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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
  • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
  • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention relates to the field of drug synthesis, and in particular to an intermediate for synthesizing Evans blue-modified FAPI (LNC1004), a preparation method and application thereof.
• 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-oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazine-1-yl)-4-oxobutanamide)-1-oxohexyl-2-yl
• radiolabeled LNC1004 can be used as a new type of long-acting cancer treatment drug.
• the above route can obtain the target product, it has the following disadvantages: 1) the Evans blue fragment with a sulfonic acid group is used as the starting material to synthesize the Evans blue-modified FAPI (LNC1004), which increases the polarity of the intermediates involved in the entire synthetic route, making it difficult to separate and purify using traditional post-treatment methods (such as extraction, column chromatography, etc.), and the use of preparative liquid phase purification increases the difficulty of separation and has low efficiency, making it unsuitable for industrial production; 2) DOTA-NHS is used for coupling the final fragment. DOTA-NHS itself has an unstable structure and is easily decomposed and ring-opened at high temperatures, resulting in more side reactions and low yields. In addition, DOTA-NHS itself is expensive, resulting in a high overall cost.
• the technical problem to be solved by the present invention is how to improve the yield of LNC1004 synthesis and reduce the cost to make it more suitable for industrial scale-up production.
• the primary purpose of the present invention is to provide a new compound as a key intermediate for synthesizing Evans blue-modified FAPI.
• the synthesis of Evans blue-modified FAPI through the key intermediate can not only reduce the production cost, but also significantly improve the production efficiency and yield, and is suitable for large-scale industrial production.
• the second object of the present invention is to provide a method for preparing the key intermediate.
• Another object of the present invention is to provide a method for synthesizing Evans blue-modified FAPI using the key intermediate, that is, the use of the key intermediate in synthesizing Evans blue-modified FAPI.
• the present invention adopts the following technical solution:
• the present invention provides an intermediate for the synthesis of Evans blue-modified FAPI (LNC1004), the structure of which is shown in formula (I):
• the present invention provides a method for preparing the above intermediate (i.e., compound of formula I), comprising the following steps:
• Step a (S)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-6-(3-(piperazin-1-yl)propoxy)quinoline-4-carboxamide (i.e., compound II) reacts with succinic anhydride to obtain compound III;
• Step b Compound III and tert-butyl (S)-(6-amino-1-((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)amino)-1-oxohexan-2-yl)carbamate (i.e., compound IV) are subjected to coupling reaction and treatment to obtain compound V;
• Step c Compound V is subjected to a deprotection reaction to obtain Compound VI, which is then subjected to a substitution reaction and treatment to obtain Compound VII;
• Step d The compound VII is subjected to hydrolysis reaction and treatment to obtain the intermediate of formula I.
• reaction route of the intermediate (i.e., compound of formula I) of the present invention is as follows:
• the step b is specifically as follows: in N,N-dimethylformamide solvent, 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-oxohexan-2-yl)carbamate (i.e., compound IV) are added, and then a condensing agent and an organic base are added, and the mixture is stirred and reacted at 20 to 45° C. for 1 to 8 hours, and compound V is obtained after treatment.
• 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-oxohexan-2-yl)carbamate (i.e., compound IV) are added,
• the condensing agent in step b) is any one of HATU, HBTU, TBTU, TSTU, PyAOP, and PyBOP, with HATU being most preferred;
• the organic base in step b) is any one of N,N-diisopropylethylamine and triethylamine, with N,N-diisopropylethylamine being most preferred.
• step c is specifically as follows: adding the compound V obtained in step b to an organic solvent, and then adding an organic acid, stirring the reaction at 20-45°C for 1-8 hours to obtain compound VI, and then adding an excess of an organic base, and then adding 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (DOTA-TRIS-TBU-ESTER NHS), stirring the reaction at 15-45°C for 1-8 hours, and obtaining compound VII after treatment.
• DOTA-TRIS-TBU-ESTER NHS 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) tri
• the organic solvent in step c is any one of dichloromethane, N,N-dimethylformamide or a mixture thereof, and N,N-dimethylformamide is most preferred;
• the organic acid in step c is any one of trifluoroacetic acid and p-toluenesulfonic acid, and trifluoroacetic acid is most preferred;
• the organic base in step c is any one of N,N-diisopropylethylamine and triethylamine, and N,N-diisopropylethylamine is most preferred.
• the step d is specifically as follows: adding the compound VII obtained in step c to an organic solvent, and then adding an organic acid, stirring the reaction at 20 to 45° C. for 1 to 8 hours, and obtaining an intermediate of the structure of formula I after treatment.
• the organic solvent in step d) is dichloromethane or acetonitrile.
• the organic acid in step d) is any one of trifluoroacetic acid and p-toluenesulfonic acid, and trifluoroacetic acid is most preferred.
• the present invention provides a method for preparing the above intermediate (i.e., compound of formula I), and the compound of formula (I) is prepared by hydrolyzing the compound of formula (VII), and the preparation route is:
• the preferred method for preparing the compound of formula (I) from the compound of formula (VII) is as follows: adding the compound (VII) to an organic solvent, and then adding an organic acid, stirring the reaction at 20-45°C for 1-8 hours, and obtaining an intermediate of formula I after treatment.
• the organic solvent described in the above preparation method is preferably dichloromethane or acetonitrile.
• the organic acid described in the above preparation method is preferably any one of trifluoroacetic acid and p-toluenesulfonic acid, and more preferably trifluoroacetic acid.
• the preferred preparation method of the compound of formula (VII) from the compound of formula (VI) is as follows: add compound VI to an organic solvent, then add an excess of an organic base, and then add tri-tert-butyl 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (DOTA-TRIS-TBU-ESTER NHS), stir the reaction at 15-45°C for 1-8 hours, and obtain compound VII after treatment.
• DOTA-TRIS-TBU-ESTER NHS tri-tert-butyl 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)tri
• the organic solvent in the above preparation method is any one of dichloromethane, N,N-dimethylformamide or a mixture thereof, and N,N-dimethylformamide is most preferred.
• the organic base described in the above preparation method is any one of N,N-diisopropylethylamine and triethylamine, and N,N-diisopropylethylamine is most preferred.
• the preferred method for preparing the compound of formula (VI) from the compound of formula (V) is as follows: Compound V is added to an organic solvent, followed by an organic acid, and the mixture is stirred at 20 to 45° C. for 1 to 8 hours to obtain compound VI.
• the organic solvent in the above preparation method is any one of dichloromethane, N,N-dimethylformamide or a mixture thereof, and N,N-dimethylformamide is most preferred.
• the organic acid described in the above preparation method is any one of trifluoroacetic acid and p-toluenesulfonic acid, and trifluoroacetic acid is most preferred.
• the preferred preparation method of the compound of formula (V) from the compound of formula (IV) and the compound of formula (III) is as follows: in N,N-dimethylformamide solvent, compound III and compound IV are added, and then a condensing agent and an organic base are added, and the reaction is stirred at 20 to 45° C. for 1 to 8 hours, and compound V is obtained after treatment.
• the condensing agent described in the above preparation method is any one of HATU, HBTU, TBTU, TSTU, PyAOP, and PyBOP, and HATU is most preferred.
• the organic base described in the above preparation method is any one of N,N-diisopropylethylamine and triethylamine, and N,N-diisopropylethylamine is most preferred.
• the compound of formula (III) is obtained by reacting the compound of formula (II) with succinic anhydride, and its preparation route is:
• the present invention provides a method for synthesizing Evans blue-modified FAPI (LNC1004) using the intermediate described in the present invention (i.e., the compound of formula I).
• the present invention also provides an application of the intermediate described above (i.e., the compound of formula I) in synthesizing Evans blue-modified FAPI (LNC1004).
• the above method or application includes the following reaction route:
• reaction steps of the above method or application are: dissolving the intermediate with a structure such as formula (I) in water, adding hydrochloric acid solution and sodium nitrite in sequence under stirring conditions at -5 to 25°C to react to generate a diazonium salt solution, then mixing 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate to undergo acid-base neutralization reaction to generate 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate aqueous solution, and dripping the diazonium salt solution into the 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt and sodium bicarbonate aqueous solution under stirring at -5 to 25°C, reacting for 1 to 8 hours, and obtaining Evans blue-modified FAPI (i.e., LNC1004) through post-treatment.
• a structure such as formula (I) in water
• the molar concentration of the hydrochloric acid solution in the above reaction step is 0.5 mol/L to 6 mol/L, more preferably 1 to 3 mol/L;
• the equivalent of hydrochloric acid used (molar ratio to the intermediate) is 1.0 to 3.0, more preferably 1.5 to 2.0;
• the equivalent of sodium nitrite used (molar ratio to the intermediate) is 1.0 to 3.0, more preferably 1.0 to 1.5;
• the equivalent of 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;
• the equivalent of sodium bicarbonate used (molar ratio to the intermediate) is 5.0 to 10.0, more preferably 8.0 to 10.0.
• the method for synthesizing Evans blue modified FAPI (LNC1004) of the present invention is based on the synthesis of the intermediate (compound of formula I) of the present invention, and finally the intermediate is coupled with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt to obtain LNC1004.
• the overall synthesis method of the present invention is mainly different in that the reaction with 1-amino-8-naphthol-2,4-disulfonic acid monosodium salt is placed in the last step; when introducing the DOTA group, the DOTA group with carboxyl protection is first coupled and then deprotected. Based on the above differences, the advantages of the present invention are:
• the DOTA group with carboxyl protection 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) triacetate tri-tert-butyl ester (DOTA-TRIS-TBU-ESTER NHS) is used instead of DOTA-NHS to participate in the reaction, and the tert-butyl ester is subsequently removed, which can make the synthesis process more efficient, higher in yield and lower in cost.
• 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate tri-tert-butyl ester (
• FIG1 is a mass spectrum of compound III described in Example 1.
• FIG. 2 is a mass spectrum of compound V described in Example 1.
• FIG3 is a mass spectrum of compound VII described in Example 1.
• FIG4 is a mass spectrum of the intermediate of Formula I described in Example 1.
• FIG. 5 is a mass spectrum of Evans blue-modified FAPI (LNC1004) described in Example 2.
• LNC1004 Evans blue-modified radiolabeled fibroblast activation protein inhibitor
• tert-butyl (4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl) carbamate (compound 1) (0.31g, 1.0mmol) and 4ml of acetonitrile were added respectively, ice-bathed, 1.5ml of 2M hydrochloric acid was added dropwise into the reaction flask, reacted for 15min, then sodium nitrite (0.068g, 1.0mmol) was dissolved in 2ml of water, and added dropwise into the reaction flask again, reacted for half an hour, and used as liquid A for standby.
• compound 1 tert-butyl (4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl) carbamate
• the reaction route is as follows:
• Comparing Examples 1 and 2 of the present invention with Comparative Example 1 it can be seen that Comparative Example 1 synthesizes LNC1004 through 7 steps of reaction, and each step of post-reaction treatment requires liquid phase purification, and its total yield is 4.09%.
• the overall synthetic route of Examples 1 and 2 of the present invention synthesizes LNC1004 through 6 steps of reaction, of which only the last two steps require liquid phase purification, and its total yield is 20.38%. It can be seen that the synthetic method of the present invention can significantly improve the yield of LNC1004.

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