FAP-alpha and MMPs dual-targeting compound as well as radiolabeled derivative and application thereof

Abstract

The invention discloses an FAP-alpha and MMPs dual-targeting compound as well as a radiolabeled derivative and application thereof, and belongs to the crossing field of pharmaceutical chemistry and radiopharmaceutical chemistry. The compound has a structure as shown in a formula I, and can be simultaneously combined with FAP-alpha (IC50 is less than or equal to 10nM) and MMPs (such as MMP-9 IC50 is less than or equal to 20nM) in a tumor microenvironment with high affinity through modular design of a conjugate planar ring system and a specific amino acid chain (Y-Z). By replacing the effect group 3R with different radionuclides / chelating agents, the diagnosis and treatment integrated nuclide probe can be constructed. According to the double-targeting strategy, the residence time of the probe in tumors is obviously prolonged (2-10 times) compared with that of a single-targeting FAPI drug, and the target / non-target ratio is improved, so that the drug dosage and toxicity are reduced. The compound and the composition are suitable for diagnosis and treatment of FAP / MMP positive solid tumors (such as ovarian cancer and lung cancer) and fibrotic diseases.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of medicinal chemistry and radiopharmaceutical chemistry, specifically relating to a dual-targeting compound of FAP-α and MMPs, its radiolabeled derivatives, and its application in the preparation of drugs for diagnosing and / or treating FAP-α and MMP-positive related diseases. Background Technology

[0002] Fibroblast activator protein-α (FAP-α) is a type II transmembrane serine protease that functions as a homodimer and possesses unique dipeptidyl peptidase and endopeptidase activities. Under physiological conditions, FAP-α is expressed at extremely low levels in most adult tissues. However, in over 90% of epithelial-derived malignancies (such as pancreatic cancer, breast cancer, and colorectal cancer), FAP-α is specifically highly expressed on the surface of tumor-associated fibroblasts (CAFs). FAP-α plays a crucial role in tumor development, progression, invasion, metastasis, and drug resistance through multiple mechanisms, including extracellular matrix degradation, angiogenesis promotion, induction of epithelial-mesenchymal transition, and immunosuppression. Therefore, FAP-α has become a highly promising molecular target for tumor diagnosis and targeted therapy.

[0003] FAPI-based targeted radiopharmaceuticals (FAPIs) using small molecule inhibitors are currently a research hotspot in the field of nuclear medicine. A series of FAPI drugs developed using UAMC-1110 as a lead compound (such as...) 68 Ga-FAPI-04, 18 F-FAPI-42 and other similar drugs have demonstrated precise imaging capabilities for various tumors in preclinical and clinical studies. However, existing FAPI drugs generally suffer from problems such as short retention time within tumor lesions and unsatisfactory target / background (especially tumor / muscle) ratios, which severely limit their application in conjunction with long-half-life therapeutic radionuclides (such as F-FAPI-42 and F-FAPI-42). 177 Lu, 22 The lack of matching with 5Ac limits its application in targeted radiotherapy.

[0004] The tumor microenvironment is a highly complex and dynamic ecosystem. Besides extracellular matrix enzymes (CAFs), various cellular components (such as tumor cells and immune cells) secrete large amounts of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9. The MMP family, along with FAP-α, also drives malignant tumor progression by degrading the extracellular matrix, promoting angiogenesis, and facilitating immune escape. Therefore, the tumor stroma region is often a region where FAP-α and multiple MMPs are co-expressed.

[0005] Currently, FAP-targeted drug development mainly focuses on improving single-target affinity and selectivity for FAP-α. Although published FAP inhibitors (such as the compound described in CN114716505B) have improved tumor uptake to some extent through structural modification, their design approach still does not break out of the single-target framework and fails to fully utilize the co-expression and functional synergy of FAP-α and MMPs in the tumor microenvironment. Therefore, developing a novel molecule that can simultaneously target and bind to both FAP-α and MMPs is crucial to achieve a dual-anchoring strategy through a single administration, significantly prolonging drug retention time at the tumor site, improving diagnostic and treatment efficiency, and reducing dosage and potential toxicity.

[0006] This invention addresses the problem of insufficient retention time and difficulty in achieving efficient integrated diagnosis and treatment of single-target FAPI drugs in existing technologies. It innovatively designs and synthesizes a class of dual-targeting compounds that possess both high FAP-α inhibitory activity and high MMP recognition ability, aiming to overcome the above-mentioned defects and provide novel drug candidates for the accurate diagnosis and efficient treatment of FAP and / or MMP-positive diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a dual-targeting compound for FAP-α and MMPs, its radiolabeled derivatives, and their applications. This compound exhibits high inhibitory activity against FAP-α and high affinity for MMPs, thus addressing the shortcomings of existing single-targeting drugs in terms of tumor retention and treatment efficiency.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a dual-targeting compound for FAP-α and MMPs, which is a compound having the structure shown in Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, or radiolabeled derivative thereof: Formula I; in, 1 R and 2 R is independently selected from hydrogen, deuterium, halogen, methyl, halomethyl, methoxy, trifluoromethoxy, hydroxy, or cyano; 3 R is selected from radionuclides, radioactive metal chelating groups, fluorescent groups, toxic groups, or contrast agents; 4 R and 5 R is independently selected from N, C, -C=C-, or -C=N-, and 4 R, 5 R, together with the atoms attached to it, forms a five-membered, six-membered, or seven-membered carbon ring or a nitrogen-containing heterocycle, and its ring system is a conjugated planar system. X is selected from O, S, -CH2- or -N(CH3)-; Y is selected from nonpolar hydrophobic amino acid residues or polar neutral amino acid residues, and Z is selected from polar neutral amino acid residues. Nonpolar hydrophobic amino acids include phenylalanine (Phe), alanine (Ala), leucine (Leu), methionine (Met), isoleucine (Ile), tryptophan (Trp), proline (Pro), and valine (Val); polar neutral amino acids include cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asn), serine (Ser), tyrosine (Tyr), and threonine (Thr). n1, n2, and n3 each represent the number of repeating units, where n1 is an integer from 0 to 10, n2 is an integer from 0 to 3, and n3 is an integer from 0 to 10; The compound has an inhibitory concentration (IC50) of 5'-to-6' against fibroblast activator protein-α. 50 It has a molecular weight of ≤10 nM and an affinity for at least one matrix metalloproteinase.

[0009] Secondly, the present invention provides a radionuclide probe targeting FAP-α and MMPs, which is a radionuclide-labeled compound of Formula I and a radionuclide labeling unit. The radionuclide labeling unit comprises a radionuclide chelating group and a radionuclide.

[0010] Radioactive metal chelating groups are selected from the following structural formulas: DOTA and its derivatives: ; DTPA and its derivatives: ; NOTA and its derivatives: ; or: .

[0011] The radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide is selected from... 11 C 18 F, 61 Cu、 64 Cu、 66 Ga、 68 Ga、 89 Zr、 99m Tc, 111 In、 113m In、 124 I or 131 At least one of I; The therapeutic radionuclide is selected from... 47 Sc、67 Cu、 90 Y、 131 I, 161 Tb, 177 Lu、 211 At、 212 Pb, 213 Bi、 225 Ac or 277 At least one of Th.

[0012] Thirdly, a pharmaceutical composition is provided, comprising a therapeutically effective amount of the compound of formula I above, or a pharmaceutically acceptable salt, hydrate, solvate, radiolabeled derivative thereof, or the aforementioned nuclide probe, and at least one pharmaceutically acceptable carrier or excipient. The pharmaceutical composition is preferably an injectable preparation, such as a sterile injection or a lyophilized powder for injection.

[0013] Fourthly, the present invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, radiolabeled derivative thereof, or the above-described radionuclide probe, or the above-described pharmaceutical composition, in the preparation of an agent for the diagnosis and / or treatment of FAP-α and MMPs-related diseases.

[0014] Furthermore, the FAP-α and MMPs-positive associated diseases are solid tumors or fibrotic diseases. The solid tumors are selected from ovarian cancer, lung cancer, pancreatic cancer, breast cancer, glioma, head and neck squamous cell carcinoma, or esophageal cancer.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) Through its unique conjugated planar ring system and tunable Y and Z amino acid chains, it achieves simultaneous high affinity binding to two key targets in the tumor microenvironment, FAP-α and MMPs; this dual-targeting strategy produces a significant synergistic effect.

[0016] (2) It exhibits a significantly prolonged retention time in tumor lesions co-expressing FAP / MMP. Animal experiments show that the retention time can be increased by 2 to 10 times, thus better matching the physical half-life of therapeutic radionuclides.

[0017] (3) While prolonging the retention time, it can obtain a higher target / non-target tissue ratio (such as tumor / muscle ratio) and better imaging contrast; it helps to locate lesions more accurately and deliver radiation dose more efficiently.

[0018] (4) Due to the improved targeting efficiency, the amount of compound and radionuclide required to achieve the same therapeutic effect can be reduced by 1 to 5 times, which is expected to significantly reduce systemic toxic side effects.

[0019] (5) Adopting a modular design, through adjustment 3The R group can be flexibly converted into diagnostics (such as linking). 68 Ga) or treatment (such as connection) 177 The Lu (Lu) application truly realizes integrated diagnosis and treatment. At the same time, its broad range of FAP / MMP positive indications covers a variety of refractory solid tumors and fibrotic diseases. Attached Figure Description

[0020] Figure 1 This is a synthesis roadmap for FAPI involved in Example 1.

[0021] Figure 2 The image shows the hydrogen nuclear magnetic resonance spectrum of FAPI-02 synthesized in Example 1.

[0022] Figure 3 The image shows the hydrogen NMR spectrum of FAPI-04 synthesized in Example 1.

[0023] Figure 4 The image shows the proton NMR spectrum of the FAPI-04 precursor synthesized in Example 1.

[0024] Figure 5 This is the synthetic route diagram of the dual-target molecule involved in Example 2.

[0025] Figure 6 The high-resolution mass spectra and proton NMR spectra of compound 13-1 synthesized in Example 2 are shown.

[0026] Figure 7 This is a high-resolution mass spectrum of compound 13-2 synthesized in Example 2.

[0027] Figure 8 This is a high-resolution mass spectrum of compound 14-1 synthesized in Example 2.

[0028] Figure 9 This is a high-resolution mass spectrum of compound 14-2 synthesized in Example 2.

[0029] Figure 10 for 68 Graph showing the efficiency and stability of radiolabeling of Ga-compound 15.

[0030] Figure 11 The graph shows the in vitro inhibitory activity and molecular docking binding energy of compounds 11, 14-1, and 14-2 against FAP-α and MMP-9.

[0031] Figure 12 for 68 Ga-compound 15 and control 68 MicroPET / CT images and time-activity curves of Ga-FAPI-04 in a tumor-bearing mouse model.

[0032] Figure 13 for 68 Bar chart showing the biodistribution data of Ga-compound 15 in tumor-bearing mice. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0034] Example 1: Synthesis of FAPI compounds.

[0035] like Figure 1 The synthetic route shown uses Fmoc-protected amino acid resin as the starting material and employs a standard Fmoc solid-phase peptide synthesis strategy, sequentially coupling the desired amino acid and quinoline carboxylic acid module. All coupling reactions are carried out under conditions where HBTU / HOBt / DIPEA is used as the condensing agent. Finally, the side-chain protecting groups are cleaved and removed in a trifluoroacetic acid (TFA) / water mixture, purified by high-performance liquid chromatography (HPLC), and lyophilized to obtain the target FAPI compound 11.

[0036] Get Figure 1 The proton NMR spectrum of compound 9 in the sample, as shown below. Figure 2 and Figure 3 As shown, Figure 2 The above is the 1H NMR spectrum of the synthesized DOTA-free FAPI-02. Figure 3 The 1H NMR spectrum of FAPI-04 with a protecting group is shown below. The 1H NMR spectrum of compound 10 is shown below. Figure 4 As shown, the molecular structure and purity of the compound in this embodiment are verified by nuclear magnetic resonance hydrogen spectrum.

[0037] Example 2: Synthesis of dual-target molecules (using compounds 13 and 14 as examples) like Figure 5 The synthetic route shown involves introducing a YZ amino acid sequence module capable of recognizing MMPs at a specific position after synthesizing the FAPI core framework via an amide coupling reaction. Key intermediates and the final product were purified by HPLC to obtain compound 13, which targets two molecules.

[0038] The YZ amino acid sequence in compound 13 was polymerized once and twice to form compounds 13-1 and 13-2, respectively. High-resolution mass spectra (HR-MS) and ¹H NMR spectra of compounds 13-1 and 13-2 were obtained for confirmation. Figure 6 and Figure 7 As shown.

[0039] Based on the dual-targeting molecule synthesized in Example 2, the chelating agent DOTA was introduced at appropriate sites (usually at the amino terminus or via an additional linker) using solid-phase or liquid-phase synthesis methods to obtain radiolabeled precursor compounds 14-1 and 14-2; high-resolution mass spectrometry (HR-MS) confirmed the presence of compounds 14-1 and 14-2. Figure 8 and Figure 9 As shown.

[0040] Subsequently, the previous body and radioactive nuclide were... 68 Ga underwent a complexation reaction in a suitable buffer system (such as sodium acetate buffer) to obtain compound 15. After optimization of the labeling conditions, a labeling rate of over 95% was achieved by reacting at 50°C for 30 minutes. The radiochemical purity of the labeled product was analyzed by thin-layer chromatography (TLC) or high-performance liquid chromatography (Radio-HPLC), such as... Figure 10 As shown, the product exhibits good stability (>2 hours).

[0041] In vitro bioactivity evaluation FAP-α inhibitory activity assay: A fluorescence substrate method was used. Recombinant human FAP-α protein, the test compound (serially diluted), and the fluorescent substrate (e.g., Z-Gly-Pro-AMC) were mixed in the detection buffer and incubated at 37°C for a specified time. Fluorescence intensity (Ex / Em = 380 / 460 nm) was measured using a microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated using the GraphPad Prism software with a sigmoidal dose-response model. 50 ).

[0042] MMPs (using MMP-9 as an example) inhibitory activity assay: The method is similar, using recombinant human MMP-9 protein and its specific fluorescent substrate (e.g., Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2). The inhibition rate of different concentrations of the compound on MMP-9 enzyme activity was measured, and the IC50 was calculated. 50 .

[0043] Using software such as Molecular Operating Environment (MOE) or AutoDock, compounds 14-1, 14-2, and 11 were docked into the active pockets of the crystal structures of FAP-α and MMP-9, and the binding free energy was calculated and predicted.

[0044] Experimental results: such as Figure 11 As shown in Table 1, the representative dual-targeting compounds in this embodiment not only maintained excellent FAP-α inhibitory activity comparable to the FAPI standard (IC50), but also demonstrated... 50(≤10 nM), while exhibiting nanomolar-level high affinity for MMP-9. Molecular docking results also support its strong binding ability to both targets.

[0045] Table 1. Biological experimental data and binding energy calculation data compound <![CDATA[FAP-α IC 50 ]]> <![CDATA[MMP-9 IC 50 ]]> FAP-α Affinity MMP-9 Affinity 11 7.5nM 7.5μM -9.2 kcal / mol / 14-1 7.8nM 10.2nM -8.7 kcal / mol -8.2 kcal / mol 14-2 8.0nM 9.2nM -9.0 kcal / mol -8.8 kcal / mol .

[0046] In vivo evaluation of radiolabeled substances MicroPET / CT imaging of tumor-bearing mouse models; establishment of subcutaneous xenograft models expressing FAP and MMPs. Approximately 5-7 MBq of [a specific drug / method] was injected via the tail vein. 68 Ga-labeled probe of the present invention ( 68 Ga-compound 15) or control probe ( 68 Ga-FAPI-04). Small animal PET / CT imaging was performed at different time points after injection (30 min, 1 h, 2 h, 4 h).

[0047] Experimental results: such as Figure 12 As shown, with 68 Compared with Ga-FAPI-04, 68 Ga-compound 15 showed faster, higher, and more sustained uptake at tumor sites. 68 Ga-FAPI-04 reaches peak tumor uptake 1 hour after injection and is then rapidly cleared from the tumor. 68 The tumor radioactivity signal of Ga-compound 15 remained at a high level throughout the observation period (up to 4 hours), and the tumor retention time was significantly prolonged. Quantitative analysis showed that... 68 The peak normalized uptake (SUV) of Ga-compound 14-1 in tumors and its retention after 2 hours are: 68 It is 2 to 5 times that of Ga-FAPI-04.

[0048] After injecting the radioactive probe, the tumor-bearing mice were sacrificed at a selected time point, and the major organs and tumor tissues were removed, weighed, and the radioactivity count was measured using a gamma counter. The percentage injection dose rate per gram of tissue (%ID / g) was calculated.

[0049] Experimental results: such as Figure 13 As shown, the biological distribution data is consistent with the imaging results, confirming... 68 Ga-compound 15 exhibited a higher %ID / g value and a slower clearance rate in tumors. Simultaneously, its clearance rate in blood and most non-target organs (such as muscle and bone) was comparable to or faster than the control, resulting in significantly improved tumor / muscle (T / M) and tumor / blood (T / B) ratios, demonstrating superior targeting specificity and therapeutic potential.

[0050] In summary, this invention provides a novel class of FAP-α / MMP dual-targeting compounds. This series of compounds achieves a dual improvement in diagnostic sensitivity and therapeutic efficacy by synergistically targeting two key proteases in the tumor microenvironment, demonstrating great application potential in the integrated diagnosis and treatment of tumors and other fibroproliferative diseases.

[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A dual-targeting compound for FAP-α and MMPs, characterized in that, The compound is a compound having the structure shown in Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, or radiolabeled derivative thereof: Equation I in, 1 R and 2 R is independently selected from hydrogen, deuterium, halogen, methyl, halomethyl, methoxy, trifluoromethoxy, hydroxy, or cyano; 3 R is selected from radionuclides, radioactive metal chelating groups, fluorescent groups, toxic groups, or contrast agents; 4 R and 5 R is independently selected from N, C, -C=C-, or -C=N-, and 4 R, 5 R, together with the atoms attached to it, forms a five-, six-, or seven-membered carbon ring or a nitrogen-containing heterocycle, and its ring system is a conjugated planar system. X is selected from O, S, -CH2- or -N(CH3)-; Y is selected from nonpolar hydrophobic amino acid residues or polar neutral amino acid residues; Z is selected from polar neutral amino acid residues; n1, n2, and n3 each represent the number of repeating units, where n1 is an integer from 0 to 10, n2 is an integer from 0 to 3, and n3 is an integer from 0 to 10; The compound has an inhibitory concentration (IC50) of 5'-to-6' against fibroblast activator protein-α. 50 It has a molecular weight of ≤10 nM and an affinity for at least one matrix metalloproteinase.

2. The FAP-α and MMPs dual-targeting compound according to claim 1, characterized in that, The Y is selected from at least one of the following amino acid residues: Nonpolar hydrophobic amino acids include phenylalanine (Phe), alanine (Ala), leucine (Leu), methionine (Met), isoleucine (Ile), tryptophan (Trp), proline (Pro), and valine (Val); polar neutral amino acids include cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asn), serine (Ser), tyrosine (Tyr), and threonine (Thr).

3. The FAP-α and MMPs dual-targeting compound according to claim 1, characterized in that, The Z is selected from at least one of the following amino acid residues: Cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asn), serine (Ser), tyrosine (Tyr), threonine (Thr).

4. The FAP-α and MMPs dual-targeting compound according to claim 1, characterized in that, The 3 The radioactive metal chelating group of R is selected from the following structural formulas: DOTA and its derivatives: ； DTPA and its derivatives: ； NOTA and its derivatives: ； or: 。 5. The FAP-α and MMPs dual-targeting compound according to claim 4, characterized in that, The radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide is selected from... 11 C 18 F, 61 Cu、 64 Cu、 66 Ga、 68 Ga、 89 Zr、 99m Tc, 111 In、 113m In、 124 I or 131 At least one of I; The therapeutic radionuclide is selected from... 47 Sc、 67 Cu、 90 Y、 131 I, 161 Tb, 177 Lu、 211 At、 212 Pb, 213 Bi、 225 Ac or 277 At least one of Th.

6. The FAP-α and MMPs dual-targeting compound according to claim 1, characterized in that, The matrix metalloproteinases include MMP-1, MMP-2, MMP-9, and MMP-14.

7. The FAP-α and MMPs dual-targeting compound according to claim 1, characterized in that, The compound is selected from at least one of the following specific compounds: 。 8. A radionuclide probe targeting FAP-α and MMPs, characterized in that, The radionuclide probe is a dual-targeting compound of FAP-α and MMPs and a radionuclide labeling unit as described in any one of claims 1 to 7, wherein the radionuclide labeling unit comprises a radionuclide chelating group and a radionuclide.

9. A pharmaceutical composition, characterized in that, The compound comprising a therapeutically effective amount of the dual-targeting compound of FAP-α and MMPs as described in any one of claims 1 to 7 or the radionuclide probe as described in claim 8, and at least one pharmaceutically acceptable carrier or excipient.

10. The use of the FAP-α and MMPs dual-targeting compound of any one of claims 1 to 7, or the radionuclide probe of claim 8, or the pharmaceutical composition of claim 9 in the preparation of an agent for the diagnosis and / or treatment of FAP-α and matrix metalloproteinase-positive related diseases; in, The diseases associated with positive FAP-α and matrix metalloproteinases are solid tumors or fibrotic diseases.

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