CN119390720A

CN119390720A - A bicyclo[1.1.1]pentane compound targeting fibroblast activation protein and its application

Info

Publication number: CN119390720A
Application number: CN202411979351.1A
Authority: CN
Inventors: 高丽梅; 曹天野
Original assignee: Hangzhou Jingjiahang Biomedical Technology Co ltd
Current assignee: Hangzhou Jingjiahang Biomedical Technology Co ltd
Priority date: 2024-12-31
Filing date: 2024-12-31
Publication date: 2025-02-07
Anticipated expiration: 2044-12-31
Also published as: CN119390720B

Abstract

The invention relates to the technical field of radiopharmaceuticals, in particular to a bicyclo [1.1.1] pentane compound combined with fibroblast activation protein in a targeting way and application thereof. The structure of the compound shown in the formula (I) is as follows: (I) According to the invention, through modifying the structure of the FAP binding compound, the compound with longer residence time in the tumor in vivo is obtained.

Description

Bicyclo [1.1.1] pentane compound for targeting and combining fibroblast activation protein and application thereof

Technical Field

The invention relates to the technical field of radiopharmaceuticals, in particular to a targeted binding fibroblast activation protein multimeric compound and application thereof.

Background

Aromatic rings are widely present in natural products and bioactive compounds, have a restricted molecular conformation, enhance the ability to bind receptors by non-covalent interactions, and build a rich library of compounds by cross-coupling reactions. But at the same time, the aromatic ring rich in pi electrons is easy to oxidize, and the metabolic stability is seriously reduced. Furthermore, aromatic molecules have reduced water solubility and increased log D (resulting in increased probability of off-target) compared to C (sp 3) molecules, hampering drug development. The C (sp 3) -based bicyclic hydrocarbon backbone acts as an isostere for the aromatic ring and can be used as a substitute for the aromatic ring (WIESENFELDT, nature, 2023, 618, 513).

Benzene rings can be said to be the module most relied upon by pharmaceutical chemists or chemists. One from FDA orange book statistics in the united states, benzene rings appear in about 45% of small molecule drugs on the market, which have various interactions with targets, such as hydrophobic interactions, pi-stacking, amide bond stacking, cation-pi interactions, etc. The spatial effect and electronic effect synergism of the benzene ring resonance structure can precisely optimize the interaction between the medicine and the target, physical and chemical characteristics and in-vivo medicine properties (such as absorption, distribution, metabolism and excretion). However, benzene rings have various drawbacks in pharmaceutical applications, such as increasing the number of aromatic rings in the compound, significantly decreasing the water solubility and significantly increasing the lipid solubility. In addition, the benzene ring affects the metabolic stability, safety of the compound, and metabolism may also occur in the benzene ring in the drug candidate, leading to stability problems, specific toxicity, and safety risks. Various benzoquinone derivatives can be produced. Such metabolites have high activity and can react with nucleophiles in vivo and crosslink to bioactivate.

Fibroblast Activation Protein (FAP) is a type II transmembrane serine protease belonging to the prolyl oligopeptidase family. FAP high expression is associated with tumor invasiveness and poor prognosis (Cohen, S.J, pancreas 2008, 37, 154-158; coto-Llerena, M, front, oncol, 2020, 10, 979), which makes it a very attractive target for tumor imaging and treatment (Lindner, T, EJNMMI radioboom, chem, 2019, 4, 16). FAP is therefore considered as a powerful therapeutic target for cancer treatment, against which the corresponding inhibitor (FAPI) molecule was recently developed globally, a strategy for the imaging and treatment of cell populations of the peri-tumor stroma (cancer-associated fibroblasts). According to the FAPI molecular structure of the referenced patent (patent application publication No. CN 118955616A), the structure of quinoline structure connected with ureido is adopted, in the biodistribution experiment of ¹⁷⁷ Lu-JYT2-401 in the example, the tumor gradually drops from high uptake (at 2 h-34.7% ID/g) to 13.64% ID/g (at 48 h), which is probably due to the fact that the quinoline-ureido structure is degraded by different enzymes in the body (such as quinoline aromatic ring and ureido mediated by cytochrome P450 enzyme under oxidation, forming benzoquinone derivatives, which leads to easy degradation (Jet Tsien, nature REVIEWS CHEMISTRY, 2024, 8, 605).

In view of this, the present invention has been made.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a bicyclo [1.1.1] pentane compound for targeting and combining fibroblast activation protein and application thereof.

Specifically, the invention hopes to enhance the stability of the medicine in tumors, and aims at the defect of benzene ring, and the substitution of bioelectrode isostere is utilized to replace the bioelectrode isostere except for improving the bioactivity, physicochemical properties, PK attribute and in vivo stability of the medicine. At present, the substitution of bioisostere of benzene ring, except the application of traditional pyridine, cyclohexane, piperidine and the like, discovers that the effect of certain polycyclic saturated frameworks is better.

The invention uses bicyclo [1.1.1] pentane to replace the quinoline structure of JYT2-401 in the patent application CN 118955616A studied in advance, and develops the synthesis research of single-chain and double-chain molecules and the in-vitro activity measurement thereof through an optimized structure, and simultaneously develops the comparison of the metabolism of drugs in vivo, tumor uptake and the like after marking different nuclides, and protects the bicyclo [1.1.1] pentane molecules of the targeting FAP.

Based on the above, the invention has the following technical scheme:

In a first aspect, the present invention provides a compound represented by formula (I) or a derivative thereof, wherein the compound represented by formula (I) has the structure:

(I);

Wherein x represents any integer of 1 to 4;

r represents one or more of-H, -CN, -F, -B (OH) ₂, alkyl substituted with F and alkylene;

L each independently, identically or differently represents a substituted or unsubstituted C ₁~C₁₀ alkylene group, a substituted or unsubstituted C ₁~C₁₀ cycloalkylene group, a substituted or unsubstituted C ₁~C₁₀ heteroalkylene group, a substituted or unsubstituted C ₁~C₁₀ heterocycloalkylene group, a substituted or unsubstituted C ₁~C₁₀ oxyalkylene group, a substituted or unsubstituted C ₁~C₁₀ aminoalkylene group, a substituted or unsubstituted C ₁~C₁₀ alkenylene group, a substituted or unsubstituted C ₁~C₁₀ cycloalkenyl group, a substituted or unsubstituted C ₁~C₁₀ heterocycloalkenylene group, a substituted or unsubstituted C ₁~C₁₀ heterocycloalkenylene group, a substituted or unsubstituted C ₁~C₁₀ alkynylene group, a substituted or unsubstituted C ₁~C₁₀ heteroarylene group, a substituted or unsubstituted C ₁~C₃₀ heteroarylene group, a dialkylene siloxane group, a carbonyl group, an imino group, an imide group, an amide group, a thioamide group, a phosphoryl group, a thioether group, a disulfide group, an ester group, a thioester group, a carbonate group, a phosphate group, a dibasic acid group, a peptide, a tripeptide group, a peptide or a group; wherein, when substituted, the substituent comprises one or more of alkyl, alkenyl, alkynyl, aryl, heteroaryl, ester and amino;

d is a single bond or any of the following structural formulas or any connected group thereof: M is any integer from 0 to 10;

C is selected from any one of (a) - (C) (a) a chelating agent group suitable for radiolabeling, (b) a radioactive group comprising a radioisotope, (C) a chelate of a radioisotope with a chelating agent;

The derivatives include pharmaceutically acceptable tautomers, racemates, hydrates, solvates or salts.

In the present invention, x may be 2, 3 or 4, and m may be 1,2, 3, 4, 5,6,7,8, 9 or 10. Specifically, m of different structural formulas in D are independent and may be the same or different.

In the invention, the structural formula of the imide group is-CO-NH-, the structural formula of the thioamide group is-CS-NH-, the structural formula of the phosphoryl group is-PO-NH ₂ -, the structural formula of the thioester group is-S-CO-, the structural formula of the carbamate group is-NHCOO-, the phosphate group can be one or more of phosphoric monoester (hydrocarbyl phosphoric acid), secondary phosphoric acid ester (phosphoric diester) and tertiary phosphoric acid ester (phosphotriester), the saccharide can be one of monosaccharide groups of five-carbon sugar or six-carbon sugar, the five-carbon sugar is selected from ribose, and the six-carbon sugar is selected from glucose, mannose or galactose.

In the present invention, the hetero atom in the alkylene group, the heterocycloalkylene group, the heteroarylene group, the heterocycloalkenylene group, the heteroarylene group and the heteroarylene group includes at least one of oxygen, sulfur and nitrogen.

As a preferred alternative to this,Selected from the group consisting of Any one of them.

It is further preferred that the composition comprises,Is that。

In the present invention, L is an intermediate linking fragment of the active fragment and the metal complex, comprising one or more linker groups, each linker group being independently selected from any one of the following groups.

Preferably, L each independently, identically or differently represents a substituted or unsubstituted alkylene group of C ₁~C₅, a substituted or unsubstituted heteroalkylene group of C ₁~C₅, a substituted or unsubstituted heterocycloalkylene group of C ₁~C₆, a substituted or unsubstituted heteroarylene group of C ₁~C₅, a substituted or unsubstituted arylene group of C ₁~C₅, a carbonyl group, an alkoxy group, a thioether group, a disulfide group, an acid anhydride group, a carbonate group, a carbamate group, a sugar, a peptide, a polyethylene glycol, an amide group, or an ester group, or a group of one or more of the foregoing groups, wherein, when substituted, the substituent includes one or more of an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an ester group, and an amine group.

In the present invention, when substituted, each L, independently, the same or different, may be an arylene group (i.e., an aralkylene group) of C ₁~C₃₀ substituted with an alkyl group, a heteroarylene group (i.e., a heteroarylene group) of C ₁~C₃₀ substituted with an alkyl group, an arylene group (i.e., an aralkenylene group) of C ₁~C₃₀ substituted with an alkenyl group, a heteroarylene group (i.e., a heteroarylene group) of C ₁~C₃₀ substituted with an alkenyl group, thiazolidine, methylenealkoxycarbamate, or the like.

More preferably, L each independently, identically or differently represents any one of the following groups:

;

Wherein n is any integer from 1 to 10;

Wherein R ₁ is selected from-H, -C _1~6 alkyl, -O-C _1~6 alkyl, -S-C _1~6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or-C _1~6 aralkyl, each of said-C _1~6 alkyl groups is optionally substituted with 1 to 3 substituents selected from-OH, oxygen, halogen, n is any integer from 1 to 10, such as 1,2, 3, 4,5, 6, 7, 8, 9 or 10.

As a preferred embodiment of the present invention, L isN is any integer from 1 to 5, such as 1,2,3, 4 or 5.

In the present invention, when two or more n are present in the group L, the different n are each independently the same or different, e.g.The values of the two n can be the same or different.

Preferably, C represents an optical dye, a photodynamic therapeutic agent, a radiological imaging agent, a radiotherapy agent, a chemotherapeutic agent, an anti-fibrosis agent or an anticancer agent.

Preferably, C is a ligand moiety, C is a chelator that forms a complex with a divalent or trivalent metal cation, preferably the chelator is selected from the group consisting of 1,4,7, 10-tetraazacyclododecane-N, N ', N, N' -tetraacetic acid, ethylenediamine tetraacetic acid, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid, triethylenetetramine, iminodiacetic acid, diethylenetriamine-N, N, N ', N', N-pentaacetic acid, bis- (carboxymethyl imidazole) glycine, or 6-hydrazinopyridine-3-carboxylic acid, preferably the ligand moiety further comprises a non-radioisotope, a radiopharmaceutical, or a combination thereof, preferably the radioisotope is selected from the group consisting of an isotope that emits alpha rays, an isotope that emits beta rays, an isotope that emits gamma rays, an isotope that emits auger electrons, and more preferably the isotope comprises one or more of ¹⁸F、¹⁸F-Al、⁵¹Cr、⁶⁷Ga、⁶⁸Ga、¹¹¹In、^99mTc、¹³⁹La、¹⁴⁰La、¹⁷⁵Yb、¹⁵³Sm、¹⁶⁶Ho、⁸⁸Y、⁹⁰Y、¹⁴⁹Pm、¹⁶⁵Dy、¹⁶⁹Er、¹⁷⁷Lu、⁴⁷Sc、¹⁴²Pr、¹⁵⁹Gd、²¹²Bi、²¹³Bi、⁷²As、⁷²Se、⁹⁷Ru、¹⁰⁹Pd、¹⁰⁵Rh、^101mRh、¹¹⁹Sb、¹²⁸Ba、¹²³I、¹²⁴I、¹³¹I、¹⁴⁹Tb、¹⁵²Tb、¹⁵⁵Tb、¹⁶¹Tb、¹⁹⁷Hg、²¹¹At、¹⁵¹Eu、¹⁵³Eu、¹⁶⁹Eu、²⁰¹Tl、²⁰³Pb、²¹²Pb、⁶⁴Cu、⁶⁷Cu、¹⁸⁸Re、¹⁸⁶Re、¹⁹⁸Au、²²⁵Ac、²²⁷Th and ¹⁹⁹ Ag;

Or C is a ligand moiety selected from xanthine, acridine, oxazine, cyanine, styryl, coumarin, porphyrin, metal ligand-complex, fluorescent protein, nanocrystal, perylene, borodipyrromethene, and phthalocyanine, or a conjugate or combination of the foregoing classes of dyes;

or C is a contrast agent comprising or consisting of a paramagnetic agent, preferably the paramagnetic agent comprises or consists of paramagnetic nanoparticles.

Preferably, the compound shown in the formula (I) has any one of the following structural formulas:

。

in a second aspect, the present invention provides a pharmaceutical composition comprising the compound of formula (I) or a derivative thereof.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In a third aspect, the invention provides a kit comprising a compound of formula (I) or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate, or salt thereof, or a pharmaceutical composition, and instructions for diagnosing or treating a disease.

In a fourth aspect, the invention provides the use of a compound of formula (I) or a derivative thereof, or the pharmaceutical composition or the kit for the manufacture of a medicament for the diagnosis and/or treatment of a disease.

Preferably, the disease comprises a disease characterized by overexpression of fibroblast activation protein in an animal or human subject; further preferably, the disease is selected from one or more of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scar disease, more preferably the cancer is selected from one or more of small intestine cancer, head and neck cancer, hepatocellular carcinoma, hypopharyngeal carcinoma, nasopharyngeal carcinoma, myeloma cells, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, primary unknown carcinoma, thymus cancer, glioma, astrocytoma, cervical cancer, eye cancer, rectal cancer, colon cancer, cervical cancer, prostate cancer, breast cancer, bladder cancer, oral cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, uterine cancer, ovarian cancer, testicular cancer, kidney cancer, brain cancer, central nervous system cancer, throat cancer, skin melanoma, acute lymphocytic leukemia, acute myeloid leukemia, ewing sarcoma, kaposi sarcoma, basal cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, angioma, renal cell tumor, neuroblastoma, esophagus cancer, laryngeal carcinoma, neuroblastoma, lymphoma, tumor of any kind of blood, and lymphoma.

In a fifth aspect, the present invention provides an inhibitor or binding agent for targeted binding to Fibroblast Activation Protein (FAP), comprising the compound of formula (I) or a derivative thereof, or the pharmaceutical composition.

In a sixth aspect, the present invention provides a therapeutic agent for tumors comprising the compound represented by the formula (I) or a derivative thereof or the pharmaceutical composition.

In a seventh aspect, the present invention provides a tumor imaging agent comprising the compound of formula (I) or a derivative thereof or the pharmaceutical composition.

In an eighth aspect, the present invention provides a tumor diagnostic agent comprising the compound represented by the formula (I) or a derivative thereof or the pharmaceutical composition.

According to the invention, by optimizing the structural formula of the compound, particularly introducing bicyclo [1.1.1] pentane into the main structure of the compound to be connected with ureido, the activity of the novel FAPI molecule is improved to a certain extent after the bicyclo [1.1.1] pentane is used for replacing the quinoline structure, and the uptake of the novel bicyclo [1.1.1] pentane FAPI molecule in tumors is not obviously reduced in a long time (> 48 h), so that the stability of the bicyclo [1.1.1] pentane in FAPI molecule in vivo can be effectively improved.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be presented as a simple description, and it is obvious that the drawings in the description below are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram ¹ HnmR of JYQ2-452 in example 1 of the present invention.

FIG. 2 shows the HPLC results of JYQ2-452 in example 1 of the present invention.

FIG. 3 shows the MS results of JYQ2-452 in example 1 of the present invention.

FIG. 4 shows the result of ¹ HnmR of JYT-4521 in example 2 of the present invention.

FIG. 5 shows the HPLC results of JYT-4521 in example 2 of the present invention.

FIG. 6 is a graph showing the MS results of JYT-4521 in example 2 of the present invention.

FIG. 7 shows the results of in vitro activity assays for JYQ2-452 and JYT-4521 in example 2 of the present invention, wherein (A) is the results of JYQ2-452 and (B) is the results of JYT-4521.

FIG. 8 is a graph showing the radioactive signal detected by HPLC of ⁶⁸ Ga-JYQ2-452 in example 1 of the present invention.

FIG. 9 is a graph of UV signal detected by HPLC of ⁶⁸ Ga-JYQ2-452 in example 1 of the present invention.

FIG. 10 is a graph showing the radioactive signal detected by HPLC of ⁶⁸ Ga-JYT-4521 in example 2 of the present invention.

FIG. 11 is a graph showing the ultraviolet signal spectrum of HPLC detection of ⁶⁸ Ga-JYT-4521 in example 2 of the present invention.

FIG. 12 is a PET/CT image of ⁶⁸ Ga-JYQ2-452 in example 1 and ⁶⁸ Ga-JYT-4521 in example 2 of the present invention.

FIG. 13 is a graph showing the radioactive signal detected by HPLC of ¹⁷⁷ Lu-JYQ2-452 in example 1 of the present invention.

FIG. 14 is a graph of UV signal from HPLC detection of ¹⁷⁷ Lu-JYQ2-452 in example 1 of the present invention.

FIG. 15 is a graph showing the radioactive signal detected by HPLC of ¹⁷⁷ Lu-JYT-4521 in example 2 of the present invention.

FIG. 16 is a graph showing the UV signal detected by HPLC of ¹⁷⁷ Lu-JYT-4521 in example 2 of the present invention.

FIG. 17 is a graph showing the result of ¹⁷⁷ Lu-JYQ2-452 biodistribution in example 1 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise indicated, the various starting materials used in the examples and comparative examples were commercially available conventional starting materials, and the technical means used were conventional means well known to those skilled in the art.

EXAMPLE 1 preparation of Small molecule inhibitors

The synthetic route of JYQ2-452 is shown as follows:

Step one (3- (methoxy (methyl) carbamoyl) bicyclo [1.1.1] pent-1-yl) carbamic acid tert-butyl ester

3- ((Tert-Butoxycarbonyl) amino) bicyclo [1.1.1] pentane-1-carboxylic acid (300 mg, 1.32 mmol) was dissolved in dichloromethane (10 mL) and dimethylamine hydrochloride (154 mg, 1.58mmol, 1.2 eq.) was added, HATU (602 mg, 1.58mmol, 1.2 eq.) and DIPEA (512 mg, 3.96mmol, 3.0 eq.). The reaction was carried out at room temperature for 2 hours. TLC detects complete reaction of starting material. Water (50 mL) was added, dichloromethane (100 mL x 2) was added, the organic phase was concentrated, and the crude product was purified by silica gel column chromatography (PE/EA=1/1) to give JYQ2-452-1 (354 mg, 1.31mmol, 99.2% yield) as a white solid. MS (ES+) M/z 271.2 [ M+H ] ⁺.

Step di (3-formyl-bicyclo [1.1.1] pent-1-yl) carbamic acid tert-butyl ester

JYQ2-452-1 (441 mg, 1.63 mmol) was dissolved in anhydrous tetrahydrofuran (8 mL) and DIBAL-H (3.26 mL, 1M, 2.0 eq.) was slowly added dropwise at-70℃under nitrogen. The reaction was carried out at-70℃for 1 hour. TLC detects complete reaction of starting material. Methanol (10 mL) was added to the reaction mixture and stirred for 10 minutes. Dilute hydrochloric acid was added to adjust the pH to 3, water (10 mL) was added, ethyl acetate (50 mL x 2) was added to extract, the organic layers were combined and washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give JYQ2-452-2 (279 mg, 1.32mmol, yield 81.0%) as a white solid. MS (ES+) M/z 212.2 [ M+H ] ⁺.

Step (E) -3- (3- ((tert-Butoxycarbonyl) amino) bicyclo [1.1.1] pent-1-yl) acrylic acid ethyl ester

JYQ2-452-2 (279 mg, 1.32 mmol) was dissolved in tetrahydrofuran (8 mL) and ethoxyformyl methylene triphenylphosphine (460 mg, 1.32mmol, 1.0 eq) was added. The reaction was carried out at room temperature for 15 hours. TLC detects complete reaction of starting material. The reaction was concentrated, and the crude product was purified by silica gel column chromatography (PE/ea=2/1) to give JYQ2-452-3 (317 mg, 1.13mmol, yield 85.3%) as a white solid. MS (ES+) M/z 282.3 [ M+H ] ⁺.

Step four methyl 3- (3- ((tert-Butoxycarbonyl) amino) bicyclo [1.1.1] pent-1-yl) propanoate

JYQ2-452-3 (317 mg, 1.13 mmol) was dissolved in methanol (8 mL) and Pd/C (100 mg, 10%) was added and the reaction was hydrogenated at room temperature for 3 hours. TLC detects complete reaction of starting material. Filtration and concentration of the mother liquor gave JYQ2-452-4 (283 mg, 1.05mmol, 93.0% yield) as a white solid.

Step five (3- (3-hydroxypropyl) bicyclo [1.1.1] pent-1-yl) carbamic acid tert-butyl ester

JYQ2-452-4 (280 mg, 1.04 mmol) was dissolved in tetrahydrofuran (10 mL) and LAH (79 mg, 2.08mmol, 2.0 eq.) was added in portions at 0deg.C. After the addition, the reaction was carried out at 0℃for 1 hour. TLC detects complete reaction of starting material. The reaction solution was stirred for 10 minutes with sodium sulfate decahydrate and filtered. The mother liquor was concentrated to give JYQ2-452-5 (246 mg, 1.02mmol, 98.0% yield) as a colorless solid. MS (ES+) M/z 186.1 [ M-56+H ] ⁺.

Step six 3- (3- ((tert-Butoxycarbonyl) amino) bicyclo [1.1.1] pent-1-yl) propylmethanesulfonate

JYQ2-452-5 (246 mg, 1.02 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (309 mg, 3.06mmol, 3.0 eq) was added. Methanesulfonyl chloride (152 mg, 1.33mmol, 1.3 eq.) was added at 0 ℃. After the addition, the reaction mixture was allowed to warm to room temperature for 1 hour. TLC detects complete reaction of starting material. The reaction mixture was extracted with water (50 mL) and dichloromethane (50 mL. Times.2), and the organic phase was concentrated to give JYQ2-452-6 (305 mg, 0.95mmol, yield: 93.6%) as a white solid.

Step seven benzyl 4- (3- ((tert-Butoxycarbonyl) amino) bicyclo [1.1.1] pent-1-yl) propyl) piperazine-1-carboxylate

JYQ2-452-6 (305 mg, 0.95 mmol) was dissolved in DMF (10 mL), benzyl-1-piperazine carbonate (210 mg, 0.95mmol, 1.0 eq.) potassium carbonate (262 mg, 1.9mmol, 2.0 eq.) and sodium iodide (142 mg, 0.95mmol, 1.0 eq.) were added. After the addition, the reaction solution was heated to 80℃and reacted for 3 hours. TLC detects complete reaction of starting material. The reaction was taken up in water (50 mL), extracted with ethyl acetate (50 mL x 2), the organic phase concentrated and the crude product purified by silica gel column chromatography (DCM/MeOH=15/1) to give JYQ2-452-7 (419 mg, 0.94mmol, 99.4% yield) as a yellow liquid. MS (ES+) M/z 444.3 [ M+H ] ⁺.

Step eight benzyl 4- (3- (3-Aminobicyclo [1.1.1] pent-1-yl) propyl) piperazine-1-carboxylate

JYQ2-452-7 (1.05 g, 2.37 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added and reacted at room temperature for 1 hour. TLC detects complete reaction of starting material. Concentrated, the crude product was basified to ph=9 with water (20 mL), extracted with dichloromethane (50 mL), the organic phase dried and concentrated to give yellow liquid JYQ2-452-8 (755 mg, 2.20mmol, 92.7% yield).

Step nine benzyl 4- (3- (3- (2-oxo-2- ((R) -2- ((3 aS,4S,6S,7 aR) -3a, 5-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxolan-2-yl) pyrrolidin-1-yl) ethyl) ureido) bicyclo [1.1.1] pent-1-yl) propyl) piperazine-1-carboxylate

Triphosgene (108 mg, 0.36mmol, 0.33 eq) was dissolved in dichloromethane (20 mL) and a solution of JYQ2-452-8 (378 mg, 1.10 mmol) and triethylamine (245 mg, 2.42mmol, 2.2 eq) in dichloromethane (20 mL) was added dropwise at 0 ℃. After the addition, the reaction was continued for 30 minutes at 0 ℃. TLC detects complete reaction of starting material. JY2-111-1-5 (303 mg, 0.99mmol, 0.9 eq.) was added to the reaction solution, and the reaction solution was warmed to room temperature and allowed to react for 1 hour. TLC detects complete reaction of starting material. The reaction was taken up in water (30 mL), extracted with dichloromethane (50 mL x 2), the combined organic layers were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product purified by silica gel column chromatography (DCM/MeOH=20/1) to give JYQ2-452-9 (320 mg, 0.47mmol, 43.1% yield) as a colorless solid. MS (ES+) M/z 676.4 [ M+H ] ⁺.

Step ten 1- (2-oxo-2- ((R) -2- ((3 aS,4S,6S,7 aR) -3a, 5-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxolan-2-yl) pyrrolidin-1-yl) ethyl) -3- (3- (3- (piperazin-1-yl) propyl) bicyclo [1.1.1] pent-1-yl) urea

JYQ2-452-9 (320 mg, 0.47 mmol) was dissolved in methanol (10 mL) and Pd/C (100 mg, 10%) was added. Hydrogenation was carried out at room temperature for 1 hour, and TLC detected complete reaction of starting materials. Filtration and concentration of the filtrate gave JYQ2-452-10 (251 mg, 0.46mmol, 98.6% yield) as a white solid.

Step eleven, 2' - (10- (2-oxo-2- (4- (3- (3- (2-oxo-2- ((R) -2- ((3 aS,4S,6S,7 aR) -3a, 5-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxolan-2-yl) pyrrolidin-1-yl) ethyl) ureido) bicyclo [1.1.1] pent-1-yl) propyl) piperazin-1-yl) ethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) tri-acetic acid tri-tert-butyl ester

Tritert-butyl 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (191 mg, 0.33mmol, 1.0 eq.) was dissolved in DMF (10 mL) and PYBOP (206 mg, 0.40mmol, 1.2 eq.) and DIPEA (128 mg, 0.99mmol, 3.0 eq.) were added. The reaction was carried out at room temperature for 30 minutes. JYQ2-452-10 (180 mg, 0.33 mmol) was added and the reaction was continued at room temperature for 3 hours. TLC detects complete reaction of starting material. The reaction was taken up in water (50 mL), extracted with ethyl acetate (50 mL x 2), the organic phase concentrated and the crude product purified by silica gel column chromatography (DCM/MeOH=15/1) to give JYQ2-452-11 (203 mg, 0.19mmol, 56.1%) as a yellow viscous liquid.

Step twelve 2,2' - (10- (2-oxo-2- (4- (3- (3- (2-oxo-2- ((R) -2- ((3 aS,4S,6S,7 aR) -3a, 5-trimethylhexahydro-4, 6-methylbenzo [ d ] [1,3,2] dioxolan-2-yl) pyrrolidin-1-yl) ethyl) ureido) bicyclo [1.1.1] pent-1-yl) propyl) piperazin-1-yl) ethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-tri-yl) triacetic acid

JYQ2-452-11 (203 mg, 0.19 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (2 mL) was added. The reaction was carried out at room temperature for 15 hours. Directly concentrating to obtain the purple solid JYQ2-452-12 (crude product) which is directly put into the next step.

Step tride (R) -2, 2'' - (10- (2- (4- (3- (3- (2- (2-bromopyrrolidin-1-yl) -2-oxoethyl) ureido) bicyclo [1.1.1] pent-1-yl) propyl) piperazin-1-yl) 2-oxoethyl) -1,4,7, 10-tetraazacyclododecane-1, 4, 7-triyl) triacetic acid

The crude JYQ2-452-12 was dissolved in water (6 mL) and acetonitrile (3 mL) and phenylboronic acid (29 mg, 0.24mmol, 1.3 eq.) was added, trifluoroacetic acid (2 d) and MTBE (18 mL). The reaction was carried out at room temperature for 3 hours. The mixture was separated, and the aqueous phase was washed with MTBE (10 mL). The aqueous phase was purified by RP-HPLC (Waters preparative chromatograph equipped with SunFire@PrepC18 OBD5um 30×250mm column using a gradient of 95:5 to 20:80 water/acetonitrile+0.1% TFA, over 20 min), the fractions were collected and lyophilized to give JYQ2-452 (5 mg, 0.0037mmol, 1.9% yield in two steps) as a white solid. MS (ES+) M/z 776.4 [ M-5 XCF ₃ COOH (molecular weight 114.02) -H ₂ O (molecular weight 18) +H ] ⁺ (90.91% purity, 210 nm).

¹HnmR (400MHz, D₂O) δ 4.54-4.42 (m, 1H), 3.99-3.64 (m, 8H), 3.60 (s, 2H), 3.55-3.25 (m, 13H), 3.21-2.85 (m, 14H), 2.09-1.94 (m, 3H), 1.92-1.73 (m, 7H), 1.71-1.43 (m, 5H).

¹ HnmR of JYQ2-452 are shown in FIG. 1.

HPLC results for JYQ2-452 are shown in Table 1 and FIG. 2.

The liquid phase detection conditions comprise a chromatographic column of YMC-Triart C, 3um of 4.6X105 mm, a flow rate of 1ml/min, a column temperature of 30 ℃, a mobile phase of 0.1% hClO ₄, and a pH=1.5 of H ₂ O solution of C: ACN.

TABLE 1

The MS results of JYQ2-452 are shown in FIG. 3.

EXAMPLE 2 preparation of Small molecule inhibitors

The synthetic route of JYT-4521 is shown below:

Step one N- [2- (4- {1- [ (2-methylpropan-2-yl) oxy ] -1-oxoeth-2-yl } -7, 10-bis {2- [ (2-methylpropan-2-yl) oxy ] -2-oxoeth-ylene } -1,4,7, 10-tetraazacyclododecane-1-yl) acetyl ] -L-glutamic acid dibenzyl ester

L-glutamic acid dibenzoyl ester hydrochloride (0.83 g, 2.27mmol, 1.3 eq) was dissolved in DCM (20 mL), TEA (0.53 g, 5.24mmol, 3.0 eq) and DOTA- (OtBu) 3 (1.00 g, 1.75 mmol) were added sequentially, stirred at room temperature for 5 min, BOPCl (0.67 g, 2.62mmol, 1.5 eq) was added at 0-5℃and the reaction was warmed to room temperature for 16h after the addition, and TLC showed the end of the reaction. The reaction mixture was washed with saturated ammonium chloride (10 mL), extracted with DCM (10 mL. Times.2) in the aqueous phase, the organic phases were combined, washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product purified by silica gel column chromatography to give JYT-4521-1 (1.04 g, 1.18mmol, 67.4%) as a colorless oil. MS (ES+) m/z 441.8[ M/2+H ] ⁺.

Step two N- [2- (4- {1- [ (2-methylpropan-2-yl) oxy ] -1-oxoeth-2-yl } -7, 10-bis {2- [ (2-methylpropan-2-yl) oxy ] -2-oxoeth-ylene } -1,4,7, 10-tetraazacyclododecane-1-yl) acetyl ] -L-glutamic acid

JYT-4521-1 (0.41 g, 0.46 mmol) was dissolved in methanol (5 mL), 10% Pd/C (40 mg) was added, hydrogen balloon was substituted 3 times, stirred at room temperature for 16 hours, LCMS detection was complete, the reaction solution was filtered, the filter cake was washed with methanol (3 mL), and the filtrate was concentrated to give JYT-4521-2 (0.32 g, 0.46mmol, 99.1% yield) as a white foamy solid. MS (ES+) m/z 702.4[ M+H ] ⁺.

Step three: [10- (2- { [ (2S) -5- {4- [3- (1- { [ ({ 2- [ (2R) -2- [ (1S, 2S,6R, 8S) -2,9,9-trimethyl-4-bora-3, 5-dioxatricyclo [6.1.1.02,6] dec-4-yl ] tetrahydro-1H-pyrrol-1-yl ] -2-oxyethyleneethyl } amino) carbonyl ] amino } bicyclo [1.1.1] pent-3-yl) propyl ] piperazin-1-yl } -1- {4- [3- (3- { [ ({ 2- [ (2R) -2- [ (1S, 2S,6R, 8S) -2,10,10-trimethyl-4-bora-3, 5 ] Dioxatricyclo [6.1.1.02,6] dec-4-yl ] tetrahydro-1H-pyrrol-1-yl ] -2-oxyethyleneethyl } amino) carbonyl ] amino } bicyclo [1.1.1] pent-1-yl) propyl ] piperazin-1-yl } -1, 5-dioxapent-2-yl ] amino } -2-oxyethyleneethyl) -4, 7-bis {2- [ (2-methylpropan-2-yl) oxy ] -2-oxyethyleneethyl } -1,4,7, 10-tetraazacyclododec-1-yl ] acetic acid 2-methylpropan-2-yl ester

JYT-4521-2 (95 mg, 0.14 mmol) was dissolved in DMF (8 mL) and DMTMM (97 mg, 0.35mmol, 2.5 eq.) and DIPEA (72 mg, 0.56mmol, 4.0 eq.) were added. JYQ2-452-10 (150 mg, 0.28mmol, 2.0 eq.) was added after stirring for 30min at room temperature. The reaction was carried out at room temperature for 5 hours. TLC detects complete reaction of starting material. Adding water (10 mL), separating out solid, filtering and drying to obtain yellow solid JYT-4521-3 (crude product), and directly throwing into the next step.

Step four [4, 7-bis (carboxymethyl) -10- (2- { [ (2S) -5- {4- [3- (1- { [ ({ 2- [ (2R) -2- [ (1S, 2S,6R, 8S) -2,9,9-trimethyl-4-oxa-3, 5-dioxatricyclo [6.1.1.02,6] dec-4-yl ] tetrahydro-1H-pyrrol-1-yl ] -2-oxoethylene } amino) carbonyl ] amino } bicyclo [1.1.1] pent-3-yl) propyl ] piperazin-1-yl } -1- {4- [3- (3- { [ ({ 2- [ (2R) -2- [ (1S, 2S,6R, 8S) -2,10,10-trimethyl-4-oxa-3, 5-dioxatricyclo [6.1.1.02,6] dec-4-yl ] tetrahydro-1H-pyrrol-1-yl ] -2-oxoethylene } amino) carbonyl ] amino } bicyclo [1.1.1] pent-1-yl) piperazin-1, 5-oxon-3-yl } -1, 5-dioxa-2-5-aza ] penta-1-yl) -acetic acid, 7-aza-2-azol-4-yl

The crude JYT-4521-3 was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (1 mL) was added. The reaction was carried out at room temperature for 15 hours. TLC detects complete reaction of starting material. The reaction solution was directly concentrated to give brown liquid JYT-4521-4 (crude product) which was directly taken to the next step.

Step five, [4, 7-bis (carboxymethyl) -10- (2- { [ (2S) -1, 5-bis {4- [3- (3- { [ ({ 2- [ (2R) -2- (dihydroxyboryl) tetrahydro-1H-pyrrol-1-yl ] -2-oxolenethyl } amino) carbonyl ] amino } bicyclo [1.1.1] pent-1-yl) propyl ] piperazin-1-yl } -1, 5-dioxopent-2-yl ] amino } -2-oxolenethyl) -1,4,7, 10-tetraazacyclododec-n-1-yl ] acetic acid

The crude JYT-4521-4 was dissolved in a mixed solution of water (4 mL), acetonitrile (2 mL) and MTBE (12 mL). Phenylboronic acid (34 mg,0.28mmol, 2.0 eq.) and trifluoroacetic acid (1 drop) were added. The reaction was carried out at room temperature for 3 hours. Water (8 mL) was added to the reaction mixture, and the mixture was separated. The aqueous phase was washed with MTBE (6 mL), and the aqueous phase was lyophilized to give JYT-4521 (6 mg, 0.003mmol, three step yield 2.1%) as a white solid by HPLC. MS (ES+) M/z 629.9 [ (M-3 XH ₂ O (molecular weight 18) -6 XCF ₃ COOH (molecular weight 114.02))/2+H ] ⁺ (99.82% purity, 210 nm).

¹HnmR (400MHz, D₂O) δ 4.58-4.22 (m, 3H), 4.19-4.02 (m, 1H), 3.96-2.80 (m, 52H), 2.61-2.44 (m, 2H), 2.08-1.92 (m, 5H), 1.88-1.73 (m, 15H), 1.73-1.41 (m, 9H).

FIG. 4 shows the ¹ HnmR results of JYT-4521.

HPLC results for JYT-4521 are shown in FIG. 5 and Table 2.

TABLE 2

FIG. 6 shows the MS results of JYT-4521.

Comparative example 1

Comparative example 1 provides JYT2-401 having the structural formula shown in Table 3.

TABLE 3 Table 3

Experimental example 1 inhibitor affinity test of the example

1.1 (Bioorg. Med. Chem. Lett. 2020, 30, 127253.). The specific procedures were that FAP protein concentration was diluted to 0.4. Mu.g/mL with assaybuffer (25mM Tris,250mM NaCl,pH 7.4), substrate GP-AMC was diluted to 40. Mu.M with assay buffer, solutions of inhibitors (compounds 1-20) of different concentrations were prepared with assay buffer, 25. Mu.L of inhibitor and 25. Mu.L of substrate were sequentially added to a 96-well blackboard, 50. Mu.L of protein was further added, the mixture was placed on an microplate reader, incubated at 37℃for 1 hour, and the fluorescence intensity was measured (Ex/Em=380/460 nm), fitting was performed using GRAPHPAD PRISM, and IC50 was calculated using a sigmoidal 11dose response model.

1.2 Determination of hFAP inhibitory Activity of Compounds:

hFAP protein was diluted to 0.3 μg/mL using assay buffer (25 mM Tris,250 mM NaCl,pH =7.4) and kept on ice. The compounds were diluted with assay buffer to a concentration gradient of 40. Mu.M, 4. Mu.M, 400nm, 40nm, 4nm, 400pM, 40pM and 4pM, respectively. The enzyme substrate Z-Gly-Pro-AMC was diluted to 40. Mu.M with assay buffer and stored in the dark. The prepared 50. Mu.L of protein solution and 25. Mu.L of inhibitor were sequentially added to a 96-well plate (Corning 96-well ELISA plate black transparent bottom, no. 3904), centrifuged, and shaken at room temperature for 30min. Then, 25. Mu.L of the above-prepared enzyme substrate was added. After incubation for 3-5 hours at room temperature, fluorescence values with excitation wavelength of 360nm and emission wavelength of 465nm are monitored by using a multifunctional enzyme-labeled instrument (Agilent BioTek Synergyh 1). IC ₅₀ values were calculated, defined as the concentration of compound that resulted in a 50% decrease in enzyme activity under the assay conditions, and the results are shown in table 4 and fig. 7.

TABLE 4 Table 4

Experimental example 2 ⁶⁸ Ga labeling example for PET imaging experiments

2. ⁶⁸ Ga label and quality control

2.1 Preparation procedure

The labeling procedure was performed in 2 portions by precisely weighing 1-4. Mu.L of 20nmol of the solution of the compound (JYQ 2-452, JYT-4521), taking 500. Mu.L of 1M acetic acid-sodium acetate solution (pH 5.0) and 200. Mu.L of 100mg/mL N-acetyl-L-tyrosine solution (1M acetic acid-sodium acetate, pH 5.0), placing in a 2mL centrifuge tube, thoroughly mixing, adding 0.75mL ⁶⁸GaCl₃ of eluent, standing in a metal bath under sealed conditions at 95℃for 15min, and taking out. The purification process comprises cooling to room temperature, passing 2 parts of the reaction solution through a C18 column, eluting ⁶⁸ Ga-labeled product (⁶⁸Ga-JYQ2-452,⁶⁸ Ga-JYT-4521) by using 1mL of 50% ethanol-physiological saline solution, collecting the eluent, adding 4mL of physiological saline into the eluent for dilution, and then using the eluent for animal administration. The radioactivity is detected before and after administration, and the sample is stored at room temperature and in a lead tank for preparation.

2.2 Quality control analysis

The radioactive purity is YMC TRIART C, 3 mu M,4.6mm multiplied by 150mm Columbn, the flow rate is 1mL/min, the Column temperature is 35 ℃, the detection wavelength is 210nm, and the sample injection volume is 20 mu L (50-100 mu Ci). The gradient elution conditions are shown in Table 5.

TABLE 5

The results are shown in FIG. 8-FIG. 11 and Table 6-Table 9. The area normalization method calculates the amplification purity, the amplification purity is more than 90%, and the test requirement is met. And (3) counting ultraviolet absorption peaks of the precursor and related impurities, wherein the ultraviolet absorption peaks of the related impurities such as auxiliary materials are not counted.

TABLE 6 ⁶⁸ results of Ga-JYQ2-452hPLC radioactive signals

Remarks are that the area normalization method calculates the amplification purity, the amplification purity is more than 90 percent, and the test requirement is met.

TABLE 7 results of ⁶⁸ Ga-JYQ2-452hPLC ultraviolet signal (absorption wavelength 210 nm)

Remarks are that ultraviolet absorption peaks of JYQ2-452 and related impurities are counted.

TABLE 8 ⁶⁸ Ga-JYT-4521hPLC results of radioactive signals

TABLE 9 results of ⁶⁸ Ga-JYT-4521hPLC ultraviolet Signal (absorption wavelength 210 nm)

Remarks are made on statistics of ultraviolet absorption peaks of JYT-4521 and related impurities.

2.3 Animal model and PET imaging process

Animal identification, namely hanging cage cards for each cage of animals, and marking animal groups, animal numbers and the like.

The feed is provided, and the growth maintenance feed for mice accords with GB14924.4-2001.

The feeding method is free feeding.

The drinking water bottle is supplied with tap water after high pressure sterilization, the tap water is inspected by a Taiyuan monitoring station of a national urban water supply water quality monitoring network, meets the requirements of sanitary standards for domestic drinking water (GB 5749-2006), and the drinking water bottle is replaced every 2 days and is used after being cleaned and high pressure sterilized.

Animal model construction is completed by Beijing Veitpoli animal technology Co., ltd, HEK293ThFAP human embryo kidney cells are inoculated subcutaneously on the right side of the back of each experimental animal to transfect human FAP high expression cell line tumor cells, and the total number of the animal is 3, and the animal model construction is used for PET/CT imaging experiments.

The experimental design includes that a disposable injector extracts liquid medicine, an activity meter detects activity (μCi) before administration (full needle), tail vein injection administration is carried out, and an activity meter detects residual radioactivity count (μCi) after administration (empty needle). PET/CT imaging is carried out on tumor-bearing mice after the mice are anesthetized for 0.5h, 2h and 4h respectively, and the PET imaging data of the mice are circled by software to obtain ⁶⁸Ga-JYQ2-452,⁶⁸ Ga-JYT-4521 tissue distribution and metabolic data% ID/cc in the mice at different time points. FIG. 12 is a PET/CT image of ⁶⁸ Ga-JYQ2-452 and ⁶⁸ Ga-JYT-4521.

Table 10 ⁶⁸ Ga-JYQ2-452 PET image values of region of interest at different time points

Table 11 ⁶⁸ Ga-JYT-4521 PET image values of the region of interest at different time points

2.4 Results and discussion

HEK293ThFAP human embryo kidney cell transfected human FAP high expression cell line Balb/c nude inoculated tumor-bearing mice were injected with ⁶⁸ Ga-JYQ2-452 and ⁶⁸ Ga-JYT-4521 compound injection by single tail vein, PET/CT imaging was carried out 0.5h, 2h and 4h after administration, and different degrees of radioactive enrichment exist in tissues and organs. ⁶⁸ Ga-JYQ2-452 and ⁶⁸ Ga-JYT-4521 have low uptake values in normal organ tissues (< 2% ID/cc), ⁶⁸ Ga-JYQ2-452 is mainly enriched in tumors, and the intra-tumor uptake tie value is 11.59 +/-1.48% ID/cc at 4 hours after administration, the maximum value is 20.73+/-2.24% ID/cc, and the uptake value in tumors is slightly higher than ⁶⁸ Ga-JYT-4521.

Experimental example 3 ¹⁷⁷ Lu labeling example for biodistribution experiments

3.1 Process for ¹⁷⁷ Lu labeling examples

Precisely measuring 1-4 mu L of solution of a compound (JYQ 2-452, JYT-4521) 20nmol, adding 10 mu L of 100mg/mL of N-acetyl-L-methionine, 40 mu L of 100mg/mL of N-acetyl-L-tyrosine and 5 mu L of 100mg/mL of Vc, placing into a 1.5mL centrifuge tube, uniformly mixing, adding 1-2 mu L ¹⁷⁷LuCl₃ (activity: 1-2 mCi,0.1MhCl solution), standing in a metal bath under a sealed condition, heating at 90 ℃ in a dark place for 15min, taking out, and adding 5 mu L of DTPA (50 mg/mL) after the reaction is completed. After cooling to room temperature, HPLC was detected. 200. Mu.L of 0.5 MhAc-NaAc (pH 5.5) and 2mL of deionized water were added to adjust the pH of the solution to 5.0 to complete the formulation (¹⁷⁷Lu-JYQ2-452,¹⁷⁷ Lu-JYT-4521) for animal administration.

3.2 Quality control analysis

The radioactive purity is YMC TRIART C, 3 mu M,4.6mm multiplied by 150mm Columbn, the flow rate is 1mL/min, the Column temperature is 35 ℃, the detection wavelength is 210nm, and the sample injection volume is 20 mu L (50-100 mu Ci). The gradient elution conditions are shown in Table 12.

Table 12

The results are shown in FIG. 13-FIG. 16 and Table 13-Table 16. The area normalization method calculates the amplification purity, the amplification purity is more than 90%, and the test requirement is met. And (3) counting ultraviolet absorption peaks of the precursor and related impurities, wherein the ultraviolet absorption peaks of the related impurities such as auxiliary materials are not counted.

TABLE 13 ¹⁷⁷ results of the radioactive signals of Lu-JYQ2-452hPLC

Table 14 ¹⁷⁷ Lu-JYQ2-452hPLC ultraviolet signal (absorption wavelength 210 nm)

Table 15 ¹⁷⁷ results of the radioactive Signal of Lu-JYT-4521hPLC

Table 16 ¹⁷⁷ Lu-JYT-4521hPLC ultraviolet signal (absorption wavelength 210 nm)

3.3 Animal model and biodistribution results

The feeding method is free feeding.

Tumor inoculation, namely, animal model construction is completed by Beijing Veitway Lihua animal technology Co., ltd, HEK293 tumor cells are inoculated subcutaneously on the right side of the back of each experimental animal, and 10 animals are used for later experiments.

The disposable syringe was used to withdraw the drug solution, and the activity (μCi) before administration (full needle) was measured by an activity meter, and the administration was by tail vein injection, and the residual radioactivity count (μCi) after administration (empty needle) was measured by an activity meter.

Sampling time is 2h,24h,48h and 144h after administration;

Tissue organs-animals were lightly anesthetized, eyeballs were exsanguinated (blood sample was retained), hearts, livers, spleens, lungs, kidneys, stomach (content removed), large intestine (content removed), small intestine (content removed), femur (hindlimb), patella, muscle, pancreas, brain, bladder (no urine), tumor, skin, urine, feces (2-3 grains in large intestine), administration site (tail), and remaining cadavers were taken.

And (3) detecting by a gamma counter, namely collecting biological samples at all time points, lightly squeezing, sucking residual blood by using water absorbing paper, weighing and recording, placing the samples in an EP tube, detecting by the gamma counter, and loading the samples in 24 hours (if the radioactivity count is too high, placing and measuring after decay). The results are expressed in CPM (CPM: radioactivity counts per minute).

Data processing and analysis:

%ID/g=A_tissue/[（A₀-A_residue）×M_tissue]×100%。

a ₀ Total radioactivity Count (CPM) of syringe prior to administration.

A _residue residual radioactivity Counts (CPM) from syringe after administration.

A _tissue tissue organ radioactivity Counts (CPM).

M _tissue detection of tissue sample weight (g).

Physical decay correction ¹⁷⁷ Lu physical decay: activity dose at time t=0.5 (T/160.8).

The dose (A ₀-A_residue) is multiplied by the decay factor of the corresponding time, i.e. the corrected CPM value, and the% ID/g is calculated.

Note that the activity meter detection (μci) versus gamma Counter (CPM) calibration curve is y= 135628X (R ² = 0.9991) over an activity range of no more than 75 μci.

Table 17 ¹⁷⁷ Lu-JYQ2-452 biodistribution detailed values at various time points

Table 18 ¹⁷⁷ Lu-JYQ2-452 and ¹⁷⁷ Lu-JYT-4521 injection comparative ¹⁷⁷ Lu-JYT2-401 tissue distribution results in HEK293 human embryonic kidney cells Balb/c nude tumor bearing mice for 48h

2.4 Results and discussion

The results are shown in tables 17 to 18 and FIG. 17.HEK293ThFAP human embryo kidney cell transfected human FAP high expression cell line inoculated Balb/c nude tumor-bearing mice are injected with ¹⁷⁷ Lu-JYQ2-452 compound injection through single tail vein, biodistribution experiments are respectively carried out at 2h, 24h, 48h and 144h after administration, different degrees of radioactivity enrichment exist in tissues and organs, small amounts of uptake (1-4% ID/g) exist in the liver, spleen and skin of normal organs after 144h administration, ¹⁷⁷ Lu-JYQ2-452 are mainly enriched in tumors, the intratumoral uptake is 20.48+/-1.06% ID/g after 2h administration, the highest 26.64 +/-1.81% ID/g is reached at 48h, and the uptake is still kept at 22.55+/-5.05% ID/g after 144h administration. Compared with the biological distribution data of 48h after injection of reference compounds ¹⁷⁷ Lu-JYT2-401 and ¹⁷⁷ Lu-JYT-4521, the uptake values of normal tissues and organs of 3 compounds are basically equivalent after injection, and the tumor uptake amount 26.64 +/-1.81% ID/g of the ¹⁷⁷ Lu-JYQ2-452 compound is higher than that of ¹⁷⁷ Lu-JYT2-401 (13.64+/-1.85% ID/g) and ¹⁷⁷ Lu-JYT-4521 (12.05+/-0.58% ID/g), so that the bicyclo [1.1.1] pentane compound series molecules designed by the invention have certain advantages for treatment.

In the invention, JYT-4521 is taken as a dimeric compound of JYQ2-452 molecules, but the uptake and the residence time of tumors are not improved, which is the characteristic of a bicyclo [1.1.1] pentane substituted quinoline structure FPAI molecule.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.

Claims

1. A compound represented by formula (I) or a derivative thereof, wherein the structure of the compound represented by formula (I) is:

(I);

Wherein x represents any integer of 1 to 4;

2. The compound of formula (I) or a derivative thereof according to claim 1,Selected from the group consisting of Any one of them;

And/or L each independently, identically or differently represents a substituted or unsubstituted alkylene group of C ₁~C₅, a substituted or unsubstituted heteroalkylene group of C ₁~C₅, a substituted or unsubstituted heterocycloalkylene group of C ₁~C₆, a substituted or unsubstituted heteroarylene group of C ₁~C₅, a substituted or unsubstituted arylene group of C ₁~C₅, a carbonyl group, an alkoxy group, a thioether group, a disulfide group, an anhydride group, a carbonate group, a carbamate group, a sugar, a peptide, a polyethylene glycol, an amide group, or an ester group, or a group of one or more of the foregoing groups, wherein, when substituted, the substituent includes one or more of an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an ester group, and an amine group;

C is a ligand moiety which is a chelator forming a complex with a divalent or trivalent metal cation, the chelator being selected from the group consisting of 1,4,7, 10-tetraazacyclododecane-N, N ', N, N ' -tetraacetic acid, ethylenediamine tetraacetic acid, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid, triethylenetetramine, iminodiacetic acid, diethylenetriamine-N, N, N ', N-pentaacetic acid, bis- (carboxymethyl imidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid, preferably the ligand moiety comprises a non-radioactive isotope, a radiopharmaceutical or a combination thereof, the radioactive isotope being selected from the group consisting of an isotope that emits alpha rays, an isotope that emits beta rays, an isotope that emits gamma rays, an isotope that emits auger electrons, an isotope that emits X rays, the isotope comprising one or more of ¹⁸F、¹⁸F-Al、⁵¹Cr、⁶⁷Ga、⁶⁸Ga、¹¹¹In、^99mTc、¹³⁹La、¹⁴⁰La、¹⁷⁵Yb、¹⁵³Sm、¹⁶⁶Ho、⁸⁸Y、⁹⁰Y、¹⁴⁹Pm、¹⁶⁵Dy、¹⁶⁹Er、¹⁷⁷Lu、⁴⁷Sc、¹⁴²Pr、¹⁵⁹Gd、²¹²Bi、²¹³Bi、⁷²As、⁷²Se、⁹⁷Ru、¹⁰⁹Pd、¹⁰⁵Rh、^101mRh、¹¹⁹Sb、¹²⁸Ba、¹²³I、¹²⁴I、¹³¹I、¹⁴⁹Tb、¹⁵²Tb、¹⁵⁵Tb、¹⁶¹Tb、¹⁹⁷Hg、²¹¹At、¹⁵¹Eu、¹⁵³Eu、¹⁶⁹Eu、²⁰¹Tl、²⁰³Pb、²¹²Pb、⁶⁴Cu、⁶⁷Cu、¹⁸⁸Re、¹⁸⁶Re、¹⁹⁸Au、²²⁵Ac、²²⁷Th and ¹⁹⁹ Ag;

or C is a contrast agent comprising or consisting of a paramagnetic agent.

3. A compound of formula (I) or a derivative thereof according to claim 1, wherein L each independently, identically or differently represents any one of the following groups:

;

Wherein n is any integer from 1 to 10;

Wherein R ₁ is selected from-H, -C _1~6 alkyl, -O-C _1~6 alkyl, -S-C _1~6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or-C _1~6 aralkyl, each of the-C _1~6 alkyl groups is optionally substituted with 1 to 3 substituents selected from-OH, oxygen and halogen, and n is any integer of 1 to 10.

4. A compound of formula (I) or a derivative thereof according to any one of claims 1 to 3, wherein the compound of formula (I) has any one of the following structural formulas:

。

5. a pharmaceutical composition comprising a compound of formula (I) or a derivative thereof as defined in any one of claims 1 to 4.

6. A kit comprising a compound of formula (I) or a derivative thereof as defined in any one of claims 1 to 4 or a pharmaceutical composition as defined in claim 5, and instructions for diagnosing or treating a disease.

7. Use of a compound of formula (I) or a derivative thereof as defined in any one of claims 1 to 4 or a pharmaceutical composition as defined in claim 5 or a kit as defined in claim 6 for the manufacture of a medicament for the diagnosis and/or treatment of a disease comprising a disease characterized by overexpression of a fibroblast activation protein in an animal or human subject.

8. A targeted binding fibroblast activation protein inhibitor or binding agent comprising a compound of formula (I) or a derivative thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 5.

9. An antitumor drug comprising a tumor therapeutic agent or a tumor diagnostic agent, wherein the tumor therapeutic agent or the tumor diagnostic agent comprises the compound represented by the formula (I) or a derivative thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.

10. A tumor imaging agent comprising the compound represented by the formula (I) or a derivative thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 5.