CN117279930A

CN117279930A - Trifunctional compounds and uses thereof

Info

Publication number: CN117279930A
Application number: CN202380010993.8A
Authority: CN
Inventors: 刘志博; 刘宇
Original assignee: Beijing Borui Chuanghe Pharmaceutical Co ltd
Current assignee: Beijing Borui Chuanghe Pharmaceutical Co ltd
Priority date: 2022-08-19
Filing date: 2023-08-18
Publication date: 2023-12-22

Abstract

The present invention relates to the biomedical field, and in particular to a trifunctional compound comprising a Fibroblast Activator Protein (FAP) targeting moiety. The invention also relates to pharmaceutical compositions comprising said trifunctional compounds and to the use of said trifunctional compounds for the diagnosis or treatment of diseases.

Description

Trifunctional compounds and uses thereof

Technical Field

Background

Fibroblast Activation Protein (FAP) -Targeted Radionuclide Therapy (TRT) and cancer diagnosis are now the most advanced techniques for radiopharmaceutical development and are effective methods for cancer therapeutic diagnostics.

Targeted Radionuclide Therapy (TRT) using high affinity radioligands that selectively bind to tumor-associated antigens, thereby effectively destroying cancer cells without damaging healthy tissue, is an attractive strategy for cancer treatment (Gill MR, falzone N, du Y and Vallis KA. Targeted radionuclide therapy in a combination mode regimen (Targeted radionuclide therapy in combined-modality regimens) & ltWilljet oncology (The Lancet Oncology) & lt2017; 18:e414-e 423). Furthermore, some radionuclides that can be used for Positron Emission Tomography (PET) can also be used for targeted cancer diagnosis and patient screening when combined with these ligands, which has become an important part of cancer precision medicine (Langbein T, weber WA and Eiber M. Future theranostics: precision oncology hope in nuclear medicine (Future of Theranostics: an Outlook on Precision Oncology in Nuclear Medicine.) (J. Nuclear medicine Med.) 2019; 60:13S-19S). Recently FDA approved therapies and diagnostics for prostate cancer ¹⁷⁷ Lu-PSMA-617(Pluvicto ^TM ) And ⁶⁸ Ga-PSMA-11is a successful paradigm for this cancer therapeutic diagnostic strategy (Sartor O, de Bono J, chi KN et al for Lutetium-177-PSMA-617 (Lutetium-177-PSMA-617 for Metastatic Castration-Resistant Prostate Cancer) J.New England medical journal (NEngl J Med.)) 2021;385:1091-1103;Gafita A,Calais J,Grogan TR et al for predicting that a male with metastatic castration-resistant prostate cancer is present in a patient ¹⁷⁷ Post Lu-PSMA therapyAlignment plot of results: an international, multicentric, retrospective study (Nomograms to predict outcomes after ¹⁷⁷ Lu-PSMAtherapy in men with metastatic castration-resistant prostate cancer: an international, multicenter, retrospective study) & lt 2021 & gt, lancet oncology; 22:1115-1125). Thus, a large number of radioligands are now being developed for a few excellent targets.

Fibroblast Activation Protein (FAP) has now become the most advanced technology and breakthrough in radiopharmaceutical development as a target for cancer. The use of diagnostic radionuclide labeled FAP inhibitors (FAPI) has shown impressive imaging results in a variety of cancers over the past few years (Kratochwil C, flechsig P, lindner T et al ⁶⁸ Ga-FAPIPET/CT: tracer uptake in 28different cancers ⁶⁸ Ga-FAPIPET/CT: tracker Uppake in 28Different Kinds of Cancer) & lt, journal of Nuclear medicine & gt 2019;60:801-805; wei Y, zheng J, ma L et al [ ¹⁸ F]AlF-NOTA-FAPI-04: FAP targeting specificity, biodistribution and PET/CT imaging of various cancers ([ solution ] ¹⁸ F]AlF-NOTA-FAPI-04:FAP-targeting specificity, biondistribution, and PET/CT imaging of various cancers) & journal of nuclear medicine and molecular imaging (Eur J Nucl Med Mol Imaging) & gt 2022. High Tumor Background Ratio (TBR), rapid renal clearance, and wide availability make these radiolabeled FAPI suitable for cancer diagnosis. However, absolute tumor uptake of these radiotracers remains to be improved. In addition, FAPI currently widely used for diagnosis is mainly obtained by ⁶⁸ Ga marks, and ¹⁸ f is still the most convenient and available PET nuclide clinically. Wang et Al developed Al with excellent imaging Properties ¹⁸ F-labeled FAPI-04 (Wang S, zhou X, xu X et Al Al ¹⁸ Clinical transformation assessment of F-NOTA-FAPI for fibroblast activation protein targeted tumor imaging (Clinical translational evaluation of Al) ¹⁸ F-NOTA-FAPI for fibroblast activation protein-targeted tumour imaging) J.European journal of Nuclear medicine and molecular imaging 2021; 48:4259-4271). Al (Al) ¹⁸ F easy marking ¹⁸ F has the inherent advantage of enabling the probe to be rapidly popularized. Some other new ¹⁸ F labelThe noted FAPI also appears in a short period of time. Although these FAPIs show good diagnostic properties, the substitution or shedding of DOTA (1, 4,7, 10-tetraazacyclododecane-N, N' -tetraacetic acid) as chelating group makes them difficult to use in TRT.

Although FAP is considered a next generation target for TRT, there are still some obstacles. Will be as follows ¹⁷⁷ Lu、 ⁹⁰ Y、 ¹³¹ I and ²²⁵ the Ac long-half-life radionuclide is directly marked on the developed diagnosis FAPI and shows a certain inhibition effect on tumors (Liu Y, watabe T, kaneda-Nakashima K et al and the like in pancreatic cancer models ²²⁵ Ac]FAPI-46 comparison, use [ [ ¹⁷⁷ Lu]FAPI-46 was subjected to fibroblast activation protein targeting therapy (Fibroblast activation protein targeted therapy using [ Fibroblast activation protein targeted therapy using ]) ¹⁷⁷ Lu]FAPI-46compared with[ ²²⁵ Ac]FAPI-46-in a pancreatic cancer model) & ltEuropean journal of Nuclear medicine and molecular imaging & gt 2022; 49:871-880). However, the pharmacokinetics of these FAPI are incompatible with long half-life radionuclides because they clear rapidly in vivo, with significant decline in radioactivity in tumors within a few hours. Efforts have been made to prolong their blood circulation and tumor retention. Among them, albumin Binder conjugated FAPI (Xu M, zhang P, ding J, chen J, huo L and Liu z. Albumin Binder conjugated fibroblast activation protein inhibitor radiopharmaceuticals for cancer therapy (album Binder-Conjugated Fibroblast Activation Protein Inhibitor Radiopharmaceuticals for Cancer Therapy) & journal of nuclear medicine 2021) and multivalent FAPI derivatives show potential for clinical transformation. In addition to prolonged circulation, the adaptation of radionuclides with shorter half-lives to match the pharmacokinetics of FAPI is also one direction (Ahenkura S, cassels I, deroose CM et al for targeting Bismuth-213for radionuclide therapy: from atomic to clinical (Bismuth-213for Targeted Radionuclide Therapy:From Atom to Bedside); pharmaceutics (pharmacies.; 2021; 13). However, limited by the availability of radionuclides and radiochemistry, few studies evaluate short half-life radionuclide-labeled FAPI. However, current methods still have high toxicity to normal tissues, low tumor uptake and/or Radionuclide treatment is difficult.

Disclosure of Invention

In one aspect, the invention provides a trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof, comprising a Fibroblast Activation Protein (FAP) targeting moiety FAPT, a chelating moiety C, and aminomethyl-BF ₃ Part a.

In a further aspect, the trifunctional compound has formula (I):

wherein FAPT moiety, C moiety and A moiety have the meanings as defined above, and L is a linking group.

In another aspect, the invention provides a method for diagnosing or treating a disease comprising administering to an individual a therapeutically effective amount of a trifunctional compound.

In another aspect, the invention provides the use of the trifunctional compound in the manufacture of a medicament for diagnosing or treating a disease in an individual.

In another aspect, the invention provides methods of diagnosing or treating a disease in an individual with a trifunctional compound.

In another aspect, the present invention refers to a pharmaceutical composition comprising a trifunctional compound as defined above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and one or more pharmaceutically acceptable carriers.

In another aspect, the invention refers to a method of diagnosing or treating a disease, which is cancer, infection or inflammation, comprising administering to a subject a therapeutically effective amount of a trifunctional compound as defined above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

Drawings

FIG. 1 shows FT-FAPI-02 ¹ H NMR analysis.

FIG. 2 shows FT-FAPI ¹ H NMR analysis.

FIG. 3 shows FT-FAPI-21 ¹ H NMR analysis.

FIG. 4 shows FT-FAPI-46 ¹ H NMR analysis.

FIG. 5 shows ¹⁸ F-FT-FAPI(a)、 ¹⁸ F-FT-FAPI-02(b)、 ¹⁸ F-FT-FAPI-21 (c) and ¹⁸ in vitro stability of F-FT-FAPI-46 (d) after 6 hours incubation in saline and human serum.

FIG. 6 shows cellular uptake F18 (a), binding affinity (b), dissociation constant (c), internalization rate (d) and efflux rate (e) of FT-FAPI.

FIG. 7 shows HT-1080-FAP cells ¹⁷⁷ Lu-FT-FAPI-02、 ¹⁷⁷ Lu-FT-FAPI-21 and ¹⁷⁷ cellular uptake of Lu-FT-FAPI-46 (a); binding affinities (b) of FT-FAPI-02, FT-FAPI-21 and FT-FAPI-46; in HT-1080-FAP cells ¹⁸ F-FT-FAPI-02、 ¹⁸ F-FT-FAPI-21 and ¹⁸ dissociation constant (c) of F-FT-FAPI-46; in HT-1080-FAP cells ¹⁷⁷ Lu-FT-FAPI-02、 ¹⁷⁷ Lu-FT-FAPI-21 and ¹⁷⁷ internalization rate (d) of Lu-FT-FAPI-46; in HT-1080-FAP cells ¹⁷⁷ Lu-FT-FAPI-02、 ¹⁷⁷ Lu-FT-FAPI-21 and ¹⁷⁷ outflow Rate (e) of Lu-FT-FAPI-46.

FIG. 8 shows 1 hour after injection ¹⁸ Ex vivo biodistribution of F-FT-FAPI in HT-1080-FAP tumor-bearing mice (n=5/group).

Figure 9 shows 0.5, 1, 2, 4 and 8 hours after intravenous injection ¹⁸ Target/non-target uptake ratio of F-FT-FAPI in HT-1080-FAP tumor bearing mice (n=5/group).

FIGS. 10a and 10b show 1 hour after injection, respectively ¹⁸ F-FT-FAPI、 ¹⁸ F-AMBF ₃ FAPI-04 ⁶⁸ In vivo PET/CT imaging and quantitative analysis of Ga-FAPI-04 (n=4/group).

FIG. 11 shows 0.5, 1 and 4 hours after injection ¹⁸ PET/CT imaging and quantitative analysis of F-FT-FAPI in HT-1080-FAP tumor bearing mice (n=4/group).

FIG. 12 shows ¹⁸ F-FT-FAPI-02、 ¹⁸ F-FT-FAPI-21 and ¹⁸ F-FT-FAPin vivo PET/CT imaging and quantitative analysis of I-46 (n=4/group).

FIG. 13 shows a direct comparison between FT-FAPI and FAPI-04. (a) 0.5, 1, 2 and 4 hours after injection ⁶⁸ Ga-FT-FAPI ⁶⁸ Head-to-head PET/CT imaging comparisons between Ga-FAPI-04 (n=4/group). 0.5 hours (b) and 4 hours (c) after injection ⁶⁸ Quantitative analysis and analysis of Ga-FT-FAPI ⁶⁸ Ga-FAPI-04PET/CT imaging (n=4/group). (d) binding affinity of FAPI-04. (e) ⁶⁸ Ga-FT-FAPI ⁶⁸ Dissociation constant of Ga-FAPI-04. (f) 1 hour after injection ⁶⁸ Ga-FT-FAPI ⁶⁸ Biodistribution study of Ga-FAPI-04 (n=3/group). ⁶⁸ Ga-FT-FAPI ⁶⁸ Internalization rate (g) and outflow rate (h) of Ga-FAPI-04. (i) molecular docking of FT-FAPI and FAPI-04 with FAP.

FIG. 14 shows ²¹³ Radionuclide therapy and toxicity of Bi-FT-FAPI. (a) 0.5, 1, 2 and 4 hours after injection ¹⁸ F- ^nat PET/CT imaging of Bi-FT-FAPI in mice bearing HT-1080-FAP tumors (n=4). (b) 1 hour after injection ¹⁸ F- ^nat Biodistribution study of Bi-FT-FAPI (n=5). (c) ²¹³ Treatment regimen for Bi-FT-FAPI. (d) With different dosages ²¹³ Bi-FT-FAPI or ²¹³ Tumor growth curves and body weight changes in HT-1080-FAP tumor-bearing mice following Bi-FAPI-04 treatment (n=6/group). With high dose (3.7 MBq. Times.6), medium dose (2.22 MBq. Times.6) or 0MBq (control) ²¹³ Whole blood count (e) and blood biochemistry data (f) of HT-1080-FAP tumor bearing mice after 8 weeks of Bi-FT-FAPI treatment (n=3/group).

Figure 15 shows 0.5, 1 and 2 hours after administration ¹⁸ F- ^nat Biodistribution of Bi-FT-FAPI in HT-1080-FAP tumor bearing mice (n=5/group).

FIG. 16 shows the administration of high doses (3.7 MBq. Times.6), medium doses (2.22 MBq. Times.6) and 0MBq (control) in HT-1080-FAP tumor bearing mice ²¹³ Whole blood count data (n=3/group) after 8 weeks of Bi-FT-FAPI treatment group.

FIG. 17 shows the administration of high doses (3.7 MBq. Times.6), medium doses (2.22 MBq. Times.6) and 0MBq (control) in HT-1080-FAP tumor bearing mice ²¹³ Blood biochemical data after 8 weeks of Bi-FT-FAPI treatment group (n=3/group).

FIG. 18 shows molecular docking of FT-FAPI-02 (a), FT-FAPI-21 (b) and FT-FAPI-46 (c) with FAP. The ligand is green and the backbone of the receptor is shown as a white surface.

Fig. 19 shows the quantitative results (SUV mean) of the PET/CT imaging study 1 hour post injection (n=4/group).

FIG. 20 shows 0.5, 1, 2 and 4 hours after injection ¹⁸ F- ^nat Pb-FT-FAPI(a)； ¹⁸ F- ^nat Pb-TCMC-FT-FAPI (b) and ¹⁸ F- ^nat in vivo PET/CT images of Tb-FT-FAPI (c) (n=4/group).

FIG. 21 shows ¹⁸ F- ^nat Pb-FT-FAPI、 ¹⁸ F- ^nat Pb-TCMC-FT-FAPI and ¹⁸ F- ^nat quantitative PET results for tumor uptake of Tb-FT-FAPI (n=4/group).

Detailed Description

Definition of the definition

Unless defined otherwise below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including obvious modifications or equivalents of such techniques by those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to provide a better explanation of the present invention.

As used herein, the terms "comprises," "comprising," "includes," "including," "containing," "includes" or "including" and other variations thereof are inclusive or open-ended, and do not exclude other, unrecited elements or method steps.

When any type of range is disclosed or claimed, it is intended to cover every possible value that the range may reasonably cover, including any subrange contained therein, unless otherwise indicated. For example, the number of groups of 1 to 6 represents an integer within this range, where 1 to 6 should be understood to include 1, 2, 3, 4, 5, 6, and also include the sub-ranges 1 to 5, 1 to 4, and 1 to 3.

The term "alkyl", as used herein, alone or as part of another group, refers to a compound containing from 1 to 12 carbon atoms (i.e., C _1-12 Alkyl) or unsubstituted, straight or branched aliphatic hydrocarbons of the indicated number of carbon atoms, e.g. C ₁ Alkyl radicals such as methyl, C ₂ Alkyl radicals such as ethyl, C ₃ Alkyl radicals such as n-propyl or isopropyl radicals, C _1-3 Alkyl groups such as methyl, ethyl, n-propyl, isopropyl, etc. In one embodiment, the alkyl group is C _1-4 An alkyl group. C (C) _1-12 Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl and decyl. C (C) _1-4 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and isobutyl.

The term "cycloalkyl", as used herein, alone or as part of another group, refers to a saturated or partially unsaturated (containing one or two double bonds) cycloaliphatic hydrocarbon comprising 1 or 2 rings (i.e., C) having 3 to 12 carbon atoms or a specified number of carbon atoms _3-12 Cycloalkyl). In one embodiment, the cycloalkyl has two rings. In one embodiment, the cycloalkyl has one ring. In another embodiment, the cycloalkyl is selected from the group consisting of C _3-8 Cycloalkyl groups. In another embodiment, the cycloalkyl is selected from the group consisting of C _3-6 Cycloalkyl groups. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalinyl, adamantyl, cyclohexenyl, and cyclopentenyl.

The term "pharmaceutically acceptable salt" as used herein includes acid addition salts and base addition salts of the compounds.

Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, benzene carbonate, bicarbonate/carbonate, bisulfate/sulfate, borate, camphorsulfonate, citrate, cyclohexylsulfamate, ethanedisulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, oxybenzoate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphtholate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, aldonate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and cinxidate.

Suitable base addition salts are formed from bases that form non-toxic salts. Examples include aluminum salts, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts.

For a review of suitable salts, see Stahl and wermth, handbook of pharmaceutically acceptable salts: properties, selection and Use (Handbook of Pharmaceutical Salts: properties, selection, and Use) "(Wiley-VCH Co., wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

The term "solvate" as used herein is a substance, such as a di-, mono-, or hemi-solvate, formed by the combination, physical association, and/or solvation of a compound of the invention with a solvent molecule, wherein the ratio of solvent molecule to compound of the invention is about 2:1, about 1:1, or about 1:2, respectively. Such physical bonding involves ionic bonding and covalent bonding (including hydrogen bonding) to varying degrees. In some cases (e.g., when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid), the solvate may be isolated. Thus, solvates include both solution phases and separable solvates. The compounds of the present invention may be in solvated form with pharmaceutically acceptable solvents such as water, methanol and ethanol, and the present application is intended to cover both solvated and unsolvated forms of the compounds of the present invention.

One type of solvate is a hydrate. "hydrate" refers to a specific subset of solvates in which the solvent molecule is water. Solvates generally act as pharmacological equivalents. The preparation of solvates is known in the art, see for example M.caira et al, journal of pharmaceutical science (J.Pharmacet. Sci.), 93 (3): 601-611 (2004), which describes the preparation of solvates of fluconazole with ethyl acetate and water. Tonder et al, journal of pharmaceutical sciences and technology of the American pharmaceutical sciences (AAPS Pharm. Sci. Tech.), 5 (1): clause 12 (2004) and A.L. Bingham et al, chemical communications (chem. Commun.)) 603-604 (2001) describe similar methods for preparing solvates, hemi-solvates, hydrates, and the like. Representative and non-limiting methods for preparing solvates involve dissolving a compound of the invention in a desired solvent (organic solvent, water, or mixtures thereof) at a temperature of greater than 20 ℃ to about 25 ℃, then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by known methods such as filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of solvent in the solvate crystals.

In the context of the present invention, a "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered, and which is suitable for contacting human and/or other animal tissue within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol may also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The pharmaceutical composition may further contain a small amount of wetting agent, emulsifying agent or pH buffering agent, as required. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences) (1990).

The pharmaceutical compositions of the present invention may act systemically and/or locally. To this end, the pharmaceutical composition may be administered by a suitable route, for example by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular administration, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, topical administration, in the form of an ophthalmic formulation or by inhalation.

For these routes of administration, the pharmaceutical compositions of the invention may be administered in a suitable dosage form.

Dosage forms include, but are not limited to, tablets, capsules, troches, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

The term "effective amount" as used herein refers to the amount of active ingredient that will alleviate to some extent one or more symptoms of the condition being treated after administration.

As used herein, "individual" includes human or non-human animals. Exemplary human individuals include human individuals (referred to as patients) or normal individuals suffering from a disease (such as the diseases described herein). "non-human animals" in the present invention include all vertebrates such as non-mammals (e.g., birds, amphibians, reptiles) and mammals such as non-human primates, domestic animals and/or domestic animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

As used herein, "linking group" refers to any chemically suitable linking group. Preferably, under physiological conditions, the linking group does not cleave or only slowly cleaves.

As used herein, when a and B are connected by a linking group, and such linking group is a covalent bond or a single bond, it means that a and B are directly connected to each other, such as by a covalent bond or a single bond.

As used herein, "radionuclide" refers to a radioisotope of an element that emits alpha, beta, and/or gamma rays. Radionuclides include, but are not limited to, the following classes: ¹⁸ F、 ⁵¹ Cr、 ⁵⁵ Co、 ⁶⁷ Ga、 ⁶⁸ Ga、 ¹¹¹ In、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ¹³⁹ La、 ¹⁴⁰ La、 ¹⁷⁵ Yb、 ¹⁵³ Sm、 ¹⁶⁶ Ho、 ⁸⁶ Y、 ⁸⁸ Y、 ⁹⁰ Y、 ¹⁴⁹ Pm、 ¹⁴⁹ Tb、 ¹⁵² Tb、 ¹⁵⁵ Tb、 ¹⁶¹ Tb、 ¹⁶⁵ Dy、 ¹⁶⁹ Er、 ¹⁷⁷ Lu、 ⁴⁷ Sc、 ¹⁴² Pr、 ¹⁵⁹ Gd、 ²¹² Bi、 ²¹³ Bi、 ⁷² As、 ⁷² Se、 ⁹⁷ Ru、 ¹⁰⁹ Pd、 ¹⁰⁵ Rh、 ^101m Rh、 ¹¹⁹ Sb、 ¹²⁸ Ba、 ¹⁹⁷ Hg、 ¹⁵¹ Eu、 ¹⁵³ Eu、 ¹⁶⁹ Eu、 ²⁰¹ Tl、 ²⁰³ Pb、 ²¹² Pb、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁸⁸ Re、186Re、 ¹⁹⁸ Au、 ²²³ Ra、 ²²⁵ Ac、 ²²⁶ Th、 ²²⁷ Th、 ²³⁰ u and ¹⁹⁹ Ag。

as used herein, a radioisotope or radionuclide-labeled bioactive compound refers to a bioactive compound modified with a radioisotope or radionuclide.

As used herein, "Fibroblast Activation Protein (FAP) targeting structure/moiety" or "FAP binding structure/moiety" refers to a molecular fragment derived from a fibroblast activation protein inhibitor, such as a molecular fragment formed by a compound disclosed in WO 2019154886 A1.

The compounds of the present invention may contain one or more asymmetric centers, depending on the desired position and nature of the various substituents. The asymmetric carbon atoms may be present in the (R) or (S) configuration, giving a racemic mixture in the case of one asymmetric center and a diastereomeric mixture in the case of multiple asymmetric centers.

Isolated, purified or partially purified isomers and stereoisomers, or racemic or diastereomeric mixtures of the disclosed compounds are included within the scope of the invention. Purification and isolation of such materials may be accomplished by standard techniques known in the art.

In a further aspect, the trifunctional compound has formula (I):

In some embodiments, the FAPT moiety has formula (II),

wherein X is selected from the group consisting of: o, S, NR _x Wherein R is _x Is H or C ₁ -C ₆ An alkyl group;

one or more R _f The radicals being present in the pyrrolidine ring shown, where each R _f The radicals are independently selected from H, F, cl, -CN and C ₁ -C ₆ An alkyl group.

In some embodiments, FAPT portionsHaving the formula (II) _A ) Or formula (II) _B )，

Wherein X is selected from the group consisting of: o, S, NR _x Wherein R is _x Is H, methyl, ethyl, n-propyl or isopropyl.

In some embodiments, the C moiety is selected from the group consisting of:

in some embodiments, part a has formula (III _A ) Or formula (III) _B )：

Wherein R is ¹ And R is ² Independently selected from the group consisting of: H. c (C) ₁ -C ₆ Alkyl and C ₃ -C ₆ Cycloalkyl;

q and r are independently integers of 1 to 6.

In certain embodiments, part A has formula (IV),

wherein R is ¹ And R is ² Independently selected from the group consisting of: H. methyl and ethylThe base group of the modified polyester resin is a modified polyester resin,

q and r are independently 1 or 2.

In certain embodiments, part A has formula (V),

in some embodiments, the L moiety has formula (VI),

wherein R is ³ Selected from the group consisting of: H. methyl and ethyl, and preferably H;

L ₁ is a covalent bond, -CH ₂ -or-NHCH ₂ -；

Cy is selected from the group consisting of:

m and n are independently 1, 2, 3, 4, 5 or 6;

* Representing a location of a connection to the FAPT portion; and is also provided with

* Represents the position of attachment to the a part.

In certain embodiments, the L moiety has formula (VIIA) or formula (VIIB),

wherein m and n are independently 3, 4 or 5; and is also provided with

* And have the meaning as defined above.

In a further aspect, the present disclosure provides a trifunctional compound selected from the group consisting of:

/>

In another aspect, the present disclosure provides a method of ¹⁸ F-labelled trifunctional compounds, pharmaceutically acceptable salts, stereoisomers or solvates thereof, wherein said ¹⁸ The F-labelled trifunctional compound has one or more fluorine moieties in part a of the trifunctional compound as defined above ¹⁸ F alternative structure.

In a further aspect, the present disclosure provides a trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein the C moiety is chelated with a radionuclide or natural isotope thereof.

In some embodiments, the C moiety is sequestered with a radionuclide. In some embodiments, the C moiety is sequestered with a natural isotope of a radionuclide.

In certain embodiments, the radionuclide may emit beta particles or alpha particles. In certain embodiments, the radionuclide is selected from the group consisting of: ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁵⁵ Co、 ¹⁴⁹ Tb、 ¹⁵² Tb、 ¹⁵⁵ Tb、 ¹⁶¹ Tb、 ¹⁷⁷ Lu、 ²¹² Pb、 ²¹² Bi、 ²¹³ Bi、 ²²⁵ Ac、 ²²⁶ Th、 ²²⁷ Th、 ²²³ ra and ²³⁰ U。

in certain embodiments, the C moiety is chelated with a radionuclide selected from the group consisting of: ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁵⁵ Co、 ¹⁴⁹ Tb、 ¹⁵² Tb、 ¹⁵⁵ Tb、 ¹⁶¹ Tb、 ¹⁷⁷ Lu、 ²¹² Pb、 ²¹² Bi、 ²¹³ Bi、 ²²⁵ Ac、 ²²⁶ Th、 ²²⁷ Th、 ²²³ ra and ²³⁰ u or a natural isotope thereof.

In certain embodiments, the C moiety is chelated with a radionuclide selected from the group consisting of: ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁶⁴ Cu、 ⁶⁷ Cu、 ¹⁴⁹ Tb、 ¹⁵² Tb、 ¹⁵⁵ Tb、 ¹⁶¹ Tb、 ¹⁷⁷ Lu、 ²¹² Pb、 ²⁰³ Pb、 ²¹² Bi、 ²¹³ Bi、 ²²⁵ ac or a natural isotope thereof.

In certain embodiments, the C moiety is chelated with a radionuclide selected from the group consisting of: ⁶⁸ Ga、 ⁸⁶ Y、 ⁶⁴ Cu、 ¹⁶¹ Tb、 ¹⁷⁷ Lu、 ²¹² Pb、 ²¹² bi. 213Bi, 225Ac or natural isotopes thereof.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a trifunctional compound as defined above, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, and one or more pharmaceutically acceptable carriers.

In another aspect, the present disclosure provides a method of diagnosing or treating a disease comprising administering to a subject a therapeutically effective amount of a trifunctional compound as defined above, a pharmaceutically acceptable salt, stereoisomer, or solvate thereof. In some embodiments, the disease is cancer, infection, or inflammation.

In some embodiments, the cancer is selected from the group consisting of: prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocellular carcinoma, chordoma, small intestine cancer, pheochromocytoma, gastric cancer, renal cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymus cancer, cervical cancer, endometrial cancer, neuroendocrine tumor, thyroid cancer, intestinal cancer, and bone metastasis.

Examples

In order to make the objects and technical solutions of the present invention more clear, the present invention will be further described with reference to specific examples. It should be understood that the examples are not intended to limit the scope of the invention. Further, the specific experimental methods not mentioned in the following examples were all carried out according to conventional experimental methods.

I. Method of

Synthesis of Compounds and radiolabeling

Examples 1 to 5 describe the synthetic pathways and chemical characterizations of the trifunctional compounds of the invention. FAPI-04 was synthesized as a control based on previous studies (Lindner T, loktev A, altmann A et al, development of quinoline-based therapeutic diagnostic ligands for targeting fibroblast activation protein (Development of Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein); journal of Nuclear medicine 2018; 59:1415-1422).

18F was generated by a CYCIAE-14 proton cyclotron (China institute of atomic energy (CIAE, china)) (Jia X, guan F, yao H et al high intensity medical cyclotron ion source and injection line (Ion source and injection line for high intensity medical cyclotron); scientific instruments review (Rev Sci Instrom.))) 2014; 85:02C102). The 18F radiolabelling is by one step ¹⁸ F- ¹⁹ The F isotope exchange reaction proceeds. The labelling and purification procedure (Liu Z, pourghiasian M, radtke MA et al, a widely applicable one-step procedure) has been previously described ¹⁸ F-labeled organic trifluoroborates (An organotrifluoroborate for broadly applicable one-step ¹⁸ F-labling), "international english edition of applied chemistry (Angew Chem Int Ed engl.))," 2014;53:11876-11880; liu Z, lin KS, benard F et al, one step (18) F labelling of biomolecules with organic trifluoroborates (One-step (18) F labeling of biomolecules using organotrifluoroborates) & Nature laboratory guidelines (Nat Protoc.) & 2015; 10:1423-1432). Evaluation after incubation in saline or human serum using high performance liquid chromatography (radioactive HPLC) ¹⁸ Radiochemical purity and in vitro stability of F-FT-FAPI. Using ⁶⁸ Ge/ ⁶⁸ 0.6M HCl elution in Ga Generator (Alternba laboratory (Alternba LABS, south Africa)) ^68G aCl ₃ 。 ¹⁷⁷ LuCl ₃ Purchased from ITG company, germany (ITG, germany). Has already reported ⁶⁸ Ga and ¹⁷⁷ radiolabeling of Lu (Xu M, zhang P, ding J, chen J, huo L and Liu z. Albumin binding agent conjugated fibroblast activation protein inhibitor radiopharmaceuticals for cancer therapy 2021 journal of nuclear medicine). According to the developed scheme, from ²²⁵ Ac/ ²¹³ Elution in Bi generator ²¹³ Bi (Mongenston A, apostondis C and Bruchermetseifer F. Actinium-225and Bismuth-213) and clinical applications (Supply and Clinical Application of Actinium-225and Bismuth-213) 2020, seminal emission Med. Actinium nitrate-225 was purchased from Oak Ridge national laboratory (Oak Ridge National Laboratory) (Oak Ridge, TN, USA) in tennessee, USA.

Cell culture and assay

Human fibrosarcoma HT-1080 cell line and HT-1080 cell line transfected with human FAP (HT-1080-FAP, ming Kande Co., wuxi AptTec, china) in Eaglet minimum basal medium (Eagle' S minimum essential medium) containing 10% Fetal Bovine Serum (FBS), 1% antibiotic-antimycotic and 4. Mu.g/mL blasticidin S at 37℃in 5% CO ₂ Culturing in an incubator. For competition assays, one will ⁶⁸ Ga-FAPI-04 was added as a 0.75nM solution to HT-1080-FAP cells with a gradient concentration of non-radiolabeled compound (1.2X10 ⁶ Individual cells/wells, cultured in fresh medium without FBS). After incubation at 37℃for 1 hour, the cells were washed with ice-cold PBS and then lysed with 0.3M NaOH/0.2% sodium dodecyl sulfate. Cell-bound radioactivity was measured in a gamma counter. For saturation binding assays, varying concentrations of the radiotracer were added to HT-1080-FAP cells. After 1 hour incubation with fresh medium without FBS, cells were washed with ice-cold PBS and lysates were collected for counting using a gamma counter. For long term internalization assays, HT-1080-FAP cells are incubated with a radiotracer for 1, 6, 14 or 24 hours, respectively. Then pass through Media was removed and washed with PBS to terminate cell uptake. The cells were then incubated with glycine-HCl (1M, pH 2.2) for 10 minutes to remove cell surface binding activity. After removal of glycine-HCl and washing with PBS, cells were lysed and collected to determine the internalization fraction. For the outflow assay, the radiotracer was incubated with HT-1080-FAP cells for 60 minutes. The medium is then removed and the cells are washed and then incubated with fresh non-radioactive medium for 1, 6, 14 or 24 hours. Media and lysed cells were collected and counted.

Molecular docking

To understand the interaction between ligand and FAP, molecular docking was performed using MOE v 2020.0901. The 3D structure of FAP is obtained from the RCSB protein database (PDB ID:1Z 68). Before docking, an implicit solvation model of the force field and reaction field (R field) of the AMBER10 EHT is selected. Ser624 and cyano were chosen as reactive sites on which a molecular docking simulation of proteins with compounds was performed using Covalent-docking (covent-Dock).

Animal model

All mice were purchased from beijing velarihua laboratory animal technologies limited (Beijing Vital River Laboratory Animal Technology co., ltd). Animal experiments were performed according to the protocol approved by the ethical committee of Beijing university (Peking University) (CCME-LiuZB-2). For the cell line-derived xenograft model, 5X 10 was subcutaneously implanted on the right anterior side of 6 week old female nu/nu mice ⁶ HT-1080-FAP or HT-1080 cells. Mice were kept under temperature and humidity controlled conditions and the room was in a 12 hour light/dark cycle.

PET/CT imaging of small animals

All PET scans were in MedisoPET 122S small animal PET/CT imaging system. With isoflurane/O ₂ The mixture anesthetized mice (induction, 4%; maintenance, 2%). Static images were acquired at different time points after injection.

Biodistribution studies

Mice were injected intravenously with radiolabeled compound and then sacrificed at different time points after injection. Major organs and tissues were harvested and weighed. Radioactivity was measured for each sample using a gamma counter, and then the% ID/g was calculated for each sample.

Radionuclide therapy and toxicity

HT-1080-FAP tumor-bearing mice were randomly divided into 5 groups (n=6/group) on day 0, and were injected intravenously with saline, 0.74MBq on days 1, 3, 5, 7, 9 and 11, respectively ²¹³ Bi-FT-FAPI、2.22MBq ²¹³ Bi-FT-FAPI、3.7MBq ²¹³ Bi-FT-FAPI and 2.22MBq ²¹³ Bi-FAPI-04. Tumor volumes and body weights were monitored for all mice every two days. For toxicity studies, saline, 2.22MBq, was injected on days 1, 3, 5, 7, 9 and 11, respectively, into 3 groups of HT-1080-FAP tumor bearing mice ²¹³ Bi-FT-FAPI and 3.7MBq ²¹³ Bi-FT-FAPI (n=3/group). Eight weeks later, mice were sacrificed and whole blood cell counts (CBC) and blood biochemical tests were performed on the mice.

Statistical analysis

All data are expressed as mean ± SD. Significance was determined by T-test (Student T test) and ANOVA using GraphPad Prism 9 and OriginPro 2021. Statistical significance was considered p <0.05.

II Synthesis

AMBF ₃ As a means of ¹⁸ The efficient radiosynthon of F has been described in detail in the prior art (Liu Z, pourghiasian M, radtke MA et al, a widely applicable one-step) ¹⁸ F-labeled organic trifluoroborates 2014 from international english edition of applied chemistry; 53:11876-11880). In the present invention, the trifunctional compound is obtained by reacting AMBF ₃ Moieties, FAP targeting moieties, and chelating moieties that can coordinate radiometals into a single molecule.

Comparative example: AMBF ₃ Synthesis of FAPI-04

AMBF ₃ FAPI-04 can be synthesized according to the following synthesis scheme:

for preparing AMBF ₃ General procedure for FAPI-1

To a solution of FT-fapi_c8 (260 mg,534.42umol,1 eq) in DMF (3 mL) at 0 ℃ was added 2-bromo-1-ethyl-pyridin-1-ium; tetrafluoroborate (219.53 mg, 801.62. Mu. Mol,1.5 eq), DIEA (207.21 mg,1.60mmol, 279.26. Mu.L, 3 eq) and 2-bromoacetic acid (185.64 mg,1.34mmol, 96.19. Mu.L, 2.5 eq). The mixture was stirred at 25℃for 2 hours. LC-MS shows that FT-fapi_c8 is completely consumed and the desired MS is detected. The residue was purified by preparative HPLC (neutral conditions) to give AMBF as a yellow solid ₃ FAPI-1 (200 mg,329.25umol,61.61% yield). LCMS [ M-H ]] ^- ＝609

For preparing AMBF ₃ General procedure for FAPI-2

To a solution of BF3-FAPI-1 (200 mg,329.25umol,1 eq.) in MeCN (3 mL) at 0deg.C was added NaN ₃ (32.11 mg,493.87umol,1.5 eq.) and KI (5.47 mg,32.92umol,0.1 eq.). The mixture was stirred at 25℃for 2 hours. LC-MS shows AMBF ₃ FAPI-1 is completely consumed and the desired MS is detected. The reaction mixture was quenched by addition of 50mL of water at 25 ℃ and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (TFA conditions) to give BF as a yellow solid ₃ FAPI-2 (80 mg,140.46umol,42.66% yield). LCMS [ M-H ]] ^- ＝570.2

For preparing AMBF ₃ General procedure for FAPI-04

To AMBF ₃ To a solution of FAPI-2 (70 mg,122.90umol,1 eq.) in water (4 mL) and MeCN (6 mL) was added (2R) -2- [ (2R) -3, 4-dihydroxy-5-oxo-2H-furan-2-yl]Sodium 2-hydroxy-ethoxide (1M, 761.99uL,6.2 eq.) and CuSO ₄ (1M, 380.99uL,3.1 eq.). Then [ dimethyl (prop-2-ynyl) ammonium]Methyl-trifluoro-boron anion (60.82 mg,368.70umol,3 eq.) was added to the above solution. Then immediately use K ₂ CO ₃ (28.71 mg,207.70umol,1.69 eq.) the mixture was adjusted to ph=7. The solution was then stirred at 45℃for 12 hours. The mixture was purified by preparative HPLC (TFA conditions) to give AMBF as a pale yellow solid ₃ FAPI-04 (28.2 mg, 37.62. Mu. Mol,30.61% yield, 98% purity). LCMS [ M-H ]] ^- ＝735.3； ¹ H NMR(400MHz,CD ₃ CN):δ＝8.08-8.26(m,3H),7.57-8.06(m,3H),5.05-5.47(m,2H),5.03-5.04(m,1H),4.02-4.50(m,9H),3.31-3.35(m,3H),3.06-3.15(m,5H),3.29(s,6H),2.78-2.86(m,4H),1.94-2.35(m,4H)。

Example 1: synthesis of FT-FAPI-02

FT-FAPI-02 can be synthesized according to synthesis schemes 1 to 4.

Synthesis scheme 1:

1.1 general procedure for the preparation of FT-FAPI_E2

2S) -pyrrolidine-2-carbonitrile; hydrochloride (1 g,7.54mmol,1 eq.) was added to a stirred mixture of FT-fapi_e1 (1.32 g,7.54mmol,1 eq.) of bicharmatech (China), HBTU (3.58 g,9.43mmol,1.25 eq.) of mink, minkand, china, HOBt (1.32 g,9.80mmol,1.3 eq.) of DIEA (4.87 g,37.71mmol,6.57mL,5 eq.) of DIEA (mink, china) in DMF (5 mL) and the mixture was stirred at 15 ℃ for 12 hours. The reaction mixture was purified by adding 5mL of H ₂ O quench, and then dilute with EtOAc and extract with EtOAc. The organic layer was washed with saturated aqueous NaCl solution, and then dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=1/0 to 3/1). Compound FT-FAPI_E2 (1.9 g, crude) was obtained as a yellow oil.

1.2 general procedure for the preparation of FT-FAPI_E3

FT-FAPI_E2 (1.9 g,7.50mmol,1 eq.) was added to ACN (1)0 mL) and HCl/dioxane (20 mL) were stirred at 20 ℃ for 12 hours. LC-MS shows that fapi_e2 is completely consumed and detected to the desired quality. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=10/1 to 1:1). Compound FT-FAPI_E3 (1.3 g,6.86mmol,91.39% yield, HCl) was obtained as a yellow solid.

Synthesis scheme 2:

1.3 general procedure for preparation of Compound 2

Compound 1 (20 g,112.89mmol,1 eq.) was purified at 25℃on H (Ming Kad Corp. Of tin-free medicine, china) ₂ KOH (150.00 g,2.67mol,23.68 eq.) and 2-oxopropionic acid (17.90 g,203.21mmol,14.32mL,1.8 eq.) were added to a solution in O (30 mL). The mixture was stirred at 40℃for 16 hours. After cooling to room temperature, 10% aqueous sulfuric acid was added until ph=1. The precipitate was isolated by filtration and dried in vacuo to give the desired compound. Compound 2 (27 g,109.22mmol,96.75% yield) was obtained as a brown solid.

1.4 general procedure for preparation of Compound 3

To H ₂ SO ₄ To a solution of (2 mL) in nitrobenzene (150 mL) was added compound 2 (10 g,40.45mmol,1 eq.) and the mixture was stirred at 200deg.C for 3.5 hours. After slowly cooling the mixture, the resulting precipitate was washed with petroleum ether (100 mL) and filtered to give white crystals, and compound 3 (5.6 g,27.56mmol,68.13% yield) was obtained as a yellow solid.

1.5 general procedure for preparation of Compound 4

For five batches: to compound 3 (440 mg,2.17mmol,1 eq.) in H ₂ HBr (1.83 g,10.83mmol,1.22mL,48% purity, 5 eq.) was added to a solution in O (2 mL) (Aladin, china) of Aladin, china. The mixture was stirred at 130℃for 12 hours. Concentrating the mixture under reduced pressure, and obtaining a product in the form of a solidCompound 4 (2.05 g, crude) as a pink solid.

1.6 general procedure for preparation of Compound 5

To a solution of compound 4 (2.8 g,14.80mmol,1 eq.) in DMF (50 mL) was added K ₂ CO ₃ (6.14 g,44.41mmol,3 eq.) and 1-bromo-3-chloro (Ming Kangde, tin-free medicine, china) propane (4.66 g,29.60mmol,2.91mL,2 eq.) of Ming Kangde, tin-free medicine, china. The mixture was stirred at 20℃for 12 hours. The reaction mixture was purified by adding H ₂ O was quenched and then diluted with EtOAc and extracted with EtOAc and DCM. The combined organic layers were washed with saturated NaCl solution and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=10/1 to 2/1). Compound 5 (2 g,7.53mmol,50.86% yield) was obtained as a yellow oil.

1.7 general procedure for preparation of Compound 6

To a solution of compound 5 (3.5 g,13.17mmol,1 eq.) in DMF (40 mL) was added K ₂ CO ₃ (2.73 g,19.76mmol,1.5 eq.) and 1-bromo-3-chloro-propane (3.11 g,19.76mmol,1.94mL,1.5 eq.). The mixture was stirred at 60℃for 12 hours. The reaction mixture was purified by adding H ₂ O quench, and then dilute with EtOAc and extract with EtOAc. The combined organic layers were washed with saturated NaCl (60 mL) solution and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. Compound 6 (4.5 g, crude) was obtained as a yellow oil.

1.8 general procedure for the preparation of FT-FAPI_C5

To compound 6 (4.5 g,13.15mmol,1 eq.) in THF (50 mL) and H ₂ To a solution in O (20 mL) was added LiOH (944.73 mg,39.45mmol,3 eq). The mixture was stirred at 20℃for 12 hours. The mixture was concentrated under reduced pressure. The residue was subjected to HPLC (TFA conditions; column Phenomenex luna C (250X 70mm,15 μm); mobile phase: [ water (TFA) -ACN) ]The method comprises the steps of carrying out a first treatment on the surface of the B%:8% -33%,32 min). Compound FAPI-C5 (3.3 g,12.42mmol, 94) was obtained as a yellow solid45% yield).

1.9 general procedure for the preparation of FT-FAPI_C6

To a solution of compound FT-fapi_c5 (1 g,3.76mmol,1 eq.) in NMP (15 mL) was added KI (312.40 mg,1.88mmol,0.5 eq.) (minkand, no tin medicine, china) and tert-butyl piperazine-1-carboxylate (3.51 g,18.82mmol,5 eq.). The mixture was stirred at 60℃for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column Welch Ultimate AQ-C18 150mm 30mm 5 μm; mobile phase: [ water (TFA) -ACN ]; B%:2% -30%,20 min). Compound FT-FAPI-C6 (1.3 g,3.13mmol,83.13% yield) was obtained as a yellow solid.

Synthesis scheme 3:

1.10 general procedure for preparation of FT-FAPI_D2

To a stirred mixture of FT-FAPI_D1 (2 g,3.49mmol,1 eq) (AikonChem, china), HBTU (1.66 g,4.37mmol,1.25 eq.), HOBt (613.41 mg,4.54mmol,1.3 eq.) and DIEA (2.26 g,17.46mmol,3.04mL,5 eq.) in ACN (30 mL) was added 1-hydroxypyrrolidine-2, 5-dione (401.89 mg,3.49mmol,1 eq.) and the reaction mixture was stirred at 20℃for 12 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (SiO ₂ Ethyl acetate can=10:1, r _f =0.45) purification. Compound FT-FAPI_D2 (1.5 g,2.24mmol,64.13% yield) was obtained as a yellow solid.

1.11 general procedure for the preparation of FT-FAPI_D3

To a solution of FT-FAPI_D2 (1 g,1.49mmol,1 eq.) in DCM (10 mL) was added TFA (8.51 g,74.65mmol,5.53mL,50 eq.). The mixture was stirred at 35 ℃ for 2 hours. The mixture was concentrated under reduced pressure. The residue was diluted with EtOAc at 35 ℃ and MTBE was then added. The solid was filtered and washed with MTBE, then dried under high vacuum. Compound FT-FAPI_D3 (600 mg,1.20mmol,80.14% yield) was obtained as a white solid.

Synthesis scheme 4:

1.12 general procedure for preparation of FT-FAPI_E4

To a solution of FT-FAPI_C6 (1.8 g,4.33mmol,1 eq.) in DMF (20 mL) was added FT-FAPI_E3 (663.63 mg,4.33mmol,1 eq.), HBTU (2.05 g,5.42mmol,1.25 eq.) and HOBt (761.00 mg,5.63mmol,1.3 eq.), DIEA (2.80 g,21.66mmol,3.77mL,5 eq.). The mixture was stirred at 25℃for 16 hours. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column Phenomenex luna C100 mm. Times.40 mm. Times.5 μm; mobile phase: [ water (TFA) -ACN) ]The method comprises the steps of carrying out a first treatment on the surface of the B%:5% -35%,8 min). Compound FAPI_E4 (550 mg, 998.82. Mu. Mol,23.06% yield) was obtained as a brown oil. ¹ H NMR(400MHz,DMSO-d6)δ＝9.20-9.06(m,1H),8.90(br d,J＝4.1Hz,1H),8.14-7.94(m,2H),7.65-7.46(m,2H),3.63-3.30(m,6H),3.22-3.08(m,1H),2.42-2.03(m,6H),1.67-1.25(m,3H)。

1.13 general procedure for the preparation of FT-FAPI_E5

To a solution of FT-FAPI_E4 (550 mg, 998.82. Mu. Mol,1 eq.) was added HCl/EtOAc (4M, 8.25mL,33.04 eq.). The mixture was stirred at 25℃for 2 hours. The solid was filtered and washed with EtOAc, then dried under high vacuum. Compound FT-FAPI_E5 (350 mg, crude, HCl) was obtained as a yellow oil.

1.14 general procedure for preparation of FT-FAPI_E6

To a solution of FT-FAPI_E5 (350 mg, 776.86. Mu. Mol,1 eq.) and FT-FAPI_J7 (509.98 mg, 776.86. Mu. Mol,1 eq.) in DMF (3 mL) was added DIEA (301.21 mg,2.33mmol, 405.94. Mu.L, 3 eq.). The mixture was stirred at 20℃for 2 hours. DMF (3 mL) containing compound FT-fapi_e6 (700 mg, crude) was a yellow liquid, which was used in the next step without further purification.

1.15 general procedure for preparation of FT-FAPI_E7

To a solution of FT-FAPI_E6 (700 mg, 705.71. Mu. Mol,1 eq.) in DMF (1 mL) was added piperidine (180.27 mg,2.12mmol, 209.08. Mu.L, 3 eq.) (Minkand, tin-free medicine, china). The mixture was stirred at 20℃for 2 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column Phenomenex Luna C, 200mm x 40mm x 10 μm; mobile phase: [ water (FA) -ACN ];: B%:1% -50%,8 min). Compound FT-FAPI_E7 (100 mg, 129.93. Mu. Mol,18.41% yield) was obtained as a white solid.

1.16 general procedure for the preparation of FT-FAPI-02

To a solution of FT-FAPI_E7 (100 mg, 129.93. Mu. Mol,1 eq.) in DMF (1.5 mL) was added FT-FAPI_D3 (97.73 mg, 194.89. Mu. Mol,1.5 eq.) and DIEA (167.92 mg,1.30mmol, 226.31. Mu.L, 10 eq.). The mixture was stirred at 20℃for 2 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column Phenomenex Luna C75 mm. Times.30 mm. Times.3 μm; mobile phase: [ water (FA) -ACN)]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -25%,8 min). Compound FT-FAPI-02 (25.74 mg, 22.27. Mu. Mol,17.14% yield) was obtained as a white solid. ¹ H NMR(400MHz,DMSO-d ₆ )δ＝9.04(br t,J＝4.7Hz,1H),8.79(br d,J＝4.1Hz,1H),8.41(s,1H),8.27(br d,J＝7.9Hz,1H),8.15(s,1H),7.97(br d,J＝9.3Hz,1H),7.89(br s,1H),7.54-7.41(m,2H),4.80(br dd,J＝3.3,7.7Hz,1H),4.73-4.60(m,1H),4.51(s,2H),4.41(br t,J＝6.4Hz,3H),4.20(br d,J＝5.3Hz,5H),3.11-2.85(m,22H),2.81-2.63(m,6H),2.42-2.30(m,6H),2.29-2.15(m,5H),2.13-1.92(m,6H),1.88-1.75(m,3H),1.67-1.51(m,3H),1.35-1.16(m,3H)。

Example 2: synthesis of FT-FAPI

FT-FAPI can be synthesized according to synthesis schemes 5 and 6.

Synthesis scheme 5:

2.1 general procedure for preparation of FT-FAPI_J2

For two batches: to a solution of N, N-dimethylpropan-2-yn-1-amine (7.05 g,84.84mmol,9.00mL,1 eq) (China's safety Chemical, china)) in MTBE (1800 mL) was added FT-fapi_j1 (25 g,93.32mmol,1.1 eq) (China's safety Zesheng, china) the mixture was stirred at 25 ℃ for 1.5 hours, the mixture was filtered to give a residue, the compound FT-fapi_j2 (19 g,84.77mmol,99.93% yield) was obtained as a white solid, 1H NMR (400 MHz, chloroform-d) delta ppm 1.33 (s, 12H) 2.92 (br s, 1H) 3.60-3.64 (m, 8H) 4.90 (d, j=2.38 hz, 2H).

2.2 general procedure for preparation of FT-FAPI_J3

To a solution of FT-FAPI_J2 (10 g,44.62mmol,1 eq.) in ACN (100 mL) was added HCl (4M, 60.01mL,5.38 eq.) and KHF ₂ (3M, 59.94mL,4.03 eq.) of Ming Kangde, tin-free medicine, china. The mixture was stirred at 60℃for 1 hour. The mixture was adjusted to ph=7. The reaction mixture was taken up in DCM (300 mL) with H ₂ O (100 mL) between partitions. The organic phase was separated over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=10/1 to 0/1). Compound FT-FAPI_J3 (4 g,24.25mmol,57.14% yield) was obtained as a yellow solid.

2.3 general procedure for preparation of FT-FAPI_J5

To NaN ₃ (19.02 g,292.57mmol,1 eq.) of Ming Kangde, a tin-free medicine of China at H ₂ To a solution of O (200 mL) and DCM (200 mL) was added Tf ₂ O (16.51 g,58.51mmol,9.65mL,0.2 eq). The mixture was stirred at 20℃for 2 hours. The mixture was removed and treated with CH ₂ Cl ₂ The aqueous phase was extracted. The organic fraction containing trifluoromethanesulfonyl azide was treated with 5% NaHCO ₃ Aqueous (100 mL) wash over Na ₂ SO ₄ Drying and use without further purification. To FT-FAPI_J4 (5 g,13.57mmol,1 eq.) in H ₂ NaHCO was added to a solution of O (4 mL) and MeOH (8 mL) ₃ (11.40 g,135.71mmol,5.28mL,10 eq.) and CuSO ₄ ·5H ₂ O (33.89 mg, 135.71. Mu. Mol,0.01 eq) (China)Tin-free Ming Kangde Co.) and DCM (10 mL) containing N-diazonium-1, 1-trifluoro-methanesulfonamide (2.38 g,13.57mmol,1 eq.) were added. The mixture was stirred at 20℃for 16 hours. LC-MS shows that FT-fapi_j4 is completely consumed and the desired quality is detected. The organic solvent was removed under high vacuum and the remaining solution was acidified at ph=2 by addition of aqueous HCl. The mixture was concentrated under reduced pressure to remove MeOH. The mixture was then extracted with EtOAc, the organic fractions were combined and washed with saturated aqueous NaCl solution, then Na ₂ SO ₄ Dried, filtered and the filtrate concentrated under high vacuum. Compound FT-FAPI_J5 (5 g, crude) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝394.0。

2.4 general procedure for preparation of FT-FAPI_J6

To FT-FAPI-J5 (5 g,12.68mmol,1 eq.) in H ₂ Sodium was added to a solution in O (50 mL) and ACN (50 mL); (2R) -2- [ (2R) -3, 4-dihydroxy-5-oxo-2H-furan-2-yl]-2-hydroxy-glycolate (15.57 g,78.60mmol,6.2 eq.) CuSO ₄ (6.27 g,39.30mmol,6.03mL,3.1 eq.) then FT-FAPI_J3 (4 g,24.25mmol,1.91 eq.) was added to the solution. Then immediately use K ₂ CO ₃ (2.98 g,21.55mmol,1.7 eq.) the mixture was adjusted to ph=7. The solution was then stirred at 45℃for 12 hours. LC-MS shows that FT-fapi_j5 is completely consumed and the desired quality is detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (basic conditions, column: xtimate C18 x 80mm; mobile phase: [ water (NH ₄ HCO ₃ )-ACN]The method comprises the steps of carrying out a first treatment on the surface of the B%:5% -35%,8 min). Compound FT-FAPI_J6 (6 g, crude) was obtained as a blue solid. LCMS: ms-F ^- ＝540.3。

2.5 general procedure for preparation of FT-FAPI_J7

To a solution of FT-FAPI_J6 (2 g,3.58mmol,1 eq.) in DCM (10 mL) and ACN (10 mL) was added DIEA (924.18 mg,7.15mmol,1.25mL,2 eq.) followed by [ dimethylamino- (2, 5-dioxopyrrolidin-1-yl) oxy-methylene]-dimethyl-ammonium; tetrafluoroborate (2.15 g,7.15mmol,2 eq.) was added to the solution. The mixture is put inStirring is carried out at 20℃for 12 hours. LC-MS shows that FT-fapi_j6 is completely consumed and the desired quality is detected. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO ₂ Dichloromethane: acetonitrile=1/0 to 1/1). Compound FT-FAPI_J7 (1.2 g,1.83mmol,51.13% yield) was obtained as a white solid. LCMS: ms-F ^- ＝637.3。

Synthesis scheme 6:

2.6 general procedure for preparation of FT-FAPI_1

To a solution of FT-FAPI_J7 (190 mg, 289.43. Mu. Mol,1 eq.) in DMF (3 mL) was added FT-FAPI-C8 (98.57 mg, 202.60. Mu. Mol,0.7 eq.) and DIEA (112.22 mg, 868.30. Mu. Mol, 151.24. Mu.L, 3 eq.). The mixture was stirred at 20℃for 2 hours. LC-MS showed that FT-fapi_j7 was completely consumed and no major peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. Compound FT-FAPI_1 (260 mg, crude) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝1028.6。

2.7 general procedure for preparation of FT-FAPI_2

To a solution of FT-FAPI_1 (260 mg, 252.95. Mu. Mol,1 eq.) in DMF (2 mL) was added piperidine (64.61 mg, 758.84. Mu. Mol, 74.94. Mu.L, 3 eq.). The mixture was stirred at 20℃for 2 hours. LC-MS showed that FT-fapi_1 was completely consumed and one main peak with the desired m/z was detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column Phenomenex Luna C75 mm. Times.30 mm. Times.3 μm; mobile phase: [ water (FA) -ACN)]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -30%,8 min). Compound FT-FAPI_2 (50 mg, 62.06. Mu. Mol,24.54% yield) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝806.5。

2.8 general procedure for preparation of FT-FAPI

To a solution of FT-FAPI_2 (50 mg, 62.06. Mu. Mol,1 eq.) in DMF (1 mL) was added DIEA (80.21 mg, 620.62. Mu. Mol, 108.10. Mu.L, 10 eq.) and FT-FAPI_D3 (31.12 mg, 62.06. Mu. Mol,1 eq.). Will be mixedThe mixture was stirred at 20℃for 2 hours. LC-MS shows that FT-fapi_2 is completely consumed and a main peak with the desired mass is detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (FA conditions: column Phenomenex Luna C200 mm. Times.40 mm. Times.10 μm; mobile phase: [ water (FA) -ACN)]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -20%,8 min). Compound FT-FAPI (17 mg, 14.26. Mu. Mol,22.98% yield) was obtained as a white solid. LCMS Ms/2=596.2; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝9.13(br t,J＝5.9Hz,1H),8.81(d,J＝4.4Hz,1H),8.42(s,1H),8.30(br d,J＝7.5Hz,1H),8.19(s,1H),7.99(d,J＝9.1Hz,1H),7.87(d,J＝2.6Hz,1H),7.51(d,J＝4.4Hz,1H),7.47(dd,J＝2.7,9.2Hz,1H),5.15(dd,J＝2.5,9.3Hz,1H),4.67(br d,J＝5.8Hz,1H),4.52(s,2H),4.47-4.28(m,6H),4.26-4.10(m,8H),3.55-3.41(m,8H),3.22(br s,4H)。

three isomers of the compound FT-FAPI were obtained similarly:

example 3: synthesis of FT-FAPI-21 may be synthesized according to FIG. 7. Synthesis scheme 7:

3.1 general procedure for the preparation of FT-FAPI-21_1

To a solution of FT-FAPI_C5 (2 g,7.53mmol,1 eq.) in NMP (20 mL) was added KI (624.79 mg,3.76mmol,0.5 eq.) and (1S, 4S) -2, 5-diazabicyclo [ 2.2.1)]Heptane-2-carboxylic acid tert-butyl ester (7.46 g,37.64mmol,5 eq.) was obtained from Ming Kangde, tin-free medicine, china. The mixture was stirred at 60℃for 12 hours. LCMS showed that the desired m/z was detected. The residue was purified by preparative HPLC (column: welch Xtime C18 mm. Times.70 mm. Times.10 μm; mobile phase: [ water (FA) -ACN) ]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -25%,20 min). Compound FT-FAPI-21_1 (1.2 g,2.81mmol,37.29% yield) was obtained as a yellow solid. LCMS: ms+H ⁺ ＝428.2。

3.2 general procedure for the preparation of FT-FAPI-21_2

To a solution of FT-FAPI-21_1 (0.9 g,2.11mmol,1 eq.), FT-FAPI_B6 (522.51 mg,2.32mmol,1.10 eq., HCl), DIEA (816.27 mg,6.32mmol,1.10mL,3 eq.), HOBt (568.94 mg,4.21mmol,2 eq.) in DMF (10 mL) was added HBTU (1.60 g,4.21mmol,2 eq.). The mixture was stirred at 20℃for 12 hours. LC-MS showed that FT-FAPI-21_1 was completely consumed and one main peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column Phenomenex luna C100 mm. Times.40 mm. Times.5 μm; mobile phase: [ water (TFA) -ACN)]The method comprises the steps of carrying out a first treatment on the surface of the B%:5% -50%,8 min). Compound FT-FAPI-21_2 (1.1 g,1.84mmol,87.28% yield) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝599.3。

3.3 general procedure for the preparation of FT-FAPI-21_3

A mixture of FT-FAPI-21_2 (1 g,1.67mmol,1 eq.) in HCl/EtOAc (4M, 1.67mL,4 eq.) was stirred at 20deg.C for 0.5 h. LC-MS showed that FT-FAPI-21_2 was completely consumed and one main peak with the expected m/z was detected. The solid was filtered, washed with EtOAc, and dried under high vacuum. Compound FT-FAPI-21_3 (700 mg, crude) was obtained as a white solid. LCMS: ms+H ⁺ ＝499.3。

3.4 general procedure for the preparation of FT-FAPI-21_4 (1)

To FT-FAPI-21_3 (700 mg,1.40mmol,1 eq.), [ [1- [ (5R) -6- (2, 5-dioxopyrrolidin-1-yl) oxy-5- (9H-fluoren-9-ylmethoxycarbonylamino) -6-oxo-hexyl]Triazol-4-yl]Methyl-dimethyl-ammonium]To a solution of methyl-trifluoro-boron anion (921.76 mg,1.40mmol,1 eq) (Ming Kangde, no. tin, china) in DMF (5 mL) was added DIEA (544.43 mg,4.21mmol, 733.73. Mu.L, 3 eq). The mixture was stirred at 20℃for 2 hours. LC-MS showed that FT-FAPI-21_3 was completely consumed and one main peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. Compound FT-FAPI-21_4 (1) (1.4 g, crude) was obtained as a white solid. LCMS: ms+H ⁺ ＝1040.5。

3.5 general procedure for the preparation of FT-FAPI-21_4 (2)

To a solution of FT-FAPI-21_4 (1) (1.3 g,1.25mmol,1 eq.) in DMF (2 mL) was added piperidine (212.89 mg,2.50mmol, 246.92. Mu.L, 2 eq.). The mixture was stirred at 20℃for 1 hour. LC-MS showed that FT-FAPI-21_4 (1) was completely consumed and one main peak with the desired m/z or the desired mass was detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column Phenomenex Luna C, 150 mm. Times.30 mm. Times.5 μm; mobile phase: [ water (FA) -ACN) ]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -30%,8 min). Compound FT-FAPI-21_4 (2) (500 mg, 611.50. Mu. Mol,48.92% yield) was obtained as a white solid. LCMS: ms+H ⁺ ＝818.4。

3.6 general procedure for the preparation of FT-FAPI-21

To a solution of FT-FAPI-21_4 (2) (200 mg, 244.60. Mu. Mol,1 eq.) in DMF (3 mL) was added DIEA (158.06 mg,1.22mmol, 213.03. Mu.L, 5 eq.) and FT-FAPI_D3 (122.66 mg, 244.60. Mu. Mol,1 eq.). The mixture was stirred at 20℃for 12 hours. LC-MS showed that FT-FAPI-21_4 (2) was completely consumed and detected to the desired quality. The mixture was concentrated under reduced pressure. The residue was subjected to preparative HPLC (FA conditions; column Phenomenex Luna C200 mm x 40mm x 10 μm; mobile phase: [ water (FA) -ACN]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -20%,8 min). Compound FT-FAPI-21 (115 mg, 95.51. Mu. Mol,23.00% yield, 100% purity) was obtained as a white solid. LCMS Ms/2= 602.7; ¹ H NMR(400MHz,DMSO-d6)δ＝9.25(br t,J＝5.6Hz,1H),9.08(br t,J＝5.9Hz,1H),8.79(d,J＝4.3Hz,1H),8.44-8.27(m,2H),7.96(d,J＝9.1Hz,1H),7.89-7.74(m,1H),7.54-7.40(m,2H),5.21-5.10(m,1H),4.55-4.07(m,16H),

3.56-3.30(m,14H),3.07-2.76(m,24H),2.24(br d,J＝4.1Hz,2H),1.96-1.74(m,5H),1.68-1.47(m,3H),1.30-1.11(m,2H)。

example 4: synthesis of FT-FAPI-46

FT-FAPI-46 can be synthesized according to synthesis schemes 8 and 9.

Synthesis scheme 8:

4.1 general procedure for the preparation of FT-FAPI-12_2

To a solution of FT-FAPI-12_1 (5 g,20.46mmol,1 eq.) in DCM (50 mL) was added TEA (4.14 g,40.93mmol,5.70mL,2 eq.). MsCl (5.690 g,49.67mmol,3.84mL,2.43 eq.) will then be added thereto at 0deg.C. The mixture was stirred at 20℃for 1 hour. LC-MS showed that FT-FAPI-12_1 was completely consumed and one main peak with the desired mass was detected. The reaction mixture was treated with H ₂ O was diluted and extracted with DCM. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. Crude compound FT-FAPI-12_2 (6 g, crude) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝323.2。

4.2 general procedure for the preparation of FT-FAPI-12_3

To a solution of FT-FAPI-12_2 (6 g,18.61mmol,1 eq.) in EtOH (70 mL) was added MeNH ₂ (28.90 g,279.14mmol,30% purity, 15 eq.). The mixture was stirred at 70℃for 16 hours. LC-MS showed that FT-FAPI-12_2 was completely consumed and one main peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (TFA conditions; column Agela DuraShell C, 250 mm. Times.70 mm. Times.10 μm; mobile phase: [ water (TFA) -ACN]The method comprises the steps of carrying out a first treatment on the surface of the B%:0% -25%,20 min). Crude compound FT-FAPI-12_3 (4 g, crude) was obtained as colorless oil. LCMS: ms+H ⁺ ＝258.2。

Synthesis scheme 9:

4.3 general procedure for the preparation of FT-FAPI-12_5

To a solution of compound 7 (5 g,19.84mmol,1 eq) (Ming Kangde, no. Hemsl., china) in DMSO (60 mL) was added Cs ₂ CO ₃ (14.22 g,43.64mmol,2.2 eq.) and CH ₃ I (14.08 g,99.18mmol,6.17mL,5 eq.) of Ming Kangde, tin-free medicine, china. The mixture is heated to 20 DEG C Stirred for 1 hour. LC-MS showed that compound 1 was completely consumed and one main peak with the desired m/z was detected. The residue was extracted with EtOAc. The combined organic layers were treated with H ₂ Washing with Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=5/1 to 0/1). Compound FT-FAPI-12_5 (4.5 g,16.91mmol,85.26% yield) was obtained as a yellow solid. LCMS: ms+H ⁺ ＝266.0。

4.4 general procedure for the preparation of FT-FAPI-12_6

At N ₂ FT-FAPI-12_3 (232.14 mg,901.95umol,1.2 eq.) Pd was added to a solution of FT-FAPI-12_5 (200 mg, 751.62. Mu. Mol,1 eq.) in 1, 4-dioxane (2 mL) ₂ (dba) ₃ (13.77 mg, 15.03. Mu. Mol,0.02 eq.) Cs (Ming Kangde Co., ltd., tin-free drug in China) ₂ CO ₃ (367.34 mg,1.13mmol,1.5 eq.) and XPhos (14.33 mg, 30.06. Mu. Mol,0.04 eq.). The mixture was stirred at 100℃for 12 hours. LC-MS showed that FT-FAPI-12_5 was completely consumed and one main peak with the expected m/z was detected. The reaction mixture was concentrated under reduced pressure to remove 1, 4-dioxane. The residue was purified by column chromatography (SiO ₂ Petroleum ether/ethyl acetate=100/1 to 1:1). Compound FT-FAPI-12_6 (1 g,2.26mmol,30.06% yield) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝443.2。

4.5 general procedure for the preparation of FT-FAPI-12_7

To a solution of FT-FAPI-12_6 (1 g,2.26mmol,1 eq.) in THF (20 mL) was added LiOH (2M, 4.52mL,4 eq.). The mixture was stirred at 20℃for 16 hours. LC-MS showed that FT-FAPI-12_6 was completely consumed and one main peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. Hydrochloric acid (1N) was added to the mixture to adjust ph=7. The mixture was concentrated under reduced pressure. Crude compound FT-FAPI-12_7 (0.9 g, crude) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝429.3。

4.6 general procedure for the preparation of FT-FAPI-12_8

To a solution of FT-FAPI-12_7 (500 mg,1.17mmol,1 eq.) in DMF (2 mL) was added DIEA (753.98 mg,5.83mmol,1.02mL,5 eq.), HOBt (315.32 mg,2.33mmol,2 eq.) and HBTU (884.99 mg,2.33mmol,2 eq.). FT-FAPI-B6 (394.89 mg,1.75mmol,1.5 eq., HCl) was then added. The mixture was stirred at 20℃for 12 hours. LC-MS showed that FT-FAPI-12_7 was completely consumed and one peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: phenomnex Luna 80mm 30mm 3 μm mobile phase: [ water (TFA) -ACN ]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -40%,8 min). Compound FT-FAPI-12_8 (1 g,1.67mmol,71.46% yield) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝600.5。

4.7 general procedure for the preparation of FT-FAPI-12_9

To a solution of FT-FAPI-12_8 (0.8 g,1.33mmol,1 eq.) was added HCl/EtOAc (4M, 1.33mL,4 eq.). The mixture was stirred at 20℃for 0.5 h. LC-MS showed that FT-FAPI-12_8 was completely consumed and one main peak with the expected m/z was detected. The solid was filtered, washed with EtOAc, and dried under high vacuum. Compound FT-FAPI-12_9 (600 mg, crude) was obtained as a white solid. LCMS: ms+H ⁺ ＝500.4。

4.8 general procedure for the preparation of FT-FAPI-46_1

To a solution of FT-FAPI-12_9 (600 mg,1.20mmol,1 eq.) in DMF (5 mL) was added FT-FAPI-J7 (788.45 mg,1.20mmol,1 eq.) DIEA (465.69 mg,3.60mmol, 627.61. Mu.L, 3 eq.). The mixture was stirred at 20℃for 2 hours. LC-MS showed that FT-FAPI-12_9 was completely consumed and one main peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. Compound FT-FAPI-46_1 (1.25 g, crude) was obtained as a yellow oil. LCMS: ms+H ⁺ ＝1041.5。

4.9 general procedure for the preparation of FT-FAPI-46_2

To a solution of FT-FAPI-46_1 (1.25 g,1.20mmol,1 eq.) in DMF (1 mL) was added piperidine (204.50 mg,2.40mmol, 237.18. Mu.L, 2 eq.). The mixture was stirred at 20℃for 2 hours. LC-MS showed that FT-FAPI-46_1 was completely consumed, anda main peak with the desired m/z is detected. The mixture was concentrated under reduced pressure. The residue was subjected to preparative HPLC (FA conditions; column Phenomenex Luna C200 mm x 40mm x 10 μm; mobile phase: [ water (FA) -ACN]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -30%,8 min). Compound FT-FAPI-46_2 (180 mg, 219.86. Mu. Mol,18.31% yield) was obtained as a yellow oil. LCMS: ms+H ⁺ 819 and (M-19)/2=400.

4.10 general procedure for the preparation of FT-FAPI-46

To a solution of FT-FAPI-46_2 (180 mg, 219.86. Mu. Mol,1 eq.) in DMF (1 mL) was added DIEA (284.16 mg,2.20mmol, 382.96. Mu.L, 10 eq.) and FT-FAPI_D3 (110.26 mg, 219.86. Mu. Mol,1 eq.). The mixture was stirred at 20℃for 12 hours. LC-MS showed that FT-FAPI-46_2 was completely consumed and one main peak with the expected m/z was detected. The mixture was concentrated under reduced pressure. The residue was subjected to preparative HPLC (FA conditions; column Phenomenex Luna C200 mm x 40mm x 10 μm; mobile phase: [ water (FA) -ACN ]The method comprises the steps of carrying out a first treatment on the surface of the B%:1% -20%,8 min). Compound FT-FAPI-46 (40 mg, 33.19. Mu. Mol,15.10% yield, 100% purity) was obtained as an orange solid. ¹ H NMR(400MHz,DMSO-d6)δ＝8.98(br t,J＝5.9Hz,1H),8.57(d,J＝4.3Hz,1H),8.40(s,1H),8.30(br d,J＝7.8Hz,1H),8.15(s,1H),7.86(d,J＝9.4Hz,1H),7.52(dd,J＝2.6,9.4Hz,1H),7.35(d,J＝4.3Hz,2H),5.12(br dd,J＝2.5,9.3Hz,1H),4.72-4.61(m,2H),4.51(s,3H),4.45-4.25(m,5H),4.23-4.08(m,5H),3.57-3.41(m,20H),3.08-2.87(m,23H),2.83-2.65(m,6H),2.42-2.19(m,8H),1.91-1.50(m,6H),1.24(br d,J＝6.6Hz,2H)。

Example 5: radiolabeling of trifunctional compounds and stability studies thereof

5.1 ¹⁸ Radiolabelling of F

To prepare ¹⁸ F-FT-FAPI、 ¹⁸ F-FT-FAPI-02、 ¹⁸ F-FT-FAPI-21、 ¹⁸ F-FT-FAPI-46 and ¹⁸ F-AMBF ₃ FAPI-04, non-Carrier added Using deionized Water ¹⁸ F ^- (18.5-37 GBq) was captured on a QMA column and then eluted with 200. Mu.L isotonic saline. The precursor dissolved in aqueous pyridazine-HCl buffer (10. Mu.L, pH 2) was added and then at 85 ℃Heating for 10 minutes. The reaction was run with 2mL of 5% NH ₄ Water quenching of OH. The reaction mixture was loaded onto a Sep-Pak light C18 column (Waters, USA) using deionized water. The product was eluted with 0.3mL ethanol. For the following ¹⁸ F- ^nat Bi-FT-FAPI, biCl ₃ Dissolved in 4M HCl with 2mM sodium ascorbate and 2mM KI. Successful marking ¹⁸ After F-FT-FAPI, biCl is then added to the unpurified mixture ₃ The solution was adjusted to pH 6 using 3M ammonium acetate. The mixture was then heated at 100℃for 20 minutes. After cooling to room temperature, the reaction mixture was captured on a Sep-Pak light C18 column using 10mL deionized water, and then eluted through 0.3mL ethanol to obtain ¹⁸ F- ^nat Bi-FT-FAPI. The product was diluted with brine for further experiments.

5.2 ⁶⁸ Radiolabelling of Ga

Preparation in this study ⁶⁸ Ga-FT-FAPI ⁶⁸ Ga-FAPI-04. From using 0.6M HCl ⁶⁸ Ge/ ⁶⁸ Elution in Ga generator ⁶⁸ GaCl ₃ The pH was then adjusted to 4 using sodium hydroxide (3M) and sodium acetate (3M). FT-FAPI or FAPI-04 was then added and the mixture was heated at 95℃for 10 minutes. After cooling to room temperature, the reaction mixture was captured on a Sep-Pak light C18 column using 10mL of deionized water, and then eluted through 0.3mL of ethanol to obtain the product. The product was diluted with brine for further experiments.

5.3 ¹⁷⁷ Radiolabelling of Lu

Preparation in this study ¹⁷⁷ Lu-FT-FAPI、 ¹⁷⁷ Lu-FT-FAPI-02、 ¹⁷⁷ Lu-FT-FAPI-21 and ¹⁷⁷ Lu-FT-FAPI-46. Using sodium acetate buffer (0.2M) will ¹⁷⁷ LuCl ₃ Is adjusted to a pH of 4.5 and then the precursor is added. The mixture was heated at 50℃for 1 hour. After cooling to room temperature, the reaction mixture was captured on a Sep-Pak light C18 column using 10mL of deionized water, and then eluted through 0.3mL of ethanol to obtain the product. The product was diluted with brine for further experiments.

5.4 ²¹³ Radiolabelling of Bi

Preparation in this study ²¹³ Bi-FT-FAPI ²¹³ Bi-FAPI-04. From using 2M HCl ²²⁵ Ac/ ²¹³ Elution in Bi generator ²¹³ BiCl ₃ The pH was then adjusted to 6 using sodium hydroxide (8M) and sodium acetate (3M). FT-FAPI or FAPI-04 was then added and the mixture was heated at 100deg.C for 20 minutes. After cooling to room temperature, the reaction mixture was captured on a Sep-Pak light C18 column using 10mL of deionized water, and then eluted through 0.3mL of ethanol to obtain the product. The product was diluted with brine for further experiments.

5.5 radioactive HPLC method

The following HPLC methods were used for quality control. For use in ¹⁸ Method a of F-labeled radiotracer: agilent C18 analytical column; solvent a, deionized water with 0.1% TFA; solvent B, meCN;0-2 minutes, 5% -5% of B;2-7 minutes, 5% -20% of B;7-15 minutes, 20% -100%;15-20 minutes, 100% -5% of B; flow rate, 1 ml/min; column temperature, 25 ℃. Method B for other radiotracers: a waters XBridge C18 column; solvent a, deionized water with 0.1% TFA; solvent B, meCN with 0.1% TFA; 0-2 minutes, 10% -10% of B;2-10 minutes, 10% -60% of B;10-12 minutes, 60% -60% of B;12-15 minutes, 60% -10% of B; flow rate, 1 ml/min; column temperature, 35 ℃.

5.6 stability studies of radiolabeled Compounds

FT-FAPI pass-through ¹⁸ F、 ⁶⁸ Ga、 ¹⁷⁷ Lu and ²¹³ and (5) marking Bi. ¹⁸ Radiolabelling of F-FT-FAPI can be accomplished within 30 minutes, with yield>30% and purity of>99%. After 6 hours incubation with saline or human serum at room temperature, ¹⁸ radiochemical purity of F-FT-FAPI still>95% (FIG. 5 a). ¹⁷⁷ Lu-FT-FAPI sum ⁶⁸ The Ga-FT-FAPI has the yield higher than 90 percent and the purity>99%. After an incubation time of 240 hours with saline, ¹⁷⁷ Purity of Lu-FT-FAPI still>95%. After the purification, the purified water is mixed with the water, ²¹³ the yield of Bi-FT-FAPI exceeds 80% and the purity thereof>95％。

For the compound FT-FAPI-02/21/46 ¹⁸ F and F ¹⁷⁷ Lu radiolabeling. ¹⁸ Yield of F-FT-FAPI-02/21/46>25% and the radiochemical purity is still higher than 95% after incubation for 6 hours in saline or human serum (FIGS. 5 b-d). ¹⁷⁷ Yield of Lu-FT-FAPI-02/21/46>90% and which is stable in saline for 240 hours.

III biological study

Example 6: characterization of FAP-expressing tumor cells by FT-FAPI

Wild-type HT-1080 tumor cells and HT-1080 (HT-1080-FAP) transduced with transmembrane human FAP cells were used. After 1 hour and 6 hours of incubation in HT-1080-FAP cells, ¹⁸ cellular uptake of F-FT-FAPI was 40.37.+ -. 5.43% and 30.03.+ -. 1.39%, respectively, whereas after 1 hour incubation only 0.55.+ -. 1.14% uptake was detected in HT-1080 cells (FIG. 6 a). Binding affinity was assessed using saturation binding and competitive inhibition assays. FT-FAPI shows high affinity for HT-1080-FAP cells (IC ₅₀ =9.73 nM and K _d =8.87 nM, fig. 6b and 6 c). The cellular internalization and efflux rates were then measured. Internalization rate is expressed as the ratio of internalized radioactivity counts to cellular uptake radioactivity counts. Outflow rate is expressed as the ratio of extracellular to total radioactivity counts (extracellular and cellular). As shown in FIG. 6d, FT-FAPI exhibited high internalization rates after 1 hour and 6 hours of incubation in HT-1080-FAP cells, values 92.02.+ -. 1.57% and 86.26.+ -. 1.47%, respectively. The internalization rate then decreased at 14 hours and 24 hours. After 1, 6, 14 and 24 hours incubation in HT-1080-FAP cells, the outflow rates of FT-FAPI were 11.00.+ -. 2.96%, 34.77.+ -. 1.24%, 56.13.+ -. 6.48 and 83.54.+ -. 2.18%, respectively (FIG. 6 e).

Example 7: characterization of FAP-expressing tumor cells by FT-FAPI-02/21/46

As shown in FIG. 7a, after 1 hour of incubation, HT-1080-FAP cells ¹⁷⁷ Lu-FT-FAPI-02、 ¹⁷⁷ Lu-FT-FAPI-21 and ¹⁷⁷ the cellular uptake of Lu-FT-FAPI-46 was 38.23.+ -. 2.22%, 62.76.+ -. 0.22% and 47.69.+ -. 4.53%, respectively, and after 6 hours of incubation, the cellular uptake was 29.69.+ -. 0.84%, 62.90.+ -. 0.89% and 39.00.+ -. 1.36%, respectively. AddingIs significantly blocked in HT-1080-FAP cells ⁶⁸ Cellular uptake of Ga-FAPI-04. IC of FT-FAPI-02, FT-FAPI-21 and FT-FAPI-46 ₅₀ 22.35, 5.34 and 8.03nM (FIG. 7 b), respectively. ¹⁸ F-FT-FAPI-02、 ¹⁸ F-FT-FAPI-21 and ¹⁸ dissociation constant (K) of F-FT-FAPI-46 _d ) 11.45, 9.36 and 10.01nM, respectively (FIG. 7 c). After 1 hour incubation in HT-1080-FAP cells, ¹⁷⁷ Lu-FT-FAPI-02、 ¹⁷⁷ Lu-FT-FAPI-21 and ¹⁷⁷ high internalization rate of Lu-FT-FAPI-46, value>90% and there is no significant difference. As the incubation time was extended, ¹⁷⁷ the internalization rate of Lu-FT-FAPI-02/21/46 gradually decreases and the outflow rate increases. Wherein, ¹⁷⁷ Lu-FT-FAPI-21 showed the most internalization and the least outflow, followed by ¹⁷⁷ Lu-FT-FAPI-46, and finally ¹⁷⁷ Lu-FT-FAPI-02 (FIGS. 7d and 7 e).

Example 8: in vivo biodistribution study of FT-FAPI in tumor-bearing mice

The present invention uses a biodistribution study to evaluate the distribution, absorption and excretion of FT-FAPI in tumor-bearing mice. 0.5, 1, 2, 4, 8 hours after injection, ¹⁸ tumor uptake of F-FT-FAPI was 4.86.+ -. 1.13, 8.90.+ -. 1.28, 8.40.+ -. 2.05, 7.98.+ -. 1.26 and 5.27.+ -. 1.58% ID/g, indicating rapid accumulation and long-term retention in tumors over 8 hours (FIG. 8). In addition, in other organs ¹⁸ Uptake of F-FT-FAPI showed a rapid increase, but the clearance rate was also fast, resulting in a high target/non-target (T/NT) ratio (FIG. 9).

Example 9: in vivo pharmacokinetic and tumor targeting performance studies of FT-FAPI, FT-FAPI-02, FT-FAPI-21 and FT-FAPI-46 in tumor-bearing mice

To further evaluate pharmacokinetic and tumor targeting properties in vivo, and also to investigate AMBF ₃ And DOTA function in FT-FAPI structure for HT-1080-FAP tumor-bearing mice ¹⁸ F-FT-FAPI、 ¹⁸ F-AMBF ₃ FAPI-04 ⁶⁸ PET/CT imaging of Ga-FAPI-04. ¹⁸ F-FT-FAPI showed high tumor accumulation and did not drop significantly within 4 hours after injection (fig. 10a and 11). In additionThere was no significant uptake in the major organs other than the kidneys and bladder, and kidney uptake was significantly reduced from 0.5 hours to 4 hours. At 0.5, 1 and 4 hours post injection ¹⁸ Quantitative tumor SUV of F-FT-FAPI _{Average value of} 1.30.+ -. 0.49, 1.35.+ -. 0.66 and 1.26.+ -. 0.22. And (3) with ¹⁸ In contrast to the F-FT-FAPI, ¹⁸ F-AMBF ₃ tumor uptake of FAPI-04 was significantly reduced (p<0.05). More importantly, there is a higher presence in non-target organs including gallbladder, liver and intestine ¹⁸ F-AMBF ₃ The accumulation of radioactivity of FAPI-04 (FIGS. 10a and 10 b) indicates that the absence of DOTA severely affects the pharmacokinetics of FT-FAPI. Is happy with ¹⁸ In contrast to the F-FT-FAPI, ⁶⁸ tumor uptake of Ga-FAPI-04 was also significantly reduced (p<0.05). In addition, more radioactivity accumulation in the background, including blood and kidneys, can be observed (FIGS. 10a and 10 b), indicating AMBF ₃ The introduction of (3) improves the tumor targeting capability of FT-FAPI.

Proceeding with ¹⁸ PET/CT imaging of F-FT-FAPI-02/21/46 and with ¹⁸ F-FT-FAPI was compared. As shown in figures 10a and 12 of the drawings, ¹⁸ F-FT-FAPI has the highest tumor uptake. In addition, in the case of the optical fiber, ¹⁸ the accumulation of radioactivity in F-FT-FAPI in non-target organs including gallbladder, liver and intestine is also minimal, followed by ¹⁸ F-FT-FAPI-21 and ¹⁸ F-FT-FAPI-46, and finally ¹⁸ F-FT-FAPI-02. All of these radiolabeled agents exhibited acceptable tumor diagnostic capability due to their high tumor uptake and TBR (fig. 19).

Example 10: direct comparison between FT-FAPI and FAPI-04

The invention is then described in ⁶⁸ Ga-FT-FAPI ⁶⁸ Head-to-head PET/CT imaging comparisons were made between Ga-FAPI-04. Due to the combination with ⁶⁸ In contrast to the Ga-FAPI-04, ⁶⁸ only one additional AMBF is present in Ga-FT-FAPI ₃ All changes in head-to-head imaging results may be attributed to AMBF ₃ Incorporated throughout the compound. As shown in figures 13a-13c, ⁶⁸ Ga-FT-FAPI shows a ratio at any point in time ⁶⁸ Significantly higher tumor uptake of Ga-FAPI-04 (p<0.05 And lower background uptake. These areThe results confirm the superior pharmacokinetic properties of FT-FAPI over FAPI-04 and allow ¹⁸ F/ ⁶⁸ Ga-FT-FAPI is promising in cancer diagnosis. In addition to imaging studies, other experiments were performed to compare the two agents. IC of FT-FAPI and FAPI-04 ₅₀ 9.73 and 7.36nM, respectively, and ⁶⁸ Ga-FT-FAPI ⁶⁸ The Kd of Ga-FAPI-04 is 5.37 and 5.89nM, respectively (FIGS. 13d and 13 e). Biodistribution studies again show ⁶⁸ In contrast to the Ga-FAPI-04, ⁶⁸ better tumor uptake and systemic distribution of Ga-FT-FAPI (FIG. 13 f). After 1 hour incubation with HT-1080-FAP cells, ⁶⁸ Ga-FT-FAPI ⁶⁸ The internalization rate and outflow rate of Ga-FAPI-04 are at the same level (FIGS. 13g and 13 h). In addition, molecular docking of FT-FAPI and FAPI-04 with FAP protein was performed. The results show that FT-FAPI can form more hydrogen bonds with residues on proteins due to its additional triazole ring, and AMBF ₃ The charge-separating groups present on the surface can also interact electrostatically with residues in the vicinity of the pocket (FIG. 13 i).

Example 11: ²¹³ radionuclide therapy and toxicity of Bi-FT-FAPI

At the position of ²¹³ Before Bi-FT-FAPI treatment, FT-FAPI is used ²⁰⁹ Bi and Bi ¹⁸ F( ¹⁸ F- ^nat Bi-FT-FAPI) label to investigate ²¹³ Pharmacokinetics of Bi-FT-FAPI. ¹⁸ F- ^nat Bi-FT-FAPI successfully labeled in 45 min, with yield>20% and purity of>99%. As shown in fig. 14 and 15, observation and measurement by in vivo PET imaging and ex vivo biodistribution studies were carried out within 2 hours after injection ¹⁸ F- ^nat High tumor uptake and rapid renal clearance of Bi-FT-FAPI. Then use ²¹³ Bi-FT-FAPI treated HT-1080-FAP tumor bearing mice (FIG. 14 c). With different doses compared to the saline-treated control group ²¹³ The Bi-FT-FAPI treated groups all showed tumor growth inhibition at the beginning. However, tumors in the low dose group resumed growth from day seven and gradually caught up with the control group, while tumors in the medium and high dose groups remained stable for nearly 30 days (fig. 14 d). And also perform ²¹³ Bi-FAPI-04 treatment, and the results show13.32MBq of cumulative dose ²¹³ Bi-FAPI-04 has no inhibition effect on tumor growth. The body weight of all mice remained stable for 30 days. In addition, one third of the mice in the medium and high dose groups were completely cured, and both remaining mice survived to the end of treatment. Finally, medium and high doses were tested ²¹³ Toxicity of Bi-FT-FAPI. In general, the composition, compared to the control group, ²¹³ Bi-FT-FAPI had no significant effect on blood cell count and biochemistry (FIGS. 14e, 14f, 16 and 17).

In summary, in the previous studies, FAP has received extensive attention as an excellent target of radiopharmaceuticals, and many efforts have been made to develop agents for FAP-targeted cancer diagnosis and radionuclide therapy. However, due to the mismatch of the pharmacokinetics of FAPI and the long half-life therapeutic radionuclides, it is often difficult to achieve the desired diagnostic and therapeutic effects on a molecule simply by replacing the diagnostic radionuclides in the chelator with the therapeutic radionuclides. To develop FAP-targeted therapeutic diagnostic agents, the present invention sequesters radioactive metals and facilitates ¹⁸ F-labelled BF ₃ Partially incorporated into a single trifunctional compound.

A series of trifunctional compounds were designed and tested in the present invention. And (3) with ¹⁸ F-AMBF ₃ In contrast to the FAPI-04, ¹⁸ F-FT-FAPI significantly reduces gastrointestinal tract uptake and increases tumor SUV, which better results in FT-FAPI with good diagnostic properties. According to the study of the present invention, ¹⁸ F-FT-FAPI also exhibits a specific ratio ⁶⁸ Ga-FAPI-04 has significantly higher tumor uptake. To illustrate AMBF ₃ Effects on pharmacokinetics, on ⁶⁸ Ga-FT-FAPI ⁶⁸ PET imaging of Ga-FAPI-04 was compared head-to-head, and the results indicate ⁶⁸ Ga-FT-FAPI has significantly better tumor uptake and TBR. These results can also explain why ²¹³ Therapeutic efficacy ratio of Bi-FT-FAPI ²¹³ The therapeutic effect of Bi-FAPI-04 is better. In addition, molecular docking was performed on the trifunctional compound (fig. 13i and fig. 18). In contrast to FAPI-04, the interaction between the ligand studied in FT-FAPI and the FAP binding site remains unchanged. However, will AMBF ₃ The interaction of the trifunctional compound with the protein is unexpectedly stronger when the molecule is introduced. This may be due, at least in part, to the fact that: AMBF ₃ Has a triazole ring that can form hydrogen bonds with residues on proteins, and AMBF ₃ The charge separating groups present thereon may also interact electrostatically with residues in the vicinity of the pocket.

Although radiolabeled FAPI exhibits excellent imaging ability in a variety of cancers, its absolute tumor uptake value is often not high enough and its clearance rate is too fast. These factors limit the development of FAP-targeted radionuclide therapies. Currently, potential strategies include modifying the structure of FAPI to extend its tumor retention time or employing short half-life radionuclides that are more compatible with FAPI pharmacokinetics. In the present invention, the different dosages are used for the first time ²¹³ Bi-labeled FAPI was evaluated and showed good therapeutic efficacy and low toxicity. Experimental results indicate that a cumulative dose of 13.32MBq is sufficient to inhibit the tumor and shows no toxicity. The trifunctional compounds of the invention may be used ¹⁸ F-labelling, simultaneously coordinating isotopes of natural bismuth or other therapeutic radiometals for which the half-life is particularly too short to be of biodistribution investigation ²¹³ Bi provides a viable solution.

Example 12: ¹⁸ F- ^nat Pb/ ^nat discussion of Tb-FT-FAPIPET imaging

Substitution of DOTA on FT-FAPI with TCMC (1, 4,7, 10-tetraaza-1, 4,7, 10-tetrakis (2-carbamoylmethyl) cyclododecane) to prepare ¹⁸ F- ^nat Pb-TCMC-FT-FAPI and on HT-1080-FAP tumor-bearing mice ⁸ F- ^nat Pb-TCMC-FT-FAPI. As shown in fig. 20-21, all compounds showed effective radioactive uptake at the tumor.

Abbreviations and terms of art

mg/kg	Milligrams per kilogram
		n	Quantity of
PBS	Phosphate buffered saline
		FAP	Fibroblast activation protein
TRT	Targeted radionuclide therapy
		TBR	Tumor to background ratio
SUV	Standard uptake value
		PET	Positron emission tomography

Claims

1. A trifunctional compound, pharmaceutically acceptable salt, stereoisomer or solvate thereof, the trifunctional compound comprising a Fibroblast Activation Protein (FAP) targeting moiety FAPT, a chelating moiety C and aminomethyl-BF ₃ Part a.

2. The trifunctional compound according to claim 1, pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound has formula (I):

wherein FAPT moiety, C moiety and A moiety have the meanings as defined in claim 1, and L is a linking group.

3. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer or solvate thereof according to claim 1 or 2, wherein the FAPT moiety has formula (II),

4. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 3, wherein the FAPT moiety has formula (II) _A ) Or formula (II) _B )，

5. The trifunctional compound according to any one of claims 1 to 4, pharmaceutically acceptable salt, stereoisomer, or solvate thereof, wherein the C moiety is selected from the group consisting of:

6. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 5, wherein the a moiety has formula (III) _A ) Or formula (III) _B )：

q and r are independently integers of 1 to 6.

7. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 6, wherein the a moiety has formula (IV),

wherein R is ¹ And R is ² Independently selected from the group consisting of: H. a methyl group and an ethyl group,

q and r are independently 1 or 2.

8. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 7, wherein the a moiety has formula (V),

9. the trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 8, wherein the L moiety has formula (VI),

L ₁ is a covalent bond, -CH ₂ -or-NHCH ₂ -；

Cy is selected from the group consisting of:

m and n are independently 1, 2, 3, 4, 5 or 6;

* Represents the position of attachment to the a part.

10. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 9, wherein the L moiety has formula (VIIA) or formula (VIIB),

wherein m and n are independently 3, 4 or 5; and is also provided with

* And has the meaning as defined in claim 9.

11. The trifunctional compound according to any one of claims 1 to 10, a pharmaceutically acceptable salt, stereoisomer or solvate thereof, wherein the trifunctional compound is selected from the group consisting of:

12. the method comprises the following steps of ¹⁸ F-labelled trifunctional compounds, pharmaceutically acceptable salts, stereoisomers or solvates thereof, wherein said ¹⁸ F-labelled trifunctional compounds having one or more fluorine-groups in the A-part of the trifunctional compounds as defined in any of claims 1 to 11 ¹⁸ F alternative structure.

13. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 12, wherein the C moiety is chelated with a radionuclide or natural isotope thereof.

14. The trifunctional compound, pharmaceutically acceptable salt, stereoisomer or solvate thereof according to claim 13, wherein the radionuclide is capable of emitting beta particles or alpha particles, and preferably is ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁵⁵ Co、 ¹⁴⁹ Tb、 ¹⁵² Tb、 ¹⁵⁵ Tb、 ¹⁶¹ Tb、 ¹⁷⁷ Lu、 ²¹² Pb、 ²¹² Bi、 ²¹³ Bi、 ²²⁵ Ac、 ²²⁶ Th、 ²²⁷ Th、 ²²³ Ra and ²³⁰ u, and the natural isotopePreferably is ⁶⁹ Ga、 ⁷¹ Ga、 ⁸⁹ Y、 ⁶³ Cu、 ⁶⁵ Cu、 ⁵⁹ Co、 ¹⁵⁹ Tb、 ¹⁷⁵ Lu、 ²⁰⁸ Pb、 ²⁰⁹ Bi、 ¹³⁹ La、 ²³² Th、 ²²⁶ Ra and ²³⁸ U。

15. a pharmaceutical composition comprising a trifunctional compound according to any one of claims 1 to 14, a pharmaceutically acceptable salt, stereoisomer or solvate thereof and one or more pharmaceutically acceptable carriers.

16. A method of diagnosing or treating a disease, the method comprising administering to a subject a therapeutically effective amount of a trifunctional compound, pharmaceutically acceptable salt, stereoisomer, or solvate thereof according to any one of claims 1 to 14.

17. The method of claim 16, wherein the disease is cancer, infection or inflammation.

18. The method of claim 17, wherein the cancer is selected from the group consisting of: prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocellular carcinoma, chordoma, small intestine cancer, pheochromocytoma, gastric cancer, renal cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymus cancer, cervical cancer, endometrial cancer, neuroendocrine tumor, thyroid cancer, intestinal cancer, and bone metastasis.