / z):328.2[M+H] + .

[0160] Step 2: Preparation of intermediate HR-03C

[0161] 260 mg of intermediate HR-03B, 776 mg of cesium carbonate, 211 mg of methyl 6-bromoquinoline-4-carboxylate, 72 mg of tris(dibenzylacetone)dipalladium, and 75 mg of 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl were added to 5 mL of dioxane. The resulting mixture was stirred at 100 °C for 1 hour under a nitrogen atmosphere. The target product was detected by LCMS. The resulting mixture was concentrated and column chromatography yielded 210 mg of intermediate HR-03C, a white solid.

[0162] LCMS(ESI,m / z):513.3[M+H] + .

[0163] Step 3: Preparation of intermediate HR-03D

[0164] 200 mg of intermediate HR-03C and 28 mg of lithium hydroxide were added to 10 mL of a 1:1 mixture of tetrahydrofuran and water. The resulting mixture was stirred at room temperature for 2 hours. The target product was detected by LCMS. The resulting mixture was concentrated under vacuum to give 205 mg of intermediate HR-03D as a white solid.

[0165] LCMS(ESI,m / z):499.2[M+H] + .

[0166] Step 4: Preparation of intermediate HR-03E

[0167] 100 mg of intermediate HR-03D, 77 mg of N,N-diisopropylethylamine, 54 mg of (2S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride, and 91 mg of HATU were added to 2 mL of N,N-dimethylformamide. The resulting mixture was stirred at room temperature for 1 hour. The target product was detected by LCMS. Column chromatography of the reaction solution yielded 103 mg of intermediate HR-03E as a white solid.

[0168] LCMS(ESI, m / z): 670.3 [M+H] + .

[0169] Step 5: Preparation of intermediate HR-03F

[0170] 100 mg of intermediate HR-03E and 1 mL of trifluoroacetic acid were added to 4 mL of dichloromethane. The resulting mixture was stirred at room temperature for 2 hours. The target product was detected by LCMS. The resulting mixture was concentrated under vacuum to give 78 mg of intermediate HR-03F as a red solid.

[0171] LCMS(ESI, m / z): 570.3 [M+H] + .

[0172] Step 6: Preparation of intermediate HR-03G

[0173] 78 mg of intermediate HR-03F, 68 mg of N,N-diisopropylethylamine, 72 mg of {4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-triazanonane-1-yl}acetic acid, and 80 mg of HATU were added to 2 mL of N,N-dimethylformamide. The resulting mixture was stirred at room temperature for 1 hour. The target product was detected by LCMS. The reaction solution was purified by reversed-phase chromatography. 60 mg of intermediate HR-03G was obtained as a white solid.

[0174] LCMS(ESI, m / z): 967.5 [M+H] + .

[0175] Step 7: Preparation of compound HR-03

[0176] 50 mg of intermediate HR-03G and 0.5 mL of trifluoroacetic acid were added to 2 mL of dichloromethane. The resulting mixture was stirred overnight at room temperature. The target product was detected by LCMS. The resulting mixture was concentrated and purified by preparative liquid chromatography to give 15 mg of compound HR-03 as a red solid.

[0177] LCMS(ESI,m / z):855.4[M+H] + .

[0178] Comparative Example 3. Preparation of Compound HR-04

[0179] Step 1. Preparation of intermediate HR-04B

[0180] 1.50 g of intermediate HR-04A, 1.50 g of 8-aminoquinoline-4-carboxylic acid, 1.39 g of EDCI hydrochloride and 1.21 g of HOBT were added to 30 mL of DMF. The mixture was stirred at room temperature for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate and concentrated. Column chromatography was used to obtain 1.31 g of intermediate HR-04B.

[0181] MS m / z(ESI): 360.35 [M+1] + .

[0182] Step 2. Preparation of intermediate HR-04C

[0183] 2.00 g of intermediate HR-04B, 613 mg of succinic anhydride and 719 mg of DIEA were added to 20 mL of DMF. The mixture was stirred at room temperature for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate and concentrated, and column chromatography was used to obtain 1.30 g of intermediate HR-04C.

[0184] MS m / z(ESI): 460.47 [M+1] + .

[0185] Step 3. Preparation of intermediate HR-04D

[0186] 700 mg of intermediate HR-04C, 391 mg of N-(4-aminocyclohexyl)carbamate tert-butyl ester, 579 mg of HATU, and 590 mg of DIEA were added to 20 mL of DMF and stirred at room temperature for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and column chromatography was used to obtain 700 mg of intermediate HR-04D.

[0187] MS m / z(ESI): 656.82 [M+1] + .

[0188] Step 4. Preparation of intermediate HR-04E

[0189] Add 500 mg of intermediate HR-04D and 10 mL of trifluoroacetic acid to 5 mL of dichloromethane, stir at room temperature for 2 hours, and concentrate the reaction solution at 25 °C to obtain 309 mg of intermediate HR-04E.

[0190] MS m / z(ESI): 556.85 [M+1] + .

[0191] Step 5. Preparation of intermediate HR-04F

[0192] 300 mg of intermediate HR-04E, 307 mg of HATU, 209 mg of DIEA and 336 mg of 2-[4,7-bis(2-tert-butoxy-2-oxoethyl)-1,4,7-triazacyclononane-1-yl]acetic acid were added to 10 mL of dichloromethane and stirred at room temperature for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate and concentrated, and column chromatography was used to obtain 400 mg of intermediate HR-04F.

[0193] MS m / z(ESI): 954.27 [M+1] + .

[0194] Step 6. Preparation of compound HR-04

[0195] 300 mg of intermediate HR-04F and 5 mL of trifluoroacetic acid were added to 3 mL of dichloromethane. The mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated at 30 °C and then purified by liquid phase separation and freeze-drying to obtain 50 mg of compound HR-04.

[0196] MS m / z(ESI): 841.43 [M+1] + .

[0197] Comparative Example 4. Preparation of Compound HR-05

[0198] Step 1. Preparation of intermediate HR-05B

[0199] Add 2.60 g of intermediate HR-05A and 25 ml of ethyl hydrochloride solution to 40 mL of ethyl acetate, stir at room temperature for 3 hours, and concentrate the reaction solution to obtain 2.08 g of intermediate HR-05B.

[0200] MS m / z(ESI): 362.26 [M+1] + .

[0201] Step 2. Preparation of intermediate HR-05C

[0202] 2.00 g of intermediate HR-05B, 3.44 g of 2-[4,7-bis(2-tert-butoxy-2-oxoethyl)-1,4,7-triazacyclononane-1-yl]acetic acid, 3.15 g of HATU, and 2.14 g of DIEA were added to 20 mL of LDMF. The mixture was stirred at room temperature for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and column chromatography was used to obtain 2.21 g of intermediate HR-05C.

[0203] MS m / z(ESI): 760.32 [M+1] + .

[0204] Step 3. Preparation of intermediate HR-05D

[0205] Add 300 mg of intermediate HR-05C and 10% Pd / C (30 mg) to 30 mL of methanol. Stir the mixture at room temperature for 6 hours under a hydrogen atmosphere. Filter the reaction solution and concentrate the filtrate at 30 °C to obtain 200 mg of intermediate HR-05D.

[0206] MS m / z(ESI): 625.66 [M+1] + .

[0207] Step 4. Preparation of intermediate HR-05F

[0208] 2.00 g of intermediate HR-05E, 1.43 g of tert-butyl 3-(methylamino)propionate, 629 mg of RuPhos PdG3 and 4.89 g of cesium carbonate were added to 30 mL of dioxane. After purging with nitrogen three times, the mixture was heated to 100 °C and reacted for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate and concentrated, and then subjected to column chromatography to obtain 2.05 g of intermediate HR-05F.

[0209] MS m / z(ESI): 345.32 [M+1] + .

[0210] Step 5. Preparation of intermediate HR-05G

[0211] Add 800 mg of intermediate HR-05F and 195 mg of lithium hydroxide to a mixed solvent of 20 mL tetrahydrofuran and 2 mL purified water. React at room temperature for 5 hours. Adjust the pH of the reaction solution to about 6-7 with 0.5 N hydrochloric acid. Extract with DCM. Dry the organic phase with anhydrous sodium sulfate and concentrate to obtain 520 mg of intermediate HR-05G.

[0212] MS m / z(ESI): 331.25 [M+1] + .

[0213] Step 6. Preparation of intermediate HR-05H

[0214] Add 500 mg of intermediate HR-05G, 690 mg of HATU, and 195 mg of DIEA to 10 mL of DMF. Stir the mixture at room temperature for 16 hours. Quench the reaction solution with water, extract with dichloromethane, dry the organic phase with anhydrous sodium sulfate, concentrate, and then concentrate by column chromatography to obtain 600 mg of intermediate HR-05H.

[0215] MS m / z(ESI): 502.64 [M+1] + .

[0216] Step 7. Preparation of intermediate HR-05I

[0217] Add 500 mg of intermediate HR-05H to a mixed solvent of 15 mL dichloromethane and 15 mL trifluoroacetic acid, stir at room temperature for 3 hours, and concentrate the reaction solution in a 30 °C water bath to obtain 320 mg of intermediate HR-05I.

[0218] MS m / z(ESI): 446.43 [M+1] + .

[0219] Step 8. Preparation of intermediate HR-05J

[0220] 128 mg of intermediate HR-05I, 120 mg of intermediate HR-05D, 73 mg of HATU and 24 mg of DIEA were added to 10 mL of dichloromethane. The mixture was stirred at room temperature for 16 hours. The reaction solution was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate and concentrated. Column chromatography was used to obtain 200 mg of intermediate HR-05J.

[0221] MS m / z(ESI): 1052.81 [M+1] + .

[0222] Step 9. Preparation of HR-05

[0223] 100 mg of intermediate HR-05J was added to a mixed solvent of 2 mL dichloromethane and 2 mL trifluoroacetic acid. The mixture was stirred at room temperature for 6 hours. The residue was slurried with 30 mL methyl tert-butyl ether for 0.5 hours, filtered, and the filter cake was purified by liquid phase separation and freeze-drying to obtain 18 mg of compound H05.

[0224] MS m / z(ESI): 941.96 [M+1] + .

[0225] Comparative Example 5. Al 18 Preparation of F-HR-03

[0226] 1 μl of a 10 mM aqueous solution of compound HR-03 was added to 50-100 μl of a 0.5 M acetate / sodium acetate buffer solution at pH 4.0-4.4. Then, 2 mM AlCl3 aqueous solution was added at a molar ratio of 2:1, followed by 50 μl of 1850 MBq QMA-purified 18F- ions. The reaction was carried out at 100 °C for 10-15 min. Purification was performed using an HLB solid-phase extraction column (HLB cartridge), followed by elution with ethanol / water (1:1) and dilution with physiological saline to below 10% ethanol content. Radiochemical purity was greater than 90% as determined by radio-HPLC, allowing for direct application in animal imaging experiments.

[0227] Comparative Example 6.A1 18 Preparation of F-HR-04

[0228] Following the labeling method of Comparative Example 5, Al was prepared. 18 F-HR-04, with a radiochemical purity greater than 90% as determined by radio-HPLC, is directly used for animal imaging experiments.

[0229] Comparative Example 7.A1 18 Preparation of F-HR-05

[0230] Following the labeling method of Comparative Example 5, Al was prepared. 18 F-HR-05, radiochemical purity greater than 90% as determined by radio-HPLC, is directly used for animal imaging experiments.

[0231] Test Example 3. FAPα Enzyme Activity Assay

[0232] Using the same method as in Test Example 1, compound 2 showed higher enzyme activity than compounds HR-03, HR-04, and HR-05; among them, the IC50 values ​​of HR-03, HR-04, and HR-05 were significantly higher than those of compound 2. 50 The ratios were >675.6 times, 61.3 times, and >675.6 times, respectively.

[0233] Test Example 4. 18 PET imaging test of F-labeled compounds

[0234] 4.1 Experimental Materials

[0235] Cell information: U-87MG cells; Culture conditions: U-87MG cell-specific medium (MEM + 10% FBS + 1% P / S); Passages: 6-9 generations;

[0236] Laboratory animals: strain, B-NDG; age, 5-8 weeks; weight: 20-24g;

[0237] Reagents: PBS (Solepro, P1020); Matrigel (ABW, 0827045); Trypsin-EDTA (Gibco, 25200-072); U-87MG special medium (Pronosai, CM-0238).

[0238] Instruments: Small animal PET / CT (ediso, nanoScan PET / CT 4-head); activity meter (Capintec); electronic balance (Changzhou Shuangjie, DT100).

[0239] 4.2 Experimental Procedure

[0240] Model building

[0241] Prepare a sufficient quantity of U-87MG cells and inoculate them into the posterior part of the right forelimb of B-NDG mice. The inoculation volume is 100 μL, containing 50% Matrigel and 5 × 10⁻⁶ cells. 6 Each cell was inoculated. Tumor volume and animal weight were monitored twice weekly after inoculation, and tumors with a volume of 200-500 mg / m³ were selected. 3 Mice from the model group were enrolled in this experiment, and each group was randomly assigned according to tumor volume.

[0242] Operating steps

[0243] 1) Wipe the work surface with 75% medical alcohol and cover it with a disposable sterile tablecloth;

[0244] 2) Prepare a 0.5mL insulin syringe, alcohol swabs, cotton swabs, and a marker in the injection room. Place the syringe in the mouse restraint and disinfect the mouse's tail with alcohol swabs;

[0245] 3) Administration via tail vein injection: 0.1 mL of test sample per dose, approximately 300 μCi / animal. Record the time of each injection, the activity of the syringe, and the activity of the empty syringe.

[0246] 4) After anesthetizing the tumor-bearing mice with isoflurane, they were placed prone on the small animal PET bed and fixed in place;

[0247] 5) PET static images were acquired at different time points after drug administration; a whole-body CT scan was performed before each static scan to obtain images of the distribution of the labeled compound throughout the tumor-bearing mice, and PET images of each experimental animal at different time points after drug administration were obtained. Major organs were selected and delineated, including: tumor, muscle, joint, liver, kidney, and gallbladder.

[0248] 6) Observe the radioactive concentration and clearance of the labeled compound in tumors (average) and non-target tissues in tumor-bearing mice.

[0249] 4.3 Experimental Results

[0250] Table 5.1

[0251] Table 5.2

[0252] Compound 7 showed the highest uptake in tumors compared to compound Al in U-87MG tumor model mice. 18 F-HR-03, Al 18 F-HR-05 was detected, and no significant uptake was observed in other organs.

[0253] Al 18 F-HR-03 and Al 18 F-HR-05 is uptaken at a low rate in tumors, but some uptake has been observed in the gallbladder and intestines.

[0254] Test Example 5. 18 PET imaging test of F-labeled compounds

[0255] Using the same method as in Test Example 4, Al 18 The radioactive concentration and clearance of F-HR-04 and compound 7 in tumor (mean) and non-target tissues in tumor-bearing mice are shown in Table 6.

[0256] Table 6

[0257] Compound 7 showed the highest uptake in tumors compared to compound Al in U-87MG tumor model mice. 18 F-HR-04 was not significantly absorbed in other organs.

Claims

1. A compound represented by formula (X), in, The mass fraction of trifluoroacetic acid is selected from 10-30%, preferably from 15-25%.

2. The compound according to claim 1, wherein it is a solid; preferably, it is a lyophilized powder.

3. A method for preparing the compound of formula (X) according to any one of claims 1 or 2, comprising the step of reacting compound 2 with 1.5-2 equivalents of trifluoroacetic acid.

4. The preparation method according to claim 3, wherein the reaction solvent is a mixed solvent of nitrile solvent and water, preferably, the nitrile solvent is acetonitrile.

5. The preparation method according to claim 4 further includes a freeze-drying step.

6. A method for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, comprising: The compound of formula (X) according to any one of claims 1 or 2, fluorine [ 18 The steps of reacting F ions with aluminum salts or their hydrates.

7. The preparation method according to claim 6, wherein the aluminum salt or its hydrate is aluminum chloride hexahydrate.

8. Use of the compound of formula (X) according to claim 1 or 2 in the preparation of the compound of formula (I) or a pharmaceutical composition thereof.

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