Compound capable of enhancing affinity of FAP inhibitor and improving pharmacokinetics and application

Abstract

There is provided a compound or a pharmaceutically acceptable salt thereof, the compound being a compound of formula (I) wherein A is a fibroblast activating protein inhibitor moiety; l is a linker part; e is a chelating agent moiety. Also provided is a radionuclide formulation comprising the compound or a pharmaceutically acceptable salt thereof, and a radionuclide chelated therewith. The invention also provides application of the compound or the pharmaceutically acceptable salt thereof in preparation of radioactive therapeutic drugs. And A-L-E (I).

Description

Technical Field

[0001] This application relates to the field of molecular imaging, and more particularly to a compound that enhances the affinity of FAP inhibitors and improves pharmacokinetics, for tumor radio-diagnosis and treatment. Background Art

[0002] Radiotheranostics of Tumors

[0003] Radiotheranostics is the only widely accepted and clinically used therapy to date. A key aspect of radiotheranostics is that patients undergoing radio-guided therapy are selected based on the imaging results of the same target area; therefore, imaging is closely related to therapeutic intervention, capable of stripping out truly crucial information, ultimately used for the auxiliary diagnosis, treatment, and grading assessment of diseases, more conducive to localizing and even qualitatively diagnosing the lesion area, with great application value in precision medicine. Radiotherapy uses the low-penetrating radiation emitted by radionuclides to accumulate a high level of energy in the cell nuclei of target cells, inducing DNA strand breaks and activating programmed cell death.

[0004] The concept of "theranostic pair" consists of probes with almost identical chemical structures, labeled with diagnostic or therapeutic radionuclides, which ensures targeting the same specific molecules in diagnostic imaging and molecular targeted therapy. Radiotherapy selects and targets tumor patients for diagnosis or treatment through the use of paired diagnostic / therapeutic probes, conforming to the ultimate concept of personalized medicine. Diagnostic radionuclides can be used for SPECT / CT or PET / CT or PET / MRI imaging. Both γ and positron rays have high tissue absorption, low energy transfer, and a relatively long radiation range, so patients undergoing imaging can be exposed to a relatively low level of radiation.

[0005] Compared with anatomical imaging such as CT or MRI, radionuclide imaging can molecularly characterize cancer cells in vivo, that is, throughout the body, for early detection and staging of diseases, evaluating tumor heterogeneity through imaging, treatment selection and planning, and subsequently formulating and timely adjusting individualized targeted treatment plans based on imaging results.

[0006] These therapeutic probes allow the diagnostic part to further early evaluate the treatment effect and detect recurrence before detectable anatomical changes occur, and play a key role in differentiating progression and pseudo-progression, capable of determining whether there is sufficient molecular target expression in tumor tissue relative to normal tissue, thus deciding whether patients can benefit from targeted radionuclide therapy.

[0007] FAP as a Cancer Target

[0008] The biological mechanisms of fibroblast activation protein α (FAP) on cancer prognosis are still unclear and inconsistent, but the presence of FAP in the malignant stroma determines its potential as a promising target for cancer imaging and treatment. Solid tumors usually consist of multiple types of stromal cells, including lymphocytes, tumor macrophages, myeloid-derived suppressor cells, endothelial cells, and fibroblasts. In most cases, these stromal cells support tumor growth and inhibit the body's immune response to eradicate them.

[0009] Like other stromal cells, cancer associated fibroblasts (CAFs) accumulate during tumor development and subsequently release growth factors, angiogenesis-stimulating cytokines, immunosuppressive agents, and extracellular matrix proteins, which harden the tumor mass, limit the entry of drugs and immune cells, promote tumor growth and metastasis, reduce immune-mediated tumor rejection, and limit the access of solid tumors to drugs.

[0010] CAFs are different from normal fibroblasts in terms of biological appearance, function, and growth pattern. FAP is a unique marker protein of them. FAP is one of the mediators of extracellular matrix remodeling, tumor progression, and metastasis in tumor cells. Stromal cells expressing FAP have also been confirmed to inhibit anti-tumor immunity. Importantly, FAP is expressed very low or not at all in normal tissues, while it is overexpressed in more than 90% of human cancers, such as breast cancer, colorectal cancer, pancreatic cancer, melanoma, myeloma, gastric cancer, brain cancer, and ovarian cancer. Therefore, FAP is highly expressed in CAFs in the tumor stroma and can be internalized rapidly and effectively, making it a potential and promising prognostic marker. Currently, FAP inhibitors based on quinolinic acid derivatives have made important progress in the field of tumor imaging after further radiolabeling.

[0011] For example, PET images obtained in multiple different tumor subtypes with FAPI-02 / 04 and FAPI-46 / 74, etc. all showed a high tumor-to-background ratio. Compared with 18 F-FDG, FAPI-based imaging agents have lower background in the brain, liver, and oropharyngeal mucosa and higher detection rates for tumor lesions. However, the high detection rate of FAPI also has the problem of high false positive rate. Therefore, it is particularly important to further improve the affinity and selectivity of FAPI.

[0012] FAPI reported currently is rapidly cleared in the blood circulation and rapidly eluted at the tumor site. Such metabolic characteristics are beneficial for imaging, which can provide a relatively clean background. However, it is extremely disadvantageous for treatment because rapid metabolism and elution result in a low effective dose and a too short retention time at the tumor site, and high doses or more frequent administration methods are required to meet the treatment needs, increasing the possibility of adverse reactions. Therefore, new strategies are needed to appropriately extend the circulating half-life of the probe, endow it with suitable pharmacokinetics, a higher tumor uptake dose, and a longer tumor retention time to meet the requirements of radionuclide therapy.

[0013] Albumin carrier strategy

[0014] Human serum albumin is a helical protein containing three homologous α-helical domains, and the three domains aggregate together to form an asymmetric heart-shaped structure. The domain is a pocket structure formed by hydrophobic and positively charged groups. Each domain contains two subdomains, and each subdomain is composed of 6 helical structures. Albumin is the most abundant plasma protein involved in the transport of substances in the body, with multiple binding sites and a circulating half-life of about 19 days. It is synthesized in hepatocytes, and about 10–15 g of albumin is produced and released into the blood circulation every day. As a drug carrier, albumin has the advantages of good biocompatibility, good stability, strong non-antigenicity, good biodegradability, and easy surface modification, so it is widely used in the field of nuclear pharmacy.

[0015] Previous studies have shown that using albumin as a delivery carrier can extend the half-life in peripheral blood, improve the uptake, enrichment, and retention time in tumors, thereby improving the therapeutic effect. At the same time, due to the rapid proliferation of tumor cells requiring a large amount of nutrients, and albumin can just provide amino acids and energy, there are albumin-binding receptors Gp60 (glycoprotein with a molecular weight of 60 kDa) on the surface of tumor cells. When albumin is used as a delivery carrier, it can targetedly bind to the Gp60 receptor, then bind to intracellular proteins (caveolin-1), and the cell membrane invaginates to form transport vesicles, thus targeting tumor cells. Therefore, this strategy can increase the tumor uptake rate of targeted probes and radionuclides.

[0016] However, due to the extremely strong affinity of existing albumin binders for albumin, the killing of normal healthy organs by radionuclides increases, resulting in corresponding side effects such as hematotoxicity and nephrotoxicity.

[0017] Therefore, it is extremely important to develop a new type of albumin binder with a suitable affinity for albumin, which can significantly increase tumor absorption while not causing serious toxic side effects to normal tissues, thereby improving the therapeutic index. Summary of the Invention

[0018] In one aspect, there is provided a compound or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (I),

[0019] A—L—E (I)

[0020] wherein A is a fibroblast activation protein inhibitor moiety; L is a linker moiety; and E is a chelator moiety.

[0021] In one aspect, there is provided a radionuclide preparation comprising the compound of the present application or a pharmaceutically acceptable salt thereof, and a radionuclide chelated therewith.

[0022] In one aspect, there is provided the use of the compound of the present application or a pharmaceutically acceptable salt thereof in the preparation of a radiotherapeutic drug.

[0023] The compound of the present application or a pharmaceutically acceptable salt thereof can effectively target tumors, so that a radiopharmaceutical comprising the same can be effectively used for tumor treatment. Description of the Drawings

[0024] Figure 1 It is the electrospray mass spectrum of FAPI in the examples of the present application.

[0025] Figure 2A 、 2B 、2C are respectively the electrospray mass spectra of FAPI-Lys(CH3)-Phe-EDA-DOTA (HX05-2), FAPI-Lys(CH3)2-Phe-EDA-DOTA (HX05-3), and FAPI-Lys(CH3)3-Phe-EDA-DOTA (HX05-4) in the examples of the present application.

[0026] Figure 3 It is the graph of the percentage of remaining drug in the stability study of HX05-2.

[0027] Figure 4 It is for 68 Ga labeling and probe 68 It is the radiochromatogram in the stability study of 68Ga-HX05-2.

[0028] Figure 5 It is the positron emission tomography of small animals.

[0029] Figure 6 It is for 68 68Ga-HX05-2, 68 68Ga-HX05-3, 68 It is the result of biodistribution analysis of 68Ga-HX05-4. Detailed Embodiments

[0030] To further elaborate on the technical means and effects adopted by this application to achieve the intended purpose, the following will, in conjunction with the accompanying drawings and preferred embodiments, elaborate in detail on the specific implementation manners, structures, features, and their effects according to this application.

[0031] Terms and Definitions

[0032] As used herein, the singular terms refer to one or more than one. For example, "element" or "an element" both refer to one element or more than one element. As used herein, the term "plurality" means at least two.

[0033] As used herein, the term "about" means approximate, within a range of approximately or near. When the term "about" is used in combination with a numerical range, it modifies the range by expanding the boundaries above or below the provided value. Generally, the term "about" as used herein causes the value to vary up and down by 10% from the provided value. On the one hand, the term "about" means plus or minus 20% of the numerical value of the number it modifies. For example, "about 50%" means within the range of 45% - 55%. Numerical ranges mentioned by endpoints herein include all integers and fractions included within that range (e.g., "1 to 5" includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all integers and fractions are considered to be modified by the term "about".

[0034] As used herein, the terms "comprising", "including", or "containing", as non-exclusive or open-ended terms, are intended to indicate that a combination (such as a device, composition, method, etc.) includes the recited elements (such as the respective units of a device, the respective components of a composition, the substantial steps of a method, etc.), but does not exclude other elements. As used herein, the term "consisting essentially of" when used to define a composition and a method means excluding other elements that have any substantial impact on the combination for the stated purpose, but does not exclude other elements that do not substantially affect the basic and novel features of the present invention. As used herein, the term "consisting of" means excluding other combinations of elements (units, components, substantial steps, etc.), but unless otherwise stated, does not mean excluding trace amounts of unavoidable impurities. Embodiments defined by each of these connecting terms are within the scope of the present invention. As a specific embodiment thereof, a technical solution disclosed including the terms "comprising", "including", or "containing" should also be regarded as simultaneously disclosing the corresponding technical solutions including the terms "consisting essentially of" and "consisting of".

[0035] As used herein, the term "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be included individually, or any combination of two or more of the listed items can be included. For example, if a group, combination, or composition, etc., is described as including (or containing) components A, B, C, and / or D, then the composition can include A individually; B individually; C individually; D individually; a combination of A and B; a combination of A and C; a combination of A and D; a combination of B and C; a combination of B and D; a combination of C and D; a combination of A, B, and C; a combination of A, B, and D; a combination of A, C, and D; a combination of B, C, and D; or a combination of A, B, C, and D.

[0036] As described herein, the term "pharmaceutical composition" means a composition comprising at least one active ingredient, which is acceptable for studying specific, effective results in mammals (such as but not limited to humans). Based on the needs of those skilled in the art, those of ordinary skill in the art will understand and know the techniques suitable for determining whether the active ingredient has the desired effective effect.

[0037] As used herein, the term "pharmaceutically acceptable salt" means a salt derived from the compounds of the present invention, retaining the biological effectiveness and properties of the compounds of the present invention, and generally not being a biologically or otherwise undesirable salt. Due to the presence of amino and / or carboxyl groups or groups similar thereto, the compounds of the present invention are capable of forming acid salts and / or base salts. The term "pharmaceutically acceptable salt" can refer to pharmaceutically acceptable addition salts prepared from pharmaceutically acceptable non-toxic acids or bases (including inorganic acids and bases and organic acids and bases).

[0038] The term "protected derivative" means a derivative of a compound (such as a compound specified herein), in which one or more functional groups of the compound (such as functional groups specified herein) are protected or blocked to avoid undesired reactions. Functional groups that can be protected are, for example, carboxyl groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups, etc., but are not limited thereto. Representative protecting groups for carboxyl groups are, for example, esters (such as p-methoxybenzyl ester), amides, and hydrazides, but are not limited thereto; representative protecting groups for amino groups are, for example, carbamates (such as tert-butoxycarbonyl) and amides, but are not limited thereto; representative protecting groups for hydroxyl groups are, for example, ethers and esters, but are not limited thereto; representative protecting groups for thiol groups are, for example, thioethers and thioesters, but are not limited thereto; representative protecting groups for carbonyl groups are, for example, acetals and ketals, but are not limited thereto.

[0039] As used herein, the term "amino acid" may be used interchangeably with "amino acid residue" and may refer to free amino acids and amino acid residues of peptides. It will be clear from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide. As described herein, "amino acid" is intended to include natural and synthetic amino acids and includes D- and L-amino acids. "Natural amino acid" refers to any of the twenty standard L-amino acids (including glycine) commonly found in naturally occurring peptides. As used herein, "D-" and "L-" amino acids, unless otherwise indicated, are not intended to exclude amino acids that do not have chirality, such as glycine. "Unnatural amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or derived from natural sources. As used herein, "synthetic amino acid" also includes chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and substituents. Amino acids contained in the peptides of the present invention may be modified by methylation, amidation, acetylation, or substitution with other chemical groups that can alter the circulatory half-life of the peptide without detrimentally affecting its activity, and particularly amino acids located at the C-terminus or N-terminus.

[0040] Embodiments

[0041] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt thereof, said compound being a compound of formula (I),

[0042] A—L—E (I)

[0043] wherein A is a fibroblast activation protein inhibitor moiety; L is a linker moiety; and E is a chelator moiety.

[0044] In some embodiments, the fibroblast activation protein inhibitor moiety is selected from the group consisting of first-generation FAP inhibitors and second-generation FAP inhibitors. In some embodiments, the fibroblast activation protein inhibitor moiety is selected from the group consisting of consisting of the group.

[0045] In some embodiments, the linker moiety is a moiety of formula (II),

[0046]

[0047] In some embodiments, n is an integer from 0 to 10, particularly 2.

[0048] In some embodiments, –Z– is composed of –Z1– and –Z2– in series, or is composed of –Z1–, –Z2–, and one or more –Z3– in series. In some embodiments, Z is composed of –Z1– and –Z2– in series, particularly Z is –Z1–Z2–. In some embodiments, Z is composed of –Z1–, –Z2–, and one or more –Z3– in series, particularly composed of –Z1–, –Z2–, and one –Z3– in series, more particularly Z is –Z1–Z2–Z3–. In some embodiments, Z is composed of –Z1–, –Z2–, and w –Z3– in series, where w is an integer from 0 to 10, particularly 0, 1, or 2, more particularly 0 or 1. In some embodiments, –Z– is –Z1–Z2– or –Z1–Z2–Z3–.

[0049] In some embodiments, the linker moiety is a moiety of formula (IIa) or a moiety of formula (IIb).

[0050]

[0051] In some embodiments, Z 1 is a basic amino acid residue, particularly a lysine residue or an arginine residue, or a derivative thereof, particularly an L-lysine residue, a D-lysine residue, an L-arginine residue, or a D-arginine residue, or a derivative thereof, more particularly, particularly an L-lysine residue or an L-arginine residue, or a derivative thereof. In some embodiments, Z 1 is selected from the group consisting of particularly consisting of more particularly consisting of wherein R 1 R 2 and R 3 are each independently selected from the group consisting of –H, halogen, –CH3, C1–C6 straight-chain alkyl, C1–C6 branched-chain alkyl, C1–C6 cycloalkyl, substituted aryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted anthracenyl, particularly selected from the group consisting of -H, halogen, –CH3, C1–C6 straight-chain alkyl, C1–C6 branched-chain alkyl, C1–C6 cycloalkyl, substituted benzyl, substituted or unsubstituted naphthalen-1-ylmethyl, and substituted or unsubstituted anthracen-9-ylmethyl. Wherein, the substitution is, for example, one or more hydrogens on the aromatic ring, more particularly the para-hydrogen on the aromatic ring, substituted by a halogen, such as fluorine, chlorine, bromine, or iodine. In some embodiments, R 3 is selected from the group consisting of

[0052] In some embodiments, Z 2 ​is a residue of an aliphatic amino acid (including alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), aspartic acid (Asp), etc.) or an aromatic amino acid (such as phenylalanine (-Phe), tryptophan (Trp), tyrosine (Tyr), etc.), or a derivative compound moiety containing an aliphatic amino acid residue or an aromatic amino acid residue, or an amino acid residue with a long-chain aliphatic structure in the side chain. In some embodiments, Z 2 is an aliphatic amino acid residue or an aromatic amino acid residue. In some embodiments, Z 2 is a residue of alanine, valine, leucine, isoleucine, methionine, aspartic acid, phenylalanine, tryptophan, or tyrosine.

[0053] In some embodiments, Z 3 is a functional group or an albumin binder.

[0054] In some embodiments, Z 3 is a functional group. In some embodiments, Z 3 or the functional group is selected from the group consisting of a polyethylene glycol chain, a rigid peptide (such as –(Ala–Pro)–, –(Pro–Ala–Pro–Ala–Pro)–, –(Glu–Ala–Ala–Ala–Lys)–, etc.), a flexible peptide (such as –(Gly)–, –(Gly–Gly–Gly–Gly–Ser)–, etc.), and a renal cleavage peptide (such as Gly–Tyr, Gly–Lys, Met–Val–Lys, Met–-Phe–Lys, Gly–-Phe–Lys, etc.). In some embodiments, m is an integer from 0 to 30, particularly 4 or 8. In some embodiments, Z 3 is selected from the group consisting of a polyethylene glycol chain, –(Ala–Pro)–, –(Pro–Ala–Pro–Ala–Pro)–, –(Glu–Ala–Ala–Ala–Lys)–, –(Gly)–, –(Gly–Gly–Gly–Gly–Ser)–, Gly–Tyr, Gly–Lys, Met–Val–Lys, Met–-Phe–Lys, Gly–-Phe–Lys, where m is an integer from 0 to 30, particularly 4 or 8.

[0055] In some embodiments, Z 3 is an albumin binder. In some embodiments, Z 3 or the albumin binder is

[0056]

[0057] where

[0058] p is an integer from 2 to 10, especially 2; and

[0059] X 1 is –H;

[0060] where y is an even number from 2 to 24; and

[0061] wherein,

[0062] X 2 is –H, –F, –Cl, –Br, –I, –(CH2)CH3, where k is an integer from 0 to 9, X 3 is H, and X 4 is H; or

[0063] X 2 is –CF3, X 3 is H or F, and X 4 is H or F.

[0064] In some embodiments,

[0065] A is

[0066] L is a moiety of formula (II), wherein

[0067] n is 2;

[0068] Z is –Z1–Z2–;

[0069] Z 1 is

[0070] where R 1 and R 2 are –CH3 and –H, respectively, or both are –CH3; or

[0071] where R 1 、R 2 、and R 3 are all –CH3; and

[0072] Z 2 is a phenylalanine residue; and

[0073] E is a 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid moiety.

[0074] In some embodiments, the chelator moiety is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA), diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 2,2′-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)hexan-2-yl)azanediyl)diacetic acid (NETA), 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A), ethylenebis(o-hydroxyphenyl)glycine (EHPG), N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED), 1,4,7,10-tetraazacyclododecane-α,α′,α″,α″′-tetramethyl-N,N′,N″,N″′-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyltetraacetic acid)11-(methyl tetraacetic acid), TETMA), ethylenediamine tetraacetic acid (EDTA), 1,3-propylenediaminetetraacetic acid (PDTA), triethylenetetraaminehexaacetic acid (TTHA), 1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM), 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), and 6-hydrazinonicotinic acid (HYNIC), and derivatives or moieties formed by reducing one or more hydrogens thereof, consisting of the group.

[0075] In one aspect, there is provided a radionuclide preparation comprising a compound of the present application or a pharmaceutically acceptable salt thereof, and a radionuclide chelated therewith. In some embodiments, the radionuclide comprises at least one selected from the group consisting of 44 Sc, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 99m Tc, 110m In, 111 In, 113m In, 114m In, 177 Lu, 188 Re, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 211 At, 223 Ra, and 225 Ac. In some embodiments, the radionuclide comprises at least one selected from the group consisting of 44 Sc,47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 90 Y, 99m Tc, 111 In, 177 Lu, 188 Re, 212 Pb, 213 Bi, 211 At, 223 Ra, and 225 at least one in the group consisting of Ac. In some embodiments, the radionuclide comprises 68 Ga or 177 Lu.

[0076] In one aspect, there is provided the use of a compound of the present application or a pharmaceutically acceptable salt thereof in the preparation of a radiotherapeutic agent. In one aspect, there is provided the use of a compound of the present application or a pharmaceutically acceptable salt thereof in radiotherapy. In one aspect, there is provided a compound of the present application or a pharmaceutically acceptable salt thereof for radiotherapy. In one aspect, there is provided a method of radiotherapy comprising administering a compound of the present application or a pharmaceutically acceptable salt thereof. In some embodiments, the radiotherapy targets tumors, fibrotic cells, or inflammation, particularly tumors, more particularly stromal fibroblasts of tumors. In some embodiments, the radiotherapeutic agent is a drug that targets tumors, fibrotic cells, or inflammation, particularly tumors, more particularly stromal fibroblasts of tumors. In some embodiments, the tumor is an epithelial-derived tumor, particularly a malignant tumor, more particularly breast cancer, gastric cancer, lung cancer, colorectal cancer, or ovarian cancer.

[0077] The present application provides a series of functional linkers and fibroblast activation protein inhibitor (FAPI) conjugates. By adding different functional linkers, the binding affinity with FAP can be effectively further enhanced, and the retention in tumors can be synergistically enhanced. Further, through the modification with a suitable albumin binder, the purpose of radiotherapy can be achieved. The functional linkers include basic amino acids, aliphatic / aromatic amino acids, functional sequences or groups.

[0078] Preliminary studies on the FAP protein structure and its molecular simulation docking with the key targeting groups of inhibitors show that the structure of the FAP active site has S1-S2 specific pockets. The S1 site in FAP is flat and can accommodate most amino acids. The S1 specific pocket in FAP is a pocket composed of Tyr 625 , Val 650 , Trp653 , Tyr 656 , Tyr 660 and Val 705 form a distinct hydrophobic pocket that can optimally accommodate a proline residue. This site is the main active site of current FAPI. The two glutamic acids (Glu 203 and Glu 204 ) at the active site of FAP protease are key amino acids for substrate recognition. These two acidic residues make the ligand-binding pocket of FAP protein negatively charged, which may contribute to substrate recognition and anchoring. Based on this, introducing basic amino acids (Arg, Lys, His, etc.) into the linker to interact with the two glutamic acid residues at the active site is beneficial for anchoring, thereby improving the affinity between the ligand and FAP protein.

[0079] Molecular docking simulation shows that the S2 active site pocket is defined by the residues Arg 123 , -Phe 350 , -Phe 351 , Tyr 541 , Pro 544 , Tyr 625 and Tyr 660 . These residues can interact with large aliphatic side chains, and large hydrophobic and aromatic residues can be modeled in the hydrophobic S2 pocket. Therefore, introducing aliphatic amino acids (Ala, Val, Leu, Ile, Met, Asp, etc.), aromatic amino acids (-Phe, Trp, Tyr, etc.), or amino acids with long-chain aliphatic side chains can improve the affinity between the ligand and FAP protein by enhancing the interaction with the S2 pocket of FAP protein.

[0080] In addition, introducing different functional sequences, such as flexible peptides, rigid peptides, renal cleavage peptides, etc., can ensure that the metal chelator / targeting group better exerts its respective functions and regulate the pharmacokinetics and pharmacodynamics of the drug.

[0081] Examples

[0082] I. Synthetic routes of HX05-2 (FAPI-Lys(CH3)-Phe-EDA-DOTA), HX05-3 (FAPI-Lys(CH3)2-Phe-EDA-DOTA), and HX05-4 (FAPI-Lys(CH3)3-Phe-EDA-DOTA)

[0083] The compound HX05-2 (FAPI-Lys(CH3)-Phe-EDA-DOTA) of this example is shown in the structure of formula (I), where:

[0084] A is

[0085] L is the moiety of formula (II), where

[0086] n is 2;

[0087] Z is –Z1–Z2–;

[0088] Z 1 is where R 1 and R 2 are –CH3 and –H, respectively; and

[0089] Z 2 is a phenylalanine residue; and

[0090] E is the 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) moiety.

[0091] The compound HX05-3 (FAPI-Lys(CH3)2-Phe-EDA-DOTA) of this example, as shown in the structure of formula (I), wherein:

[0092] A is

[0093] L is the moiety of formula (II), where

[0094] n is 2;

[0095] Z is –Z1–Z2–;

[0096] Z 1 is where R 1 and R 2 are both –CH3; and

[0097] Z 2 is a phenylalanine residue; and

[0098] E is the 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) moiety.

[0099] The compound HX05-4 (FAPI-Lys(CH3)3-Phe-EDA-DOTA) of this example, as shown in the structure of formula (I), wherein:

[0100] A is

[0101] L is the moiety of formula (II), where

[0102] n is 2;

[0103] Z is –Z1–Z2–;

[0104] Z1 where R 1 , R 2 , and R 3 are all –CH3; and

[0105] Z 2 is a phenylalanine residue; and

[0106] E is a 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) moiety.

[0107] 1. FAPI Synthesis

[0108]

[0109] 1.1 In a 25 mL round-bottom flask, dissolve 8-aminoquinoline-4-carboxylic acid (100 mg, 0.531 mmol, 1 eq), (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile salt (132 mg, 0.585 mmol, 1.1 eq), and HATU (202 mg, 0.531 mmol, 1 eq) in 900 μL of DMF and 4 mL of DCM.

[0110] 1.2 Add dropwise DIEA (371 μL, 2.127 mmol, 4 eq), and stir until the reaction is complete.

[0111] 1.3 Dilute the stock solution with DCM, wash with water, dry over Na2SO4, filter, and evaporate the solvent to obtain the dried product.

[0112]

[0113] 1.4 In a 25 mL round-bottom flask, dissolve the above product (48 mg, 0.134 mmol, 1 eq), succinic anhydride (669 mg, 6.683 mmol, 50 eq), and DMAP (8 mg, 0.067 mmol, 0.5 eq) in 3 mL of THF, and heat the reaction at 60 °C for 6 h.

[0114] 1.5 The reaction product is dried in vacuo, diluted with water, extracted with DCM, dried over Na2SO4, filtered, and concentrated. The dried product is separated by preparative separation using high-performance liquid chromatography (HPLC). The stationary phase is a semi-preparative C 18 column, and the mobile phase is eluted using a gradient elution method at a flow rate of 10 mL / min, changing from 20% acetonitrile to 60% acetonitrile in 40 minutes. The product is identified by electrospray ionization mass spectrometry (ESI-MS): m / z [M+H] + = chemical formula: 460.16, calculated molecular weight 459.14). The electrospray mass spectrum is asFigure 1 The horizontal axis is the mass-to-charge ratio (m / z), and the vertical axis is the relative intensity (%).

[0115] 2. Synthesis of FAPI-Lys(CH3)-Phe-EDA-DOTA

[0116] 2.1 Add Fmoc-EDA-(2-chlorotrityl chloride resin) (100 mg, 0.04 mmol) and 3 - 5 mL of dimethylformamide (DMF) into a solid-phase peptide synthesis tube, and place it on an oscillator to swell for 30 min.

[0117] 2.2 Add 20% piperidine (160 mL of DMF, 40 mL of piperidine, 3 - 5 mL) into the tube, and shake and wash the resin for about 1 h.

[0118] 2.3 Add HPLC-grade DMF (3 - 5 mL), shake and wash, and then filter by suction. Repeat this three times.

[0119] 2.4 Add Fmoc-L-phenylalanine (46.44 mg, 0.12 mmol) and DMF (5 mL) into the tube. Use N,N′-diisopropylethylamine (DIEA, 73.7 μL) to adjust the pH of the reaction system to 8 - 9, shake and react for 3 h, and then filter by suction.

[0120] 2.5 Repeat steps 2.2 and 2.3.

[0121] 2.6 Vacuum-dry and bake the above resin, and divide it into 3 tubes according to mass on average. Add Fmoc-L-lysine(CH3)(Boc) (57.84 mg, 0.12 mmol) and DMF (5 mL), Fmoc-L-lysine(CH3)2 (47.52 mg, 0.12 mmol) and DMF (5 mL), Fmoc-L-lysine(CH3)3 (49.32 mg, 0.12 mmol) and DMF (5 mL) into tubes 1, 2, and 3 (synthesizing HX05-2, HX05-3, and HX05-4 respectively). Use N,N′-diisopropylethylamine (DIEA, 73.7 μL) to adjust the pH of the reaction system to 8 - 9, shake and react for 3 h, and then filter by suction.

[0122] 2.7 Repeat steps 2.2 and 2.3.

[0123] 2.8 Add the product FAPI (55.2 mg, 0.12 mmol) from step 1 and DMF (5 mL) into tubes 1, 2, and 3 respectively. Use N,N′-diisopropylethylamine (DIEA, 73.7 μL) to adjust the pH of the reaction system to 8 - 9, shake and react for 3 h, and then filter by suction.

[0124] 2.9 Repeat step 2.3.

[0125] 2.10 Add 1% TFA / dichloromethane solution (3 - 5 mL) into tubes 1, 2, and 3 respectively to remove the resin. Shake and react for 1 - 3 h, then filter by suction, collect the filtrate, dry the dichloromethane with nitrogen, and then add cold ether. A white precipitate rapidly forms in the reaction flask. After ultrasonic treatment, transfer the turbid liquid in the reaction flask to a centrifuge tube and centrifuge at 1200 r / min for 15 min. Collect the bottom precipitate and dry it under vacuum to obtain compounds FAPI-Lys(CH3)(Boc)Phe-EDA, FAPI-Lys(CH3)2-Phe-EDA, and FAPI-Lys(CH3)3-Phe-EDA;

[0126] 2.11 Weigh FAPI-Lys(CH3)(Boc)Phe-EDA (8.9 mg, 0.01 mmol), FAPI-Lys(CH3)2-Phe-EDA (8 mg, 0.01 mmol), and FAPI-Lys(CH3)3-Phe-EDA (8.2 mg, 0.01 mmol) respectively, dissolve them in DMF (200 μL) and place them in a reaction flask. Add the metal chelator DOTA-NHS (10 mg, 0.02 mmol), and then use DIEA to adjust the pH of the system to 8 - 9. Stir and react at room temperature for 3 h under nitrogen protection. After the reaction is completed, rotary evaporate the liquid in the reaction flask with an oil pump, then add cold ether, centrifuge to collect the bottom precipitate, and dry it under vacuum to obtain compounds FAPI-Lys(CH3)(Boc)Phe-EDA-DOTA, FAPI-Lys(CH3)2-Phe-EDA-DOTA, and FAPI-Lys(CH3)3-Phe-EDA-DOTA.

[0127] 2.12 Remove the Boc protecting group: Take the compound FAPI-Lys(CH3)(Boc)Phe-EDA-DOTA, add 20% TFA / dichloromethane solution, shake and react for 0.5 h, dry the dichloromethane with nitrogen, then add cold ether, centrifuge to collect the bottom precipitate, and dry it under vacuum to obtain the compound FAPI-Lys(CH3)-Phe-EDA-DOTA.

[0128] 2.13 Subsequently, purify FAPI-Lys(CH3)-Phe-EDA-DOTA, FAPI-Lys(CH3)2-Phe-EDA-DOTA, and FAPI-Lys(CH3)3-Phe-EDA-DOTA by HPLC, freeze-dry to obtain the products, and identify the products by electrospray mass spectrometry (ESI-MS).

[0129] -FAPI-Lys(CH3)-Phe-EDA-DOTA (HX05 - 2): m / z [M + H] + = 1177.63 (chemical formula: C55 H 74 F2N 14 O 13 , calculate molecular weight: 1176.55);

[0130] -FAPI-Lys(CH3)2-Phe-EDA-DOTA (HX05-3): m / z [M+H] + = 1191.64 (chemical formula: C 56 H 76 F2N 14 O 13 , calculate molecular weight: 1190.57);

[0131] -FAPI-Lys(CH3)3-Phe-EDA-DOTA (HX05-4): m / z [M+H] + = 1205.63 (chemical formula: C 57 H 79 F2N 14 O 13 , calculate molecular weight: 1205.59).

[0132] The electrospray mass spectra are respectively as Figure 2A , Figure 2B , Figure 2C , with the horizontal axis being the mass-to-charge ratio (m / z) and the vertical axis being the relative intensity (%).

[0133] II. Stability study

[0134] 1. Stability Study of Precursor HX05-2

[0135] The stability of Example HX05-2 (1.0 μg / mL) in rat plasma was studied by the in vitro constant temperature (37 °C) incubation method. HX05-2 was dissolved in 1 mL of rat serum and incubated in vitro at a constant temperature for 0 h, 4 h, and 24 h. After reaching the specified time points, 300 μL of rat serum was taken, 300 μL of methanol was added, the supernatant was taken by centrifugation, and the volume was concentrated with nitrogen. Subsequently, LC-MS analysis was performed. The peak areas of the drug and the internal standard were measured by LC-MS / MS, and the ratio was used to replace the drug concentration for calculation to determine the remaining percentage of the drug. The results are as Figure 3 , and the LC-MS results showed that after incubation at 37 °C for 24 h, there were no obvious impurity peaks in the sample compared with the 0 h sample, the retention time was the same, the molecular weight was correctly verified by MS, and the remaining percentage of the drug could reach more than 95%.

[0136] 2. For HX05-2 68 Ga labeling and probe 68 Stability study of Ga-HX05-2

[0137] Use a syringe to take 0.05 mol / L HCl (about 4 - 5 mL) to leach 68 Ga3+ , 1.4 mL of the core segment with the highest radioactive dose was taken and placed in a reaction tube for subsequent labeling. The pH of the solution was adjusted to 4.0 using the prepared 0.25 mol / L sodium acetate solution (about 340 - 370 mL). The labeling precursor HX05-2 (1 nmol / mCi) was added to the reaction tube and incubated at 90 °C for 15 min to obtain 68 the Ga-HX05 conjugate. The product was monitored and quantitatively labeled by radio-high performance liquid chromatography, with a purity > 95%. The radioactive chromatogram is specifically as shown in Figure 4 .

[0138] The probe 68 Ga-HX05-2 (7.4×10 6 Bq) was dissolved in 200 μL of PBS solution or 200 μL of mouse serum, and then incubated at 37 °C for 2 h respectively. Subsequently, samples were taken for detection by Radio-HPLC. The Radio-HPLC results showed ( Figure 4 ), 68 when Ga-HX05-2 was incubated in normal saline and fetal bovine serum for 2 h, no obvious impurity peaks appeared, the retention time of the chromatographic peak remained at the same position, and the percentage of the parent compound was greater than 95%. Therefore, it was preliminarily considered that 68 Ga-HX05-2, 68 Ga-HX05-3, and 68 Ga-HX05-4 were stable in normal saline and fetal bovine serum.

[0139] III. Animal experiments

[0140] 1. Establishment of FAP-Transfected Human Fibrosarcoma (HT-1080-hFAP) Xenograft Tumor Model

[0141] BALB / C nude mice, 4 - 5 weeks old, with a body weight of about 20 g, were raised in a specific pathogen-free environment. 5×10 6 FAP-transfected human fibrosarcoma HT-1080-hFAP cells were collected, resuspended in an appropriate volume of PBS buffer, and then inoculated subcutaneously into the right axilla of the nude mice using an insulin needle. The tumor volume was monitored, and the calculation formula was: V = p / 6 × L × W × H (L represents the major axis of the tumor; W represents the minor axis perpendicular to the major axis; H represents the height of the tumor). When it grew to about 150 - 200 mm 3 , it could be used for the experiment.

[0142] 2. Positron Emission Tomography-Computed Tomography (PET-CT) of Small Animals

[0143] Tumor-bearing nude mice Tumor-bearing mice (n = 4) were injected via the tail vein with 68 Ga-HX05-2 (5×10 6After 0.5, 1, and 2 h at (Bq), anesthesia was induced with isoflurane / O₂ (2% V / V, flow rate 2 mL / min). Whole-body PET-CT imaging was obtained by static 15-min scanning using a small-animal NovelMedcal PET-CT scanner (Beijing Yongxin). The maximum tangential and radial full-width at half maximum in the center of the field of view was 1.5 mm, and the maximum tangential and radial full-width at half maximum at the edge of the field of view was 1.8 mm.

[0144] PET and CT images were acquired using NMSoft workstation software (Beijing Yongxin). The data were given as the percentage of the injected dose per gram of tissue or organ (ID / g) and were determined by decay correction for each sample (normalized to a known weight representing the injected dose). Normalization was performed during statistics, and standardized uptake values (SUVs) were calculated according to the following formula:

[0145] SUV = ([Bq / mL] × [animal body weight (g)] / [injected dose (Bq)])

[0146] The PET-CT results were as Figure 5 shown, 68 Ga-HX05-2, 68 Ga-HX05-3, and 68 Ga-HX05-4 could all rapidly identify tumor tissues with high FAP expression, and clear tumor contours were still visible at 2 h. At 0.5 h after injection, 68 Ga-HX05-2, 68 Ga-HX05-3, and 68 Ga-HX05-4 showed rapid and high uptake in xenograft tumors with high FAP expression, and the radioactive signal in the tumor could persist for more than 2 h. ROI analysis showed that 68 the SUV value of Ga-HX05-2 uptake in the tumor could reach 0.6, 68 Ga-HX05-2 could rapidly identify FAP-positive tumors and be rapidly cleared from non-target tissues, showing a good tumor-to-background ratio in HT-1080-hFAP tumor-bearing mice, and the T / M value was about 6.

[0147] 3. Biodistribution Study

[0148] HT-1080-hFAP tumor-bearing mice (n = 5) were weighed separately and injected via the tail vein with 68 Ga-HX05-2, 68 Ga-HX05-3, or 68 Ga-HX05-4 (5 × 10 6(Bq), and an equal volume of blank liquid medicine was aspirated as the control group. Two hours after drug injection, the nude mice were euthanized, and the corresponding organs (blood, heart, liver, spleen, lung, kidney, stomach, intestine, bone, muscle, tumor, etc.) were washed and weighed, and placed in radioimmunoassay tubes. The CPM values of each organ and the control group were measured with a γ counter, and the %ID was calculated as the ratio of the CPM of the experimental group to the CPM of the control group. The results of biodistribution were expressed as %ID / g.

[0149] The results of biodistribution were almost consistent with those of PET-CT ( Figure 6 ), 68 Ga-HX05-3 had a relatively high tumor uptake, but its background uptake was also relatively slightly higher; 68 Ga-HX05-3 and 68 Ga-HX05-4 had similar uptakes in the blood, both significantly higher than 68 Ga-HX05-2; 68 The uptake of Ga-HX05-4 in muscle was significantly higher than that of 68 Ga-HX05-2 and 68 Ga-HX05-3, but 68 Ga-HX05-2, 68 Ga-HX05-3, and 68 Ga-HX05-4 had no significant difference in the uptake in the kidneys of tumor-bearing mice.

[0150] 4. Statistical Analysis

[0151] GraphPad Prism 5 was used for data and image processing, and SPSS 17.0 was used for statistical analysis. One-way ANOVA and least significant difference t-test were used for data analysis. A p-value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001.

[0152] The above embodiments are only the preferred embodiments of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantive changes and substitutions made by those skilled in the art based on the present application belong to the scope of protection required by the present application.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is a compound of formula (I), A—L—E (I) wherein A is a fibroblast activation protein inhibitor moiety; L is a linker moiety; E is a chelator moiety, wherein the linker moiety is a moiety of formula (II), and wherein n is an integer from 0 to 10; –Z– is composed of –Z1– and –Z2– in series, or is composed of –Z1–, –Z2–, and one or more –Z3– in series; Z 1 is a basic amino acid residue; Z 2 is an aliphatic amino acid residue, an aromatic amino acid residue, a derivative compound moiety containing an aliphatic amino acid residue or an aromatic amino acid residue, or an amino acid residue with a long-chain aliphatic structure in the side chain; and Z 3 is a functional group or an albumin binder.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The fibroblast activation protein inhibitor moiety is selected from the group consisting of and the group consisting of.

3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, characterized in that Z 1 selected from the group consisting of wherein R 1 、R 2 、 and R 3 are each independently selected from the group consisting of –H, halogen, –CH3, C1–C6 straight-chain alkyl, C1–C6 branched-chain alkyl, C1–C6 cycloalkyl, substituted aryl, substituted or unsubstituted naphthyl, and substituted or unsubstituted anthracenyl, wherein said substitution is that one or more hydrogens on the aromatic ring are substituted by halogen.

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, characterized in that Z 2 is a residue of alanine, valine, leucine, isoleucine, methionine, aspartic acid, phenylalanine, tryptophan, or tyrosine.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, characterized in that the –Z– is composed of –Z1–, –Z2–, and one or more –Z3– in series; and Z 3 selected from the group consisting of a polyethylene glycol chain, –(Ala–Pro)–, –(Pro–Ala–Pro–Ala–Pro)–, –(Glu–Ala–Ala–Ala–Lys)–, –(Gly)–, –(Gly–Gly–Gly–Gly–Ser)–, Gly–Tyr, Gly–Lys, Met–Val–Lys, Met–Phe–Lys, and Gly–Phe–Lys, wherein m is an integer from 0 to 30.

6. The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, characterized in that the –Z– is composed of –Z1–, –Z2–, and one or more –Z3– in series; and Z 3 For wherein p is an integer from 2 to 10, especially 2; and X 1 is –H; wherein y is an even number from 2 to 24; and wherein, X 2 is –H, –F, –Cl, –Br, –I, –(CH2)CH3, where k is an integer from 0 to 9, X 3 is H, and X 4 is H; or X 2 is –CF3, X 3 is H or F, and X 4 is H or F.

7. The compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, characterized in that the chelator moiety is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid, 1,4,7-triazacyclononane-N,N′,N″-triacetic acid, diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid, 2,2′-((6-amino-1-(4,7-bis(carboxymethyl)-1,4,7-triazane-1-yl)hexane-2-yl)azanediyl)diacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, ethylenebis(orthohydroxyphenyl)glycine, N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid, 1,4,7,10-tetraazacyclododecane-α,α′,α″,α″′-tetramethyl-N,N′,N″,N″′-tetraacetic acid, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyltetraacetic acid), ethylenediaminetetraacetic acid, 1,3-propanediaminetetraacetic acid, triethylenetetraminehexaacetic acid, 1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tripyrogallol, 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene, and 6-hydrazinonicotinic acid, and derivatives or moieties formed by reducing one or more hydrogens thereof.

8. The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, characterized in that A is L is a moiety of formula (II), wherein n is 2; Z is –Z1–Z2–; Z 1 For wherein R 1 and R 2 are –CH3 and –H, respectively, or both are –CH3; or wherein R 1 , R 2 , and R 3 are all –CH3; and Z 2 is a phenylalanine residue; and E is a 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid moiety.

9. A radioactive nuclide preparation, characterized in that, Comprising: A compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof; and a radionuclide chelated with the compound or a pharmaceutically acceptable salt thereof. Optionally, the radionuclide includes at least one selected from the group consisting of 44 Sc, 47 Sc, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 99m Tc, 110m In, 111 In, 113m In, 114m In, 177 Lu, 188 Re, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 211 At, 223 Ra, and 225 Ac; Optionally, the radionuclide includes at least one selected from the group consisting of 44 Sc, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 90 Y, 99m Tc, 111 In, 177 Lu, 188 Re, 212 Pb, 213 Bi, 211 At, 223 Ra, and 225 Ac; Optionally, the radionuclide includes 68 Ga or 177 Lu.

10. Use of a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, or a radionuclide preparation according to claim 9, in the manufacture of a radiotherapeutic agent. Optionally, the radiotherapeutic agent is a drug targeting tumors, fibrotic cells, or inflammation, particularly tumors, more particularly stromal fibroblasts of tumors; Optionally, the tumor is an epithelial-derived tumor, particularly a malignant tumor, more particularly breast cancer, gastric cancer, lung cancer, colorectal cancer, or ovarian cancer.

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