COMPOUND, PHARMACEUTICAL COMPOSITION AND KIT

专利摘要:
the present invention relates to a compound of formula (i), a pharmaceutical composition comprising or consisting of said compound, a kit comprising or consisting of said compound or pharmaceutical composition and use of the compound or pharmaceutical composition in the diagnosis or treatment of a disease characterized by overexpression of fibroblast activating protein (fap).
公开号:BR112020015985A2
申请号:R112020015985-9
申请日:2019-02-06
公开日:2020-12-15
发明作者:Uwe Haberkorn；Anastasia Loktev；Thomas Lindner；Walter Mier；Frederik Giesel；Clemens KRATOCHWIL
申请人:Universität Heidelberg；
IPC主号:

专利说明:

[001] The present invention relates to a compound, a pharmaceutical composition comprising or consisting of said compound, a kit comprising or consisting of said compound or pharmaceutical composition and use of the compound or pharmaceutical composition in the diagnosis or treatment of a disease characterized by overexpression of fibroblast activating protein (FAP). BACKGROUND OF THE INVENTION
[002] [002] The growth and spread of the tumor is determined not only by the cancer cells, but also by the non-malignant constituents of the malignant lesion, which are included in the term stroma. The stroma can represent more than 90% of the tumor mass in tumors with desmoplastic reaction such as breast, colon and pancreatic carcinoma. In particular, it is known that a subpopulation of fibroblasts called cancer-associated fibroblasts (CAFs) is involved in tumor growth, migration and progression. Therefore, these cells represent an attractive target for antitumor diagnosis and therapy.
[003] [003] A distinguishing feature of CAFs is the expression of seprase or α-fibroblast activating protein (FAP-α), a type II membrane-bound glycoprotein belonging to the dipeptidyl peptidase 4 (DPP4) family. FAP-α has dipeptidyl peptidase and endopeptidase activity. Endopeptidase activity distinguishes FAP-α from other members of the DPP4 family. The substrates identified for endopeptidase activity to date are denatured Type I collagen, α 1-antitrypsin and various neuropeptides. FAP-α plays a role in normal developmental processes during embryogenesis and tissue modeling. It is not, or only at negligible levels, expressed in normal adult tissues. However, high expression occurs in wound healing, arthritis, arterosclerotic plaques,
[004] [004] The appearance of FAP-α in CAFs in many epithelial tumors and the fact that overexpression is associated with a worse prognosis in cancer patients led to the hypothesis that FAP-α activity is involved in the development of cancer, as well as in the migration and spread of cancer cells. Therefore, targeting this enzyme for imaging and endoradiotherapy can be considered a promising strategy for the detection and treatment of malignant tumors. The present inventors developed a small molecule based on a specific inhibitor of FAP-α and were able to show specific uptake, rapid internalization and successful imaging of tumors in animal models, as well as in tumor patients. A comparison with the commonly used 18F-fluorodeoxyglucose radioactive tracker (18F-FDG) revealed a clear superiority of the new FAP-α ligand in patients with locally advanced pulmonary adenocarcinoma. Thus, the present invention provides, among others: (i) detection of minor primary tumors and, therefore, the possibility of early diagnosis, (ii) detection of minor metastases and, therefore, a better assessment of the tumor stage, (iii) precise intraoperative guidance facilitating complete surgical removal of tumor tissue, (iv) better differentiation between inflammation and tumor tissue, (v) more accurate staging of patients with tumors, (vi) better monitoring of tumor lesions after antitumor therapy, (vii ) the opportunity to use the molecules as teranostic agents for diagnosis and therapy. In addition, the molecules can be used for the diagnosis and treatment of non-malignant diseases, such as chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorders. BRIEF DESCRIPTION OF THE INVENTION
[005] [005] In a first aspect, the present invention provides a compound of Formula (I)
[006] [006] In a second aspect, the present invention relates to a pharmaceutical composition comprising or consisting of at least one compound of the first aspect and, optionally, a pharmaceutically acceptable carrier and / or excipient.
[007] [007] In a third aspect, the present invention relates to the compound of the first aspect or the pharmaceutical composition of the second aspect for use in the diagnosis or treatment of a disease characterized by overexpression of fibroblast activating protein (FAP) in an animal or human being.
[008] [008] In a fourth aspect, the present invention relates to a kit that comprises or consists of the compound of the first aspect or the pharmaceutical composition of the second aspect and instructions for the diagnosis or treatment of a disease. LIST OF FIGURES
[009] [009] The following describes the content of the figures included in this specification. In this context, see also the detailed description of the invention above and / or below.
[010] [010] Figure 1: In vitro characterization of 125I-FAPI-01 and 177Lu- FAPI-02: A. Binding of radiolabelled FAPI-01 and FAPI-02 to different human cancer cell lines, as well as cell lines transfected with FAP- human α (HT-1080-FAP), murine FAP-α (HEK-muFAP) and human CD26 (HEK-CD26) after 60 minutes of incubation. B.
[011] [011] Figure 2: Specificity of binding and relative rates of internalization of FAPI derivatives: A-C. Connection and internalization rates from FAPI-03 to FAPI-15 in relation to FAPI-02 (defined as 100%). Internalization rates after 1, 4 and 24 hours of incubation are shown in gray; the extracellular bound fraction is represented by the white bars. D. Binding of selected FAPI derivatives to HEK cells that express murine FAP-α and human CD26 after 60 minutes of incubation. Right side: Reason for connecting muFAP to CD26. E. Competitive binding of selected FAPI derivatives to HT-1080-FAP cells after adding increasing concentrations of unlabeled compound.
[012] [012] Figure 3: Imaging of FAPI-02 and -04 in mice having human tumor xenografts positive for FAP (HT-1080-FAP) and negative (Capan-2, SK-LMS-1): A + C, E + G. PET imaging of small animals was performed after intravenous administration of 4 nmol 68Ga-FAPI-02 and -04 (10 MBq resp.) At the indicated times. The radioactive tracker is rapidly enriched within the tumor (indicated by the red arrow) while it does not accumulate in non-cancerous tissue. In addition, rapid elimination is seen through the kidneys and bladder. B + D, F + H. The quantification of PET images demonstrates a solid clearance (clearance) of 68Ga-FAPI-02 and -04 from the cardiovascular system and a constant uptake in the tumor.
[013] [013] Figure 4: Blocking experiments to analyze the specificity of binding in vivo: A + D. Blocking of 68Ga-FAPI-02 and -04 tumor accumulation by co-administration of 30 nmol unlabeled compound in mice having HT-1080-FAP tumor. B + C, E + F. 68Ga- FAPI-02 and -04 time-activity curves in selected organs after intravenous administration with and without a compound not marked as competitor.
[014] [014] Figure 5: Distribution of 177Lu-FAPI-02 and -04 organs in nude mice with HT-1080-FAP: A-C tumor. The biodistribution of 177Lu-FAPI-02 and -04 was measured ex vivo at the indicated times after intravenous administration of 1 MBq to mice having FAP-positive human HT-1080 tumor xenografts; n = 3 for each instant. The indicated values are expressed as a percentage of injected dose per gram of tissue (% ID / g). Radioactive trackers have been shown to accumulate within the tumor that expresses FAP, showing the greatest enrichment after 1 hour for FAPI-02 (4.5% ID / g) and 2 hours for FAPI-04 (5.4% ID / g). D-F. Tumor / normal tissue ratios of 177Lu-FAPI-02 and -04 1, 4 and 24 hours after intravenous administration.
[015] [015] Figures 6-9: PET / CT imaging of FAPI-02 in cancer patients: 6A-C. Maximum intensity projections (MIP) of PET / CT scans in a patient suffering from metastatic breast cancer. D.
[016] [016] Figures 10-16: PET / CT imaging of FAPI-04 in cancer patients:
[017] [017] Figure 17: Competitive binding of selected FAPI derivatives to HT-1080-FAP cells after adding increasing concentrations of unlabeled compound (10-10 to 10-5 M, incubation for 60 minutes, n = 3).
[018] [018] Figure 18: Binding of FAPI derivatives to HEK cells that express murine FAP and human CD26 after 60 minutes of incubation, n = 3. Values are expressed as percentage of applied dose (% ID) per 1 million cells.
[019] [019] Figure 19: Biodistribution of selected FAPI derivatives in HT-1080-FAP xenotransplants 1, 4 and 24 hours after intravenous administration of the radioactive trackers, n = 3. Values are expressed as percentage of dose injected per gram of tissue ( % ID / g).
[020] [020] Figure 20: TAP / blood ratio of FAPI derivatives selected in HT-1080-FAP xenografts 1, 4 and 24 hours after intravenous administration of radioactive scanners, n = 3.
[021] [021] Figure 21: PET imaging of FAPI-21 and FAPI-46 labeled with Ga-68 in mice having HT-1080-FAP tumor; n = 1.
[022] [022] Figure 22: Maximum standardized uptake values (SUV) of selected FAPI derivatives in mice with HT-1080-FAP tumor; n = 1.
[023] [023] Figure 23: Maximum (Max SUV, Figure 23 A) and average (Medium SUV, Figure 23 B) uptake values of FAPI-02 and FAPI-04 marked with Ga-68 in cancer patients; n = 25.
[024] [024] Figure 24: Intraindividual comparison of 6 patients with 6 different tumor entities submitted to FDG-PET and FAPI-PET imaging within <9 days.
[025] [025] Figure 25: PET / CT imaging of FAPI-04 marked with Ga-68 in patients with carcinomatous peritonitis (A), myocarditis (B) and arthrosis of the hip joint (C) 1 hour p.i.
[026] [026] Figure 26: PET / CT imaging of FAPI-21 labeled with Ga-68 in cancer patients 1 hour p.i.
[027] [027] Figure 27: PET / CT imaging of FAPI-46 labeled with Ga-68 1 hour p.i. and Sm-153-labeled FAPI-46 intra-therapeutic imaging 30 minutes p.i. in cancer patients.
[028] [028] Figure 28: Intra-therapeutic imaging of Sm-153-labeled FAPI-46 up to 20 hours p.i.
[029] [029] Figure 29: A. Projection of maximum intensity (MIP) 1 hour after intravenous administration of 68Ga-FAPI-46 to a patient with metastatic colorectal carcinoma. B. Bremsstrahlung imaging 2 hours after therapeutic treatment with 90Y-FAPI-46 from the same patient.
[030] [030] Figure 30: PET / CT imaging of Ga-68 labeled FAPI-46 1 hour p.i. in patients with lung cancer with idiopathic pulmonary fibrosis. A, B. The maximum uptake of the tracker in tumor tissue is significantly greater than in non-exacerbated fibrotic lesions. C. The maximum uptake of the tracker in tumor tissue is slightly less than in exacerbated fibrotic tissue.
[031] [031] Figure 31: A. Tc-99m-labeled FAPI-19 binding to HT-1080-FAP cells, n = 3. B. Competitive Tc-99m-labeled FAPI-19 binding to HT-1080-FAP cells after adding increasing concentrations of unlabeled compound (10-10 to 10-5 M, incubation for 60 minutes, n = 3). Ç.
[032] [032] Figure 32: A. Tc-99m-labeled FAPI-34 binding to HT-1080-FAP cells, n = 3. B. Tc-99m-labeled FAPI-34 scintigraphy in HT-1080-FAP xenografts, n = 1.
[033] [033] Figure 33: Tc-99m-labeled FAPI-34 scintigraphy in a patient with metastatic pancreatic carcinoma.
[034] [034] Figure 34: A. Binding of Pb-203-labeled FAPI derivatives to HT-1080-FAP cells, n = 3. B. Efflux kinetics of derivatives of Pb-203
[035] [035] Figure 35: Scintigraphy of FAPI-04 and FAPI-46 labeled with Pb-203 in HT-1080-FAP xenografts, n = 1.
[036] [036] Figure 36: Biodistribution of FAPI-04 and FAPI-46 labeled with Pb-203 in HT-1080-FAP xenografts 1, 4, 6 and 24 hours after intravenous administration of the radioactive scanners, n = 3. Values are expressed as a percentage of injected dose per gram of tissue (% ID / g).
[037] [037] Figure 37: A. Binding of FAPI-42 and FAPI-52 labeled with Cu-64 to HT-1080-FAP cells, n = 3. B. Competitive binding of FAPI-42 and FAPI-52 labeled with Cu- 64 to HT-1080-FAP cells after adding increasing concentrations of unlabeled compound (10 -10 to 10-5 M, incubation for 60 minutes, n = 3). C. Kinetics of the efflux of FAPI-42 and FAPI-52 labeled with Cu-64 after incubation of HT-1080-FAP cells with radiolabeled compound for 60 minutes and consequent incubation with non-radioactive medium for 1 to 24 hours, n = 3.
[038] [038] Figure 38: PET imaging of FAPI-42 and FAPI-52 labeled with Cu-64 in mice with HT-1080-FAP tumor; n = 1.
[039] [039] Figure 39: PET imaging of FAPI-42 and FAPI-52 labeled with AlF-18 in mice with HT-1080-FAP tumor; n = 1.
[040] [040] Figure 40: a. PET imaging of small animal of FAPI-02 labeled with 68Ga in nude mice having U87MG tumor up to 140 minutes after intravenous administration of the radioactive tracker. The tumor is indicated by the red arrow. B. Biodistribution of 177Lu-labeled FAPI-02 and FAPI-04 in nude mice having U87MG tumor 1, 4 and 24 hours after intravenous administration of radioactive scanners; n = 3.
[041] [041] Figure 41: Tumor-organ ratios of FAPI-02 and -04 marked with 177Lu in mice with U87MG tumor 1, 4 and 24 hours after intravenous administration.
[042] [042] Figure 42: Projection of maximum intensity (MIP) of PET / CT scans in a patient with glioblastoma 10 minutes, 1 and 3 hours after the administration of 68Ga-FAPI-02.
[043] [043] Figure 43: Exemplary images (T1-weighted MRI with increased contrast, FAPI-PET and fused images of both embodiments) HDI of glioblastomas in weight, gliomas with HDI mutant WHO grade II and glioblastomas with HDI- mutant.
[044] [044] Figure 44: Absolute SUVmax values for all 18 gliomas.
[045] [045] Figure 45: Statistical analysis of SUVmax / BG values.
[046] [046] Figure 46: Dose-dependent inhibition of FAP enzyme activity by FAPI-04 and Talabostat. Unlike Talabostat, a potent DPP4 inhibitor with marginal FAP activity, FAPI-04 demonstrates robust and dose-dependent FAP inhibition.
[047] [047] Figure 47: Recapture of FAPI-04 and FAPI-46 labeled with 177Lu in HT-1080-FAP cells. After incubating the cells with the radioactive scanners for 60 minutes at 37 ° C, the compounds are removed and the non-radioactive medium with (+ Comp.) And without (- Comp.) Unlabeled compound is added and incubated for 10 minutes at 6 hours. In the first ten minutes of incubation, a new uptake of unmarked FAPI derivatives occurs, displacing parts of the radiolabeled fraction, which results in significantly lower radioactivity values compared to the pure medium without a competitor. After 6 hours of incubation, there was an almost complete displacement of the radiolabelled FAPIs. These findings indicate a continuous reuptake of intact FAP molecules back to the cell membrane after initial internalization, allowing renewed binding and internalization of FAP ligands.
[048] [048] Figure 48: Organ distribution of 177Lu-labeled FAPI-04 after single and multiple injection in nude mice with HT-1080-FAP tumor. Administration of two equal doses of 177Lu-FAPI-04 at 4-hour intervals results in increased total organ activities, including the tumor, measured 8 and 24 hours after the first injection. On the other hand, the administration of three doses (higher initial dose, lower subsequent doses) does not reveal changes in general organ activities.
[049] [049] Figure 49: Binding of F-18-FAPI derivatives to HT1080 cells that express human FAP after 10, 30, 60 and 90 minutes of incubation, n = 3. Values are expressed as percentage of applied dose (% ID ) per 1 million cells.
[050] [050] Figure 50: PET imaging of FAPI-74 and FAPI-52 labeled with AlF-18 in mice having HT-1080-FAP tumor; n = 1.
[051] [051] Figure 51: Biodistribution of FAPI-75 in HT-1080-FAP xenotransplants 1, 4 and 24 hours after intravenous administration of the radioactive scanner, n = 3. Values are expressed as percentage of injected dose per gram of tissue (% ID / g).
[052] [052] Figure 52: PET imaging of a patient with non-small cell lung cancer: Robust accumulation of FAPI-74 marked with F18 in various metastases.
[053] [053] Figure 53: Time-activity curves of the cardiac region
[054] [054] Figure 54: FAPI-02 and FAPI-04 at different times of imaging (10 minutes, 1 hours and 3 hours p.i.) in two patients with metastatic breast cancer. Rapid tumor targeting and rapid blood clearance are followed by a long plateau phase with no relevant change in the image contrast (top). In comparison with FAPI-02, the ligand FAPI-04 is characterized by a prolonged tumor retention time (bottom).
[055] [055] Figure 55: The effective dose of FAPI-02 was 1.80E-02 mSv / MBq calculated with OLINDA (1.82E-02 with IDAC1 / ICRP60, 1.79E-02 with IDAC2 / ICRP103). The effective dose for FAPI-04 PET / CT was 1.64E-02 mSv / MBq calculated with OLINDA (1.66E-02 with IDAC1 / ICRP60, 1.35E-02 with IDAC2 / ICRP103). If the scan is delayed at 3 pm p.i. were omitted in clinical practice, the routine activity for a FAPI exam could be reduced to 200 MBq 68Ga; consecutively, the radiation dose for this FAPI-PET / CT scan would be 3-4 mSv.
[056] [056] Figure 56: A) 68Ga-FAPI-04 after 1 hour post injection in different tumor entities in PET / CT. The highest mean SUVmax (> 12) was found in sarcoma, esophageal cancer, breast, cholangiocellular carcinoma and lung cancer. The lowest FAPI uptake (mean SUVmax <6) was observed in renal cell carcinoma, differentiated from the thyroid, adenoid-cystic, gastric carcinoma and pheochromocytoma. The average SUVmax of hepatocellular carcinoma, colorectal carcinoma, head-neck cancer, ovarian carcinoma and pancreatic carcinoma was intermediate (SUV 6 <x <12). Within all tumor entities, a high inter-individual variation was observed. Due to low background activity (SUV 2), tumor / background ratios are> 2 times in the intermediate and> 4 times in the high intensity uptake group. B) Primary tumor entities showed similar SUV absorption compared to tumor entities using FAPI-04.
[057] [057] Figure 57: Exemplary PET images of different tumor entities that were used for the quantifications shown in Figure 56 A-B. DETAILED DESCRIPTION OF THE INVENTION
[058] [058] Before the present invention is described in detail below, it should be understood that this invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary.
[059] [059] Preferably, the terms used here are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
[060] [060] Throughout this specification and the following claims, unless the context otherwise requires, the word "understand" and variations such as "understand" and "comprising" will be understood to imply the inclusion of an integer declared, step or group of whole numbers or steps, but not the exclusion of any other whole number or step or group of whole numbers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect thus defined can be combined with any other aspect or aspects, unless clearly indicated to the contrary. In particular, any characteristic indicated as being optional, preferred or advantageous can be combined with any other characteristic or characteristics indicated as being optional, preferred or advantageous.
[061] [061] Several documents are cited throughout the text of this specification. Each of the documents cited here (including all patents, patent applications, scientific publications, manufacturer specifications, instructions etc.), above or below, are hereby incorporated by reference in their entirety. Nothing in this document should be construed as an admission that the invention has no right to precede such disclosure by virtue of the previous invention. Some of the documents cited here are characterized as being "incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings cited in this specification, the text of this specification will take precedence.
[062] [062] In the following, the elements of the present invention will be described.
[063] [063] Below are some definitions of terms frequently used in this specification. These terms will have, in each case of their use in the rest of the specification, the meaning defined respectively and the preferred meanings.
[064] [064] As used in this specification and the appended claims, the singular forms "one", "one" and "o", "a" include plural referents, unless the content clearly indicates otherwise.
[065] [065] In the following definitions of the terms: alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, alkenyl and alkynyl are provided. These terms, in each case of their use in the rest of the specification, will have the meaning defined respectively and the preferred meanings.
[066] [066] The term "alkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain comprises from 1 to 10 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, methyl, ethyl methyl, ethyl, propyl, isopropyl , butyl, isobutyl, tert-butyl, pentyl, hexyl, pentyl or octyl. Alkyl groups are optionally substituted.
[067] [067] The term "heteroalkyl" refers to a saturated straight or branched carbon chain. Preferably, the chain comprises 1 to 9 carbon atoms, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, octyl, which is interrupted one or more times, for example, 1, 2, 3, 4, 5, with the same or different heteroatoms. Preferably, heteroatoms are selected from O, S and N, for example -O-CH3, -S-CH3, -CH2-O-CH3, -CH2-O-C2H5, -CH2-S-CH3, - CH2-S-C2H5, -C2H4-O-CH3, -C2H4-O-C2H5, -C2H4-S-CH3, -C2H4-S-C2H5 etc. Heteroalkyl groups are optionally substituted.
[068] [068] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, represent, unless otherwise stated,
[069] [069] The term "aryl" preferably refers to an aromatic monocyclic ring containing 6 carbon atoms, an aromatic bicyclic ring system containing 10 carbon atoms or an aromatic tricyclic ring system containing 14 carbon atoms. Examples are phenyl, naphthyl or anthracenyl. The aryl group is optionally substituted.
[070] [070] The term "aralkyl" refers to an alkyl portion, which is replaced by aryl, where alkyl and aryl have the meaning described above. An example is the benzyl radical. Preferably, in this context, the alkyl chain comprises from 1 to 8 carbon atoms, that is, 1, 2, 3, 4, 5, 6, 7 or 8, for example, methyl, ethyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butenyl, tert-butyl, pentyl, hexyl, pentyl, octyl. The aralkyl group is optionally substituted on the alkyl and / or aryl part of the group.
[071] [071] The term "heteroaryl" preferably refers to a five or six membered aromatic monocyclic ring, in which at least one of the carbon atoms is replaced by 1, 2, 3 or 4 (for the five membered ring) or 1, 2, 3, 4 or 5 (for the six-membered ring) of the same or different heteroatoms, preferably selected from O, N and S; an aromatic bicyclic ring system in which 1, 2, 3, 4, 5 or 6 carbon atoms of 8, 9, 10, 11 or 12 carbon atoms have been replaced with the same or different hetero atoms, preferably selected from O, N and S; or an aromatic tricyclic ring system in which 1, 2, 3, 4, 5 or 6 carbon atoms of the 13, 14, 15 or 16 carbon atoms have been replaced with the same or different hetero atoms, preferably selected from O , N and S. Examples are oxazolyl, isoxazolyl, 1,2,5-oxadiazolyl, 1,2,3-oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, thiazolyl, isothiazolyl, 1,2,3 -thiadiazolyl 1,2,5-thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1-benzofuranyl, 2-benzofuranyl,
[072] [072] The term "heteroaralkyl" refers to an alkyl portion, which is replaced by heteroaryl, where alkyl and heteroaryl have the meaning described above. An example is 2-alkylpyridinyl, 3-alkylpyridinyl or 2-methylpyridinyl. Preferably, in this context, the alkyl chain comprises from 1 to 8 carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7 or 8, for example, methyl, ethyl methyl, ethyl, propyl, isopropyl , butyl, isobutyl, sec-butenyl, tert-butyl, pentyl, hexyl, pentyl, octyl. The heteroaralkyl group is optionally substituted on the alkyl and / or heteroaryl part of the group.
[073] [073] The terms "alkenyl" and "cycloalkenyl" refer to olefinic unsaturated carbon atoms containing chains or rings with one or more double bonds. Examples are propenyl and cyclohexenyl. Preferably, the alkenyl chain comprises from 2 to 8 carbon atoms, that is, 2, 3, 4, 5, 6, 7 or 8, for example, ethylene, 1-propenyl, 2-propenyl, isopropenyl, 1 -butenyl, 2-butenyl, 3-butenyl, iso-butenyl, sec-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, hexenyl, pentenyl, octenyl. Preferably, the cycloalkenyl ring comprises 3 to 8 carbon atoms, i.e. 3, 4, 5, 6, 7 or 8, for example, 1-cyclopropenyl, 2-cyclopropenyl, 1-cyclobutenyl, 2-cilcobutenyl, 1 -cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, cyclohexenyl, cyclopentenyl, cyclooctenyl.
[074] [074] The term "alkynyl" refers to unsaturated carbon atoms containing chains or rings with one or more triple bonds. An example is the radical propargila. Preferably, the alkynyl chain comprises from 2 to 8 carbon atoms, that is, 2, 3, 4, 5, 6, 7 or 8, for example, ethynyl, 1-
[075] [075] In one embodiment, the carbon atoms or hydrogen atoms in the alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals can be replaced independently of each other with one or more elements selected from the group consisting of O, S, N or with groups containing one or more elements selected from the group consisting of O, S, N.
[076] [076] Embodiments include alkoxy, cycloalkoxy, aricoxy, aralkoxy, alkenyloxy, cycloalkenyloxy, alkynyloxy, alkylthio, cycloalkylthio, arylthio, aralkylthio, alkenylthio, cycloalkenylthio, alkynylthioalkylamino, cycloalkylamino, cycloalkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, aralkylamino, arylaminoamino,
[077] [077] Other embodiments include hydroxyalkyl radicals hidroxicicloalquila, hidroxiarila, hidroxiaralquila, hidroxialquenila, hidroxicicloalquenila, hidroxialinila, mercaptoalquila, mercaptocicloalquila, mercaptoarila, mercaptoaralquila, mercaptoalquenila, mercaptocicloalquenila, mercaptoalquinila, aminoalkyl, aminocicloalquila, aminoarila, aminoaralquila, aminoalquenila, aminocicloalquenila, aminoalquinila .
[078] [078] In another embodiment, the hydrogen atoms in alkyl, heteroalkyl, cycloalkyl, aryl, aralkyl, alkenyl, cycloalkenyl, alkynyl radicals can be substituted independently of one another with one or more halogen atoms. A radical is the trifluoromethyl radical.
[079] [079] If two or more radicals or two or more residues can be selected independently of each other, then the term "independently" means that the radicals or residues can be the same or different.
[080] [080] As used herein, a formulation that defines the limits of a length range, such as, for example, "from 1 to 6" means any integer from 1 to 6, that is, 1, 2, 3, 4 , 5 and 6. In other words, any interval defined by two integers explicitly mentioned is intended to understand and reveal any integer that defines said limits and any integer included in that interval.
[081] [081] The term “halo”, as used here, refers to a halogen residue selected from the group consisting of F, Br, I and Cl. Preferably, the halogen is F.
[082] [082] The term "binder", as used herein, refers to any chemically suitable binder. Preferably, the ligand is not or is only slowly cleaved under physiological conditions. Thus, it is preferred that the linker does not comprise recognition sequences for proteases or recognition structures for other degrading enzymes. Since it is preferred that the compounds of the invention are administered systemically to allow ample access to all compartments of the body and subsequently enrich the compounds of the invention in any part of the body where the tumor is located, it is preferable that the ligand is chosen over so that it is not or is just slowly cleaved into the blood. Cleavage is considered to be slow if less than 50% of the ligands are cleaved 2 hours after administration of the compound to a human patient. Suitable binders, for example, comprise or consist of optionally substituted alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, sulfonyl, amine, ether phosphines, phosphoramidates, carboxamides, esters, imidoesters, amidines, thioesters, sulfonamides, 3-thiopyrrolidine-2,5-dion, carbamates,
[083] [083] The term "optionally substituted" refers to a group in which one, two, three or more hydrogen atoms may have been substituted independently of each other by the respective substituents.
[084] [084] As used herein, the term "amino acid" refers to any organic acid containing one or more amino substituents, for example, a-, β- or γ-amino, derived from aliphatic carboxylic acids. In the polypeptide notation used here, for example, Xaa5, that is, Xaa1Xaa2Xaa3Xaa4Xaa5, where Xaa1 to Xaa5 are each and independently selected from amino acids, as defined, the left direction is the direction of the amino terminus and the right direction is the direction of the carboxy terminal, according to standard usage and convention.
[085] [085] The term "conventional amino acid" refers to the twenty naturally occurring amino acids and covers all stereoisomeric isoforms, that is, amino acids D, L, D and L of them. These conventional amino acids can also be referred to here by their conventional three letter or one letter abbreviations and their abbreviations follow conventional usage (see, for example, Immunology - A Synthesis, 2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates , Sunderland Mass.
[086] [086] The term “unconventional amino acid” refers to unnatural amino acids or chemical amino acid analogs, for example, α, α-disubstituted amino acids, N-alkyl amino acids, homo-amino acids, dehydroamino acids, aromatic amino acids (except phenylalanine, tyrosine and tryptophan) and ortho-, meta- or para-aminobenzoic acid.
[087] [087] The term "aromatic or non-aromatic mono or bicyclic heterocycle containing N", as used herein, refers to a saturated or unsaturated cyclic hydrocarbon compound that contains at least one nitrogen atom as a constituent of the cyclic chain.
[088] [088] The term "radioactive moiety", as used herein, refers to a molecular assembly that carries a radioactive nuclide. The nuclide is linked by covalent or coordinated bonds that remain stable under physiological conditions. Examples are [131I] -3-iodobenzoic acid or 68Ga- DOTA.
[089] [089] A "fluorescent isotope", as used here, emits electromagnetic radiation after excitation by electromagnetic radiation of a shorter wavelength.
[090] [090] A “radioisotope”, as used herein, is a radioactive isotope of an element (included by the term “radioactive nuclide”) that emits α, β and / or γ radiation.
[091] [091] The term "radioactive drug" is used in the context of the present invention to refer to a biological active compound that is modified by a radioisotope. Especially intercalating substances can be used to provide radioactivity for direct DNA proximity (for example, a derivative of Hoechst-33258 that contains 131I).
[092] [092] The term "chelating agent" or "chelate" is used interchangeably in the context of the present invention and refers to a molecule, often organic, and often a Lewis base, with two or more electron pairs not available for donation to a metal ion. The metal ion is usually coordinated by two or more electron pairs for the chelating agent. The terms "bidentate chelating agent", "tridentate chelating agent" and "four-chelating agent" refer to chelating agents having, respectively, two, three and four electron pairs readily available for simultaneous donation to a metal ion coordinated by the chelating agent . Generally, the electron pairs of a chelating agent form coordinated bonds with a single metal ion; however, in certain instances, a chelating agent can form coordinated bonds with more than one metal ion, a variety of bonding modes being possible.
[093] [093] The term "fluorescent dye" is used in the context of the present invention to refer to a compound that emits visible or infrared light after excitation by shorter and more appropriate wavelength electromagnetic radiation. It is understood by the person skilled in the art that each fluorescent dye has a predetermined excitation wavelength.
[094] [094] The term "contrast agent" is used in the context of the present invention to refer to a compound that increases the contrast of structures or fluids in medical imaging. Enhancement is achieved by absorbing electromagnetic radiation or by changing electromagnetic fields.
[095] [095] The term “paramagnetic”, as used here, refers to paramagnetism induced by unpaired electrons in a medium. A paramagnetic substance induces a magnetic field if an external magnetic field is applied. Unlike diamagnetism, the direction of the induced field is the same as the external field and, unlike ferromagnetism, the field is not maintained in the absence of an external field.
[096] [096] The term "nanoparticle", as used here, refers to particles preferably of spherical shape, with diameters in sizes between 1 and 100 nanometers. Depending on the composition, nanoparticles may have magnetic, optical or physico-chemical qualities that can be evaluated. In addition, surface modification is possible for many types of nanoparticles.
[097] [097] The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention. Suitable pharmaceutically acceptable salts of the compound of the present invention include acid addition salts which can, for example, be formed by mixing a solution of choline or derivative thereof with a solution of a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, where the compound of the invention has an acidic portion, suitable pharmaceutically acceptable salts thereof can include alkali metal salts (for example, sodium or potassium salts); alkaline earth metal salts (for example, calcium or magnesium salts); and salts formed with suitable organic binders (for example, ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate,
[098] [098] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
[099] [099] In addition to salt forms, the present invention provides compounds that are in the form of a prodrug. The prodrugs of the compounds described herein are those compounds that easily undergo chemical changes under physiological conditions to provide a compound of formula (I). A prodrug is an active or inactive compound that is chemically modified by physiological action in vivo, such as hydrolysis, metabolism and the like, in a compound of this invention after administration of the prodrug to a patient. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme. The suitability and techniques involved in the manufacture and use of prodrugs are well known to those skilled in the art. For a general discussion of prodrugs involving esters, see Svensson and Tunek, Drug Metabolism Reviews 16.5 (1988) and Bundgaard, Design of Prodrugs, Elsevier (1985). Examples of a masked carboxylate anion include a variety of esters, such as alkyl (eg, methyl, ethyl), cycloalkyl (eg, cyclohexyl), aralkyl (eg, benzyl, p-methoxybenzyl) and alkylcarbonyloxyalkyl (eg example, pivaloyloxymethyl). The amines were masked as substituted arylcarbonyloxymethyl derivatives that are cleaved by esterases in vivo releasing the free drug and formaldehyde
[0100] [0100] The compounds according to the invention can be synthesized according to one or more of the following methods. It should be noted that the general procedures are shown with respect to the preparation of compounds with unspecified stereochemistry. However, these procedures are generally applicable to compounds of a specific stereochemistry, for example, where the stereochemistry over a group is (S) or (R). In addition, compounds with a stereochemistry (e.g., (R)) can often be used to produce those with opposite stereochemistry (i.e., (S)) using well-known methods, for example, by inversion.
[0101] [0101] Certain compounds of the present invention can exist in unsolvated forms, as well as in solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are intended to be within the scope of the present invention.
[0102] [0102] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers should all fall within the scope of the present invention.
[0103] [0103] The compounds of the present invention may also contain unnatural proportions of atomic isotopes in one or more of the atoms that constitute such compounds. For example, the compounds can be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, should be included in the scope of the present invention.
[0104] [0104] The term "pharmaceutical composition", as used in this application, refers to a substance and / or a combination of substances being used for the identification, prevention or treatment of a tissue condition or disease. The pharmaceutical composition is formulated to be suitable for administration to a patient, in order to prevent and / or treat diseases. In addition, a pharmaceutical composition refers to the combination of an active agent with a vehicle, inert or active, making the composition suitable for therapeutic use. The pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalation, rectal, sublingual, transdermal, subcutaneous or vaginal routes, according to their chemical and physical properties. The pharmaceutical compositions comprise solid, semi-solid, liquid transdermal therapeutic systems (TTS). The solid compositions are selected from the group consisting of tablets, coated tablets, powder, granules, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also included are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention. The semi-solid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, oral tablets and suppositories.
[0105] [0105] "Pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the US Pharmacopoeia or other pharmacopoeia generally recognized for use in animals and, more particularly, in humans.
[0106] [0106] The term "vehicle", as used herein, refers to a diluent, adjuvant, excipient or carrier with which the therapeutic agent is administered. Such pharmaceutical vehicles can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, of animal, vegetable or synthetic origin, such as peanut oil, soy oil, mineral oil, sesame oil and the like. A saline solution is a preferred vehicle when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.
[0107] [0107] The term "fibroblast activating protein (FAP)", as used herein, is also known under the term "seprase". Both terms can be used interchangeably here. The fibroblast activating protein is an integral homodimeric protein with dipeptidyl peptidase type IV (DPPIV) fold, presenting an alpha / beta-hydrolase domain and an eight-blade beta helix domain. WAYS OF ACCOMPLISHMENT
[0108] [0108] In the following, the different aspects of the invention are defined in more detail. Each aspect thus defined can be combined with any other aspect or aspects, unless clearly indicated to the contrary. In particular, any characteristic indicated as being preferred or advantageous can be combined with any other characteristic or characteristics indicated as being preferred or advantageous.
[0109] [0109] In a first aspect, the present invention provides a compound of Formula (I) (I), in which Q, R, U, V, W, Y, Z are individually present or absent, provided that at least three of Q, R, U, V, W, Y, Z are present; Q, R, U, V, W, Y, Z are selected independently from the group consisting of O, CH2, NR4, C = O, C = S, C = NR4, HCR4 and R4CR4, with the proviso that two O's are not directly adjacent to each other; preferably, of the six, four groups are present, two of which are C = O, one is CH2 and one is NH; more preferably, four groups are present, two of which are C = O, one is CH2 and one is NH; more preferably, V, W, Y and Z are present, of which V and Z are C = O and W and Y are selected independently from CH2 and NH; R1 and R2 are independently selected from the group consisting of -H, -OH, halo, C1-6 alkyl, -O-C1-6 alkyl, S-C1-6 alkyl; R3 is selected from the group consisting of -H, -CN, - B (OH) 2, -C (O) -alkyl, -C (O) -aryl-, -C = CC (O) -aryl, -C = CS (O) 2-aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2 and 5-tetrazolyl; R4 is selected from the group consisting of -H, -C1- alkyl
[0110] [0110] In a preferred embodiment, A and E together form a group selected from the group consisting of a monocyclic heterocycloalkyl C3, C4, C5, C6, C7 and C8, preferably monocyclic C5 or C6 or bicyclic C7 , C8, C9, C10, C11 or C12, preferably bicyclic C7, C8, C9 and C10, comprising 1, 2, 3 or 4, preferably 1 or 2 hetero atoms independently selected from the group consisting of N, O and S, preferably N and O, more preferably 1 or 2 N.
[0111] [0111] In a preferred embodiment of the first aspect of the present invention, a compound of Formula (I) is provided: (I), wherein Q, R, U, V, W, Y, Z are individually present or absent ,
[0112] [0112] In another preferred embodiment of the first aspect of the present invention A and E together form a group consisting of a monocyclic heterocycloalkyl C3, C4, C5, C6, C7 and C8, preferably monocyclic C5 or C6 or bicyclic C7 , C8, C9, C10, C11 or C12, preferably bicyclic C7, C8, C9 and C10, preferably comprising 1, 2, 3 or 4, more preferably 1 or 2 hetero atoms selected independently from the group consisting of N, O and S, preferably N and O, more preferably 1 or 2 N. The preferred monocyclic heterocycloalkyls are selected from the group consisting of pyrrolidinyl, piperidinyl, imidazolidinyl, 1,2-diazacyclohexanyl, 1,3-diazacyclohexanyl, piperazinyl, 1-oxo -2-azacyclohexanil, 1-oxo-3-azacyclohexanyl or morpholinyl, preferably piperidinyl, piperazinyl and pyrrolidinyl. Preferred bicyclic heterocycloalkyls are selected from the group consisting of bicycles
[0113] [0113] The bond between the heterocycle formed by A and E and B, on the one hand, and / or R6 or R7, on the other, is preferably through the heteroatom, preferably through N.
[0114] [0114] In particular, preferred examples of the heterocycle formed by A and E are selected from the group consisting of,,,, and.
[0115] [0115] In a preferred embodiment of the first aspect of the present invention, Q, R, U are CH2 and are individually present or absent; preferably, Q and R are absent; V is CH2, C = O, C = S or C = NR4; preferably, V is C = O; W is NR4; preferably, W is NH; Y is HCR4; preferably, Y is CH2; and Z is C = O, C = S or C = NR4, preferably, Z is C = O.
[0116] [0116] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is CH2; W is NH; Y is CH2; and
[0117] [0117] In another preferred embodiment of the first aspect of the present invention, R1 and R2 are selected independently from the group consisting of -H and halo; preferably, R1 and R2 are halo; more preferably, R1 and R2 are F; R3 is selected from the group consisting of -H, -CN and - B (OH) 2; preferably, R3 is -CN or -B (OH) 2; more preferably, R3 is - CN; R4 is selected from the group consisting of -H and -C1-6alkyl, where -C1-6alkyl is optionally substituted with 1 to 3 substituents selected from -OH. Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0118] [0118] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is CH2; W is NH; Y is CH2; Z is C = O; R1 and R2 are selected independently from the group consisting of -H and halo; preferably, R1 and R2 are halo; more preferably, R1 and R2 are F; R3 is selected from the group consisting of -H, -CN and - B (OH) 2; preferably, R3 is -CN or -B (OH) 2; more preferably, R3 is - CN; R4 is selected from the group consisting of -H and -alkyl
[0119] [0119] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is CH2; W is CH2; Y is NH; Z is C = O; R1 and R2 are selected independently from the group consisting of -H and halo; preferably, R1 and R2 are halo; more preferably, R1 and R2 are F; R3 is selected from the group consisting of -H, -CN and - B (OH) 2; preferably, R3 is -CN or -B (OH) 2; more preferably, R3 is - CN; R4 is selected from the group consisting of -H and -C1-6alkyl, where -C1-6alkyl is optionally substituted with 1 to 3 substituents selected from -OH. Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0120] [0120] In another preferred embodiment of the first aspect of the present invention, it is selected from the group consisting of,
[0121] [0121] In another preferred embodiment of the first aspect of the present invention, it is optionally further comprising 1 or 2 heteroatoms selected from O, N and S.
[0122] [0122] In another preferred embodiment of the first aspect of the present invention, it is selected from the group consisting of,,,,,,,,,,,,
[0123] [0123] In a preferred embodiment, it is selected from the group consisting of,, and.
[0124] [0124] In another preferred embodiment it is.
[0125] [0125] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is linked to the 6-quinolyl position, where
[0126] [0126] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is,
[0127] [0127] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is absent; A is S;
[0128] [0128] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is absent; A is CH2; E is C1-6 alkyl or, where m is 1, 2 or 3; preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl,
[0129] [0129] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is absent; A is NH; E is C1-6 alkyl or, where m is 1, 2 or 3; preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C1-6 alkyl, more preferably, E is C3 or C4 alkyl; B is NR4-C1-6 alkyl or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10-membered N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C1-6 alkyl, aryl, C1-6 aralkyl. Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0130] [0130] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is an amino acid, preferably containing a charged side chain; A is O; E is C1-6 alkyl or, where m is 1, 2 or 3; preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C1-6 alkyl, more preferably, E is C3 or C4 alkyl; B is NR4-C1-6 alkyl or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10-membered N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C1-6 alkyl, aryl, C1-6 aralkyl. Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0131] [0131] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is an amino acid, preferably containing a charged side chain; A is S; E is C1-6 alkyl or, where m is 1, 2 or 3; preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C1-6 alkyl, more preferably, E is C3 or C4 alkyl; B is NR4-C1-6 alkyl or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10-membered N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C1-6 alkyl, aryl, C1-6 aralkyl. Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0132] [0132] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is an amino acid, preferably containing a charged side chain; A is CH2; E is C1-6 alkyl or, where m is 1, 2 or 3; Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C1-6 alkyl, more preferably, E is C3 or C4 alkyl; B is NR4-C1-6 alkyl or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10-membered N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C1-6 alkyl, aryl, C1-6 aralkyl. Preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0133] [0133] In another preferred embodiment of the first aspect of the present invention,
[0134] [0134] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is,
[0135] [0135] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is attached to the 5- or 6- quinolyl position; more preferably, R7 is attached to the 6-quinolyl position, where D is absent; A is O; E is C3 or C4 alkyl; more preferably, E is propyl or butyl; B is an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10 membered N, preferably also comprising 1 or 2 nitrogen atoms.
[0136] [0136] In another preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle:, wherein the heterocycle optionally further comprises 1 or 2 heteroatoms selected from of O, N and S, optionally also comprises 1 nitrogen; it is connected to position 1, 2 or 3, preferably in position 2; l is 1 or 2.
[0137] [0137] In another preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle:, wherein the heterocycle optionally further comprises 1 or 2 heteroatoms selected from of O, N and S, optionally also comprises 1 nitrogen; it is connected to position 1, 2 or 3, preferably in position 2; l is 1 or 2; wherein the N-containing heterocycle is replaced with C1-6 alkyl.
[0138] [0138] In another preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:,,,,,
[0139] [0139] In another preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:,,,,,,,,,,, and, where if the N-containing heterocycle comprised in B is, the heterocycle optionally further comprises 1 or 2 heteroatoms selected from O, N and S, optionally further comprises 1 nitrogen, optionally comprises one or more side chains (for example, derived from amino acids); it is connected to position 1, 2 or 3, preferably in position 2; o is 1 or 2; preferably, if the N-containing heterocycle comprised in B is
[0140] [0140] In another preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:, and.
[0141] [0141] In another preferred embodiment of the first aspect of the present invention, the N-containing heterocycle comprised in B is selected from the group consisting of:, and, in which B is substituted with a C1-3 alkyl.
[0142] [0142] In another preferred embodiment of the first aspect of the present invention, R5 and R6 are H; R7 is preferably R7 is linked to the 6-quinolyl position, where
[0143] [0143] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is C = O; W is NH; Y is CH2; Z is C = O; R1 and R2 are selected independently from the group consisting of -H and halo; preferably, R1 and R2 are selected independently from the group consisting of -H and F; more preferably, R1 and R2 are the same and are selected from the group consisting of -H and F; R3 is -CN; R5 and R6 are H; R7 is preferably R7 is linked to the 6-quinolyl position, where D is absent; A is O; E is C1-6 alkyl or, where m is 1, 2 or 3; preferably, E is C1-6 alkyl; preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; more preferably, E is C1-6 alkyl, more preferably, E is C3 or C4 alkyl; B is NH-C 1-6 alkyl,,, or; preferably, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl; preferably B is; and is.
[0144] [0144] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is C = O; W is NH; Y is CH2; Z is C = O; R1 and R2 are equal and are selected from the group consisting of -H and F; R3 is -CN; R5 and R6 are H; R7 is,
[0145] [0145] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is C = O; W is NH; Y is CH2; Z is C = O; R1 and R2 are equal and are selected from the group consisting of -H and F; R3 is -CN; R5 and R6 are H; R7 is preferably R7 is linked to the 6-quinolyl position, where D is absent; A is O; And it is methyl, ethyl, propyl or butyl; B is,, or; preferably B is; and is.
[0146] [0146] In another preferred embodiment of the first aspect of the present invention, Q, R, U are absent; V is C = O; W is NH; Y is CH2; Z is C = O; R1 and R2 are equal and are selected from the group consisting of -H and F; R3 is -CN; R5 and R6 are H;
[0147] [0147] In another preferred embodiment of the first aspect of the present invention, C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl.
[0148] [0148] In another preferred embodiment of the first aspect of the present invention, C1-3 alkyl is selected from the group consisting of methyl, ethyl, propyl and i-propyl.
[0149] [0149] In another preferred embodiment of the first aspect of the present invention, C1-6 aralkyl is selected from the group consisting of benzyl, phenyl-ethyl, phenyl-propyl and phenyl-butyl.
[0150] [0150] In a preferred embodiment of the first aspect of the present invention, the compound of the first aspect of the invention is selected from the compounds of Table 1. More preferably, the compound of the first aspect of the invention is selected from the compounds of Table 2. More preferably, the compound of the first aspect of the invention is selected from the group consisting of FAPI-02 and FAPI-04.
[0151] [0151] In a preferred embodiment of the first aspect of the present invention, the compound of the first aspect of the invention is selected from the compounds of Table 1 and / or Table 3. More preferably, the compound of the first aspect of the invention is selected from the compounds of Table 2 and / or Table 4. More preferably, the compound of the first aspect of the invention is selected from the group consisting of FAPI-02, FAPI-04, FAPI-46, FAPI-34, FAPI- 42, FAPI-52, FAPI-69, FAPI-70, FAPI-71, FAPI-72 and FAPI-73.
[0152] [0152] § fluorescent compounds; $ chelators of 99mTc; * Pb chelators; R1 and R2 are located at the 4-pyrrolidine position; Q, R, U are absent; is, R5 is H; R6 is linked to the 7-quinolyl position; R7 is linked to the 6-quinolyl position; ‘-’ indicates that R6 or R7 is H; ‘+’ Indicates R6 or R7 being; V is C = O; W is NH; Y is CH2; Z is C = O; R3 is –CN; A is O (except FAPI-01: A is absent, R7 is linked to the 5-quinolyl position). Name R1 / R R R R8 D B E 2 6 7 FAPI-01 H / H - + I - - - FAPI-02 H / H - + - n-C3H6
[0153] [0153] Q, R, U, D are absent; R1 and R2 are located at the 4-pyrrolidine position; is, R5, R6 are H; R7 is linked to the 6-quinolyl position; V is C = O; W is NH; Y is CH2; Z is C = O; R3 is –CN; B is 1.4-
[0154] [0154] § fluorescent compounds; $ chelators of 99mTc; * precursors for 18F marking; Q, R, U are absent; R1 and R2 are located at the 4-pyrrolidine position; is, R5 and R6 are H; R7 is linked to the 6-quinolyl position and is; V is C = O; W is NH; Y is CH2; Z is C = O; R3 is –CN. Name R1 / R R8 D B E A 2 FAPI-39 F / F - n- CH2 C3H6 FAPI-40 F / F - n- S C3H6 FAPI-41 F / F - n- NH C3H6
[0155] [0155] Q, R, U, D are absent; R1 and R2 are fluorine atoms located at the 4-pyrrolidine position; is, R5, R6 are H; R7 is linked to the 6-quinolyl position; V is C = O; W is NH; Y is CH2; Z is C = O; R3 is –CN; B is 1,4-piperazine; E is 1,3-propane; A is O. Name Purpose R8 188Re-FAPI-60 Radiotherapy (β‾) Al18F-FAPI-42 PET 18F-FAPI-73 PET TABLE 5: PREFERRED PRECURSORS FOR RADIOACTIVE MARKING WITH § F-18; $ CU-64; € GA-68; £ TC-99M, RE-188; * Y-90, SM-153, LU-177. FAPI- 02 €, *
[0156] [0156] In another preferred embodiment of the first aspect of the present invention, R8 is a radioactive moiety, wherein the radioactive moiety is a fluorescent isotope, a radioisotope, a radioactive drug or combinations thereof. Preferably, the radioactive portion is selected from the group consisting of alpha radiation emitting isotopes, beta radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescence emitting isotopes, such as 11C, 18F, 51Cr, 67Ga, 68Ga, 111In, 99mTc, 186Re, 188Re, 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, 197Hg, 211At, 151Eu, 153Eu, 169Eu, 201Tl, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au, 186Re, 198Au, 225Ac, 227Th and 199Ag. Preferably 18F, 64Cu,
[0157] [0157] In another preferred embodiment of the first aspect of the present invention, R8 is a fluorescent dye selected from the group consisting of the following classes of fluorescent dyes: xanthenes, acridines, oxazins, kinins, stirila dyes, coumarins, porphins, ligand-metal complexes, fluorescent proteins, nanocrystals, perylenes, borosipirometenes and phthalocyanines, as well as conjugates and combinations of these dye classes.
[0158] [0158] In another preferred embodiment of the first aspect of the present invention, R8 is a chelating agent that forms a complex with divalent or trivalent metal cations. Preferably, the chelating agent is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N, N ', N, N'-tetraacetic acid (DOTA), ethylene diaminetetraacetic acid (EDTA), acid 1, 4,7-triazacyclononane-1,4,7-triacetic (NOTE), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N, N ', N', N ”-pentaacetic acid (DTPA), bis- (carboxymethylimidazole ) glycine and 6-hydrazinopyridine-3-carboxylic acid (HYNIC).
[0159] [0159] In another preferred embodiment of the first aspect of the present invention, R8 is a contrast agent that comprises or consists of a paramagnetic agent, preferably, wherein the paramagnetic agent comprises or consists of paramagnetic nanoparticles.
[0160] [0160] In another preferred embodiment of the first aspect of the invention, R8 is selected from any R8 in Tables 1 to
[0161] [0161] In a second aspect, the present invention relates to a pharmaceutical composition comprising or consisting of at least one compound of the first aspect and, optionally, a pharmaceutically acceptable carrier and / or excipient.
[0162] [0162] In a third aspect, the present invention relates to the compound of the first aspect or the pharmaceutical composition of the second aspect for use in the diagnosis or treatment of a disease characterized by the overexpression of fibroblast activating protein (FAP) in an animal or human being. Preferably, the disease characterized by overexpression of fibroblast activating protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorder.
[0163] [0163] Preferably, if the disease characterized by overexpression of fibroblast activating protein (FAP) is cancer, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer , rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, renal carcinoma clear cell, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (hidden primary carcinoma), thymus carcinoma, desmoid tumors, glioma, astrocytoma, cervical carcinoma and prostate cancer. Preferably, the cancer is glioma, breast cancer, colon cancer, lung cancer, head and neck cancer, liver cancer or pancreatic cancer. Most preferably, cancer is glioma.
[0164] [0164] Preferably, if the disease characterized by overexpression of fibroblast activating protein (FAP) is chronic inflammation, chronic inflammation is selected from the group consisting of rheumatoid arthritis, osteoarthritis and Crohn's disease. Preferably, the chronic inflammation is rheumatoid arthritis.
[0165] [0165] Preferably, if the disease characterized by overexpression of fibroblast activating protein (FAP) is fibrosis, fibrosis is selected from the group consisting of pulmonary fibrosis, such as idiopathic pulmonary fibrosis and liver cirrhosis.
[0166] [0166] Preferably, if the disease characterized by overexpression of fibroblast activating protein (PAF) is tissue remodeling, tissue remodeling will occur after myocardial infarction.
[0167] [0167] Preferably, if the disease characterized by overexpression of fibroblast activating protein (FAP) is a keloid disorder, the keloid disorder is selected from the group consisting of scar formation, keloid tumors and keloid scar.
[0168] [0168] In a fourth aspect, the present invention relates to a kit that comprises or consists of the compound of the first aspect or the pharmaceutical composition of the second aspect and instructions for the diagnosis or treatment of a disease. Preferably, the disease is a disease as specified above.
[0169] [0169] Based on a specific FAP-α inhibitor (Jansen et al., ACS Med Chem Lett, 2013), two radioactive trackers were synthesized. The radioiodine-labeled FAPI-01 was obtained by means of an organotin-stanylated precursor, which was prepared by palladium-catalyzed bromine / tin exchange. FAPI-02 is a precursor for the chelation of radio-metals, which was synthesized in five stages. By applying the same procedures or slightly modified procedures, additional compounds were prepared. The structures of these compounds are listed in Tables 1 and 2. The radioiodinations of the stannylated precursor were performed with peracetic acid. For chelation with Lu-177 and Ga-68, the pH of the reaction mixture was adjusted with sodium acetate and heated to 95 ° C for 10 minutes. The stability in human serum was analyzed by precipitation and radio-HPLC analysis of the supernatant. REAGENTS
[0170] [0170] All solvents and non-radioactive reagents were obtained in reagent grade from ABCR (Karlsruhe, Germany), Sigma-Aldrich (München, Germany), Acros Organics (Geel, Belgium) or VWR (Bruchsal, Germany) and were used without additional purification. Atto 488 NHS-ester was obtained from AttoTec (Siegen, Germany). 2,2 ', 2' '- (10- (2- (4-nitrophenyl) oxy) -2-oxoethyl) -1,4,7,10-tetraazacyclo-dodecane-1,4,7-triyl) triacetic (DOTA-PNP) was synthesized following the protocol of Mier et al. (Mier et al., Bioconjug Chem, 2005). The intermediates 6-methoxyquinoline-4-carboxylic acid (7), 5-bromoquinoline-4-carboxylic acid (3) and (S) -1- (2-aminoacetyl) pyrrolidine-2-carbonitrile 4-methylbenzenesulfonate were synthesized following the protocols de Jansen et al. (Jansen et al., ACS Med Chem Lett, 2013). The substance (S) -N- (2- (2-cyanopyrrolidin-1-yl) -2-oxoethyl) -5-bromoquinoline carboxamide was synthesized by a modified HBTU amidation protocol. COMPOUND SUMMARY
[0171] [0171] Scheme 1 represents the initial synthesis of FAPI-01 that was achieved by performing a Br / Li exchange with n-butyl lithium in 5-bromoquinoline-4-carboxylic acid (3) and quenching with elemental iodine for obtain iodoquinoline 4. This compound was coupled to the Gly-Pro-CN fragment by activation of HBTU / HOBt to provide non-radioactive reference material of FAPI-01 (1).
[0172] [0172] Scheme 1. Synthesis of non-radioactive FAPI-01. i) nBuLi, then I2, THF; ii) HBTU / HOBt, DIPEA, H-Gly-Pro-CN, DMF.
[0173] [0173] For the synthesis of radioactive FAPI-01 (1 *), the stanylated precursor 6 was obtained by palladium-catalyzed stanylation of inhibitor 5 in dioxane at 80 ° C (Scheme 2).
[0174] [0174] Scheme 2. Synthesis of radioactive FAPI-1 through the stanylated precursor 4. i) (Me 3Sn) 2; (PPh3) 2PdCl2; dioxane 80 ° C; ii) I-125 or I-131; AcOOH; 1M HCl; MeOH.
[0175] [0175] To allow radioactive labeling by incorporation of radio-metals, the DOTA chelator has been chemically linked to the basic support of the FAP inhibitor. As shown in Jansen et al. (Jansen et al., ACS Med Chem Lett, 2013), changes in the 6-position of quinoline-4-carboxylic acid are well tolerated without impairing the target affinity and specificity. Therefore, a bifunctional ligand was attached to the hydroxyl group of 8 by means of an ether bond, leading to the synthesis shown in Scheme 3. The ready 1-bromo-3-chloropropane available was chosen to create a spacer, which is unharmed during saponification of the ester bond formed simultaneously at the end of the pot process. Compound 9 was converted to quinoline carboxylic acid 10 protected by N-Boc, which was further coupled to H-Gly-Pro-CN by HBTU. Due to the high hygroscopicity of the free amine, compound 11 was converted directly to FAPI-02 (2) after Boc removal, solvent exchange and neutralization of excess p-toluenesulfonic acid.
[0176] [0176] Scheme 3. Chemical synthesis of FAPI-02. i) 48% aqueous HBr, 130 ° C; ii) 1-bromo-3-chloropropane, Cs2CO3, DMF, then 6 M NaOH; iii) 1-Boc-piperazine, KI, DMF; iv) HBTU / HOBt, DIPEA, H-Gly-Pro-CN, DMF; v) TosOH, MeCN, then DOTA-PNP, DIPEA, DMF.
[0177] [0177] In the case of compounds that incorporate the group A ≠ O, the quinoline-4-carboxylic acid intermediates were synthesized by a different reaction scheme. The main step in this approach is a palladium-catalyzed coupling reaction (eg Buchwald-Hartwig cross-coupling), which requires additional protection before and deprotection of the carboxylic acid function after the cross-coupling reaction (scheme 4).
[0178] [0178] Scheme 4. Synthesis of the 6- (3- (4- Boc-piperazin-1-yl) propyl-1- (methyl) amino) quinoline-4-carboxylic acid building block for the synthesis of FAPI-46. i) DCC, tBuOH, CuCl; ii) 3-methylamino-1-propanol, Cs2CO3, Pd2 (dba) 3, BINAP; iii) MsCl, NEt3, DCM and then 1-Boc-piperazine, KI, DMF; iv) TFA then Boc2O, NEt3, DMF.
[0179] [0179] 3.88 mg (10.0 µmol) of (S) -N- (2- (2-cyanopyrrolidin-1-yl) -2-oxoethyl) -5-bromoquinoline-caboxamide, 20 µL (32 mg; 96 µmol) of hexamethylditine and 0.75 mg (1.07 µmol) of bis (triphenylphosphine) palladium (II) dichloride in 1 ml of dry dioxane are stirred at 80 ° C overnight under an inert atmosphere. The volatiles are removed and the residue is taken up in 2 ml of 50% acetonitrile / water and filtered through a light C18 cartridge before purification by HPLC. 2.78 mg (5.90 µmol; 59%) of the product are obtained after freeze-drying.
[0180] [0180] LC-MS Rt 14.77 minutes, m / z 473.0786 [M (120Sn) + H] + 5-IODOQUINOLINE-4-CARBOXYLIC ACID (4)
[0181] [0181] 5.42 mg (136 µmol) of sodium hydride suspension (60% in mineral oil) is added to a solution of 30.27 mg (120 µmol) of 5-bromoquinoline-4-carboxylic acid (3) in 3 ml of dry THF under Ar at 0 ° C. The ice bath is removed and the reaction mixture is cooled to -78 ° C before 100 µL (160 µmol) nBuLi (1.6 m in hexanes) is added dropwise. After 15 minutes, 64.71 mg (254 µmol) of iodine in 2 ml of THF are added dropwise and the reaction is stirred for 30 minutes at -78 ° C before reaching room temperature. After 1 hour, the reaction is quenched by the addition of 1 ml of 0.5 M NaHCO3 and about 30 mg (170 µmol) of sodium dithionite to remove excess iodine. After removing the THF under reduced pressure, the mixture is acidified to pH 2 and extracted three times with ethyl acetate (25 ml). The combined organic phases are evaporated to dryness and purified by HPLC. 18.14 mg (60.7 µmol; 45%) of the title compound are obtained after freeze-drying.
[0182] [0182] 1H NMR (500 MHz, DMSO-d6) 13.95 (br, 0.3H), 8.93 (s, 1H), 8.34 (d, J = 7.2 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.60 (s, 1H), 7.52 (t, J = 7.9 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) 168.8, 150.3, 148.8, 141.3, 130.6, 121.0, 109.5; LC-MS Rt 8.65 minutes, m / z 299.9383 [M + H] + (S) -N- (2- (2-CYANOPIRROLIDIN-1-IL) -2-OXOETYL) -5- TRIMETYL STANYLINKINOLINE CABOXAMIDE (1 ; FAPI-01)
[0183] [0183] 9.07 mg (23.9 µmol) of HBTU in 50 µL of DMF are added to a solution of 6.21 mg (20.8 µmol) of 5-iodoquinoline-4-carboxylic acid, 7.45 mg (55.2 µmol) of HOBt and 10 µL of DIPEA in 50 µL of DMF.
[0184] [0184] 1H NMR (600 MHz, DMSO-d6) 9.06, 8.97, 8.33, 8.13, 7.56, 7.51, 4.81, 4.34, 4.06, 3.74, 3.56, 2.21, 2.17, 2.09, 2.05; 13C NMR (150
[0185] [0185] 105 mg (477 µmol) of crude 6-methoxyquinoline-4-carboxylic acid (7) are dissolved in 3 ml of 48% hydrobromic acid in water. The solution is heated to 130 ° C for 4 hours. The solution is brought to a slightly basic pH with 6 M NaOH after reaching room temperature. 79.2 mg (419 µmol; 88%) of the product are obtained after purification by HPLC and lyophilization.
[0186] [0186] 1H NMR (500 MHz, DMSO-d6) 13.65 (br, 0.6H) 10.24 (s, 1H), 8.78 (d, J = 4.4 Hz, 1H), 8 , 06 (d, J = 2.6 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 4.4 Hz, 1H), 7.37 (dd, J = 9.1, 2.6 Hz, 1H), 13C NMR (125 MHz, DMSO-d6) 167.7, 156.9, 146.5, 144.1, 133.4, 131, 2, 126.2, 122.3, 122.6, 106.5; LC-MS Rt 6.66 minutes, m / z 190.0415 [M + H] + 6-BROMOCHINOLIN-4-TERC-BUTYL CARBOXYLATE
[0187] [0187] 98.3 mg (390 µmol) of 6-bromoquinoline-4-carboxylic acid (crude) were suspended in 5 ml of tetrahydrofuran and 25.0 µL (18.3 mg; 181 µmol) of triethylamine and added to O-tert-butyl-N, N'-dicyclohexylisourea (prepared the previous day from 426 mg (2.07 mmol) of pure dicyclohexylcarbodiimide, 173 mg (2.33 mmol) of tert-butanol and 10.2 mg (103 µmol) of copper (I) chloride). The mixture was heated to 50 ° C overnight. The mixture was filtered, the solvents evaporated and the product isolated by HPLC. 49.7 mg (161 µmol; 41%) of the title compound were obtained after freeze-drying.
[0188] [0188] LC-MS Rt 20.40 minutes, m / z 251.9642 [M-tBu] + 6- (3-CHLORINE-1-PROPOXI) QUINOLINE-4-CARBOXYLIC ACID (9)
[0189] [0189] 42.4 µl (67.4 mg; 430 µmol) of 1-bromo-1-chloropropane are added to a suspension of 23.2 mg (123 µmol) of 6-hydroxyquinoline-4-carboxylic acid (8) and 190 mg (1.38 µmol) of potassium carbonate in 250 µL of DMF and heated to 60 ° C overnight. The reaction mixture is cooled to room temperature, diluted with 500 µL of water and 500 µL of acetonitrile before adding 100 µL of 6M NaOH. The reaction mixture is purified directly by HPLC (5-40%) after hydrolysis of the ester to be carried out. 26.45 mg (99.4 µmol; 81%) of the product is obtained after lyophilization.
[0190] [0190] 1H NMR (500 MHz, DMSO-d6) 13.75 (br, 0.4H), 8.88 (d, J = 4.4 Hz, 1H), 8.19 (d, J = 2 , 0 Hz, 1H), 8.04 (d, J = 9.2 Hz, 1H), 7.94 (d, J = 4.4 Hz, 1H), 7.52 (dd, J = 9.2 , 2.0 Hz, 1H), 4.24 (t, J = 5.95 Hz, 2H), 3.85 (t, J = 6.5 Hz, 2H), 2.27 (m, 2H); 13C NMR (125 MHz, DMSO-d6) 167.6, 157.5, 147.6, 144.8, 134.0, 131.2, 125.9, 122.7, 122.2, 104.5 , 64.7, 41.9, 31.6; LC-MS Rt 11.46 minutes, m / z 266.0461 [M + H] + 6- (3-HYDROXYPROPYLMETHYLAMINE) QUINOLIN-4-CARBOXYLATE TERC-BUTYL
[0191] [0191] 204.6 mg (664 µmol) of tert-butyl 6-bromoquinoline-4-carboxylate, 34.10 mg (54.7 µmol) of BINAP, 21.51 mg (23.5 µmol) of Pd 2 (dba) 3 and 480.3 mg (1.47 mmol) of cesium carbonate were dissolved in 6 mL of toluene, and 128.0 µL (118 mg; 1.32 mmol) of N-methyl-1 was added, 3- propanolamine. The mixture was stirred at 90 ° C overnight before removing the solvents, the residue was suspended in water / acetonitrile 1: 1 and filtered before purification by HPLC. 172.7 mg (547 µmol; 82%) of the title compound were obtained after freeze-drying.
[0192] [0192] LC-MS Rt 13.41 minutes, m / z 261.1213 [M-tBu + H] + 6- (3- (4-BOC-PIPERAZIN-1-IL) PROPIL-1- (METHIL) AMINO) QUINOLINE -4-TERC-BUTYL CARBOXYLATE
[0193] [0193] 62.8 mg (199 µmol) of tert-butyl 6- (3-hydroxypropylmethylamino) quinoline-4-carboxylate were dissolved in 5 ml of dichloromethane and 90.0 µL (66.6 mg; 659 µmol) of triethylamine. 20.0 µL (29.6 mg; 258 µmol) of methanesulfonyl chloride was added at 0 ° C and the mixture reacted for 60 minutes. 194.6 mg (1.05 mmol) of 1-Boc-piperazine were added prior to removal of the volatiles. 500 µL of dimethylformamide and 47.4 mg (286 µmol) of potassium iodide were added to the residue. The mixture was stirred at 60 ° C for 120 minutes before the product was isolated by HPLC.
[0194] [0194] LC-MS Rt 13.99 minutes, m / z 485.3086 [M + H] +
[0195] [0195] 15.13 mg (56.9 µmol) of 6- (3-chloro-1-propoxy) quinoline-4-carboxylic acid (9), 55.43 mg (298 µmol) of N-tert-butoxycarbonylpiperazine and 51.05 mg (30.8 µmol) of potassium iodide are dissolved in 250 µL of DMF. The reaction is stirred at 60 ° C overnight. The resulting suspension is diluted with 750 µL of water before the product is purified by HPLC. After freeze-drying, 28.73 mg (54.3 µmol; 95%) of the product is obtained as the corresponding TFA salt.
[0196] [0196] 1H NMR (500 MHz, D2O) 8.93 (d, J = 5.5 Hz, 1H), 8.17 (d, J = 9.3 Hz, 1H), 7.94 (d, J = 5.5 Hz, 1H), 7.79 (dd, J = 9.3, 2.5 Hz, 1H), 7.65 (d, J = 2.5 Hz, 1H), 4.36 ( t, J = 5.6 Hz, 2H), 4.27 (d, J = 13.55 Hz, 2H), 3.67 (d, J = 11.95 Hz), 3.47 (t, J = 15.5 Hz, 2 H), 3.27 (t, J = 12.7 Hz), 3.12 (td, J = 12.2, 2.65 Hz), 2.37 (m, 2 H) , 1.47 (s, 9H); 13C NMR (125 MHz, D2O) 155.5, 153.5, 149.0, 141.4, 134.4, 127.9, 126.6, 122.3, 118.4, 110.0, 105 , 1, 82.8, 65.5, 54.3, 51.5, 48.6, 40.7, 29.6, 27.4; LC-MS Rt 10.62 minutes, m / z 416.1997 [M + H] + ACID 6- (3- (4-BOC-PIPERAZIN-1-IL) PROPIL-1- (METHIL) AMINO) QUINOLINE-4- CARBOXYLIC
[0197] [0197] 100.12 mg (206 µmol) of tert-butyl 6- (3- (4-Boc-piperazin-1-yl) propyl- 1- (methyl) amino) quinoline-4-carboxylate were treated with 900 µL of trifluoroacetic acid, 25 µL of triisopropylsilane, 25 µL of water and 50 µL of trifluoromethanesulfonic acid for 60 minutes. The unprotected compound was precipitated with diethyl ether, dried and reacted with 60.83 mg (279 µmol) of di-tert-butyldicarbonate and 50.0 µL (36.5 mg; 361 µmol) of triethylamine in 1 ml of dimethylformamide for more 60 minutes. 55.42 mg (129 µmol;
[0198] [0198] LC-MS Rt 10.52 minutes, m / z 429.2463 [M + H] + (S) -N- (2- (2-CYANOPIRROLIDIN-1-IL) -2-OXOETYL) -6- (3 - (4-TERC- BUTOXICARBONYLPIPERAZIN-1-IL) -1-PROPOXI) QUINOLIN-4-CARBOXAMIDE (11)
[0199] [0199] 9.43 mg (24.9 µmol) of HBTU in 50 µL of DMF are added to a solution of 10.56 mg (19.9 µmol) of 6- (3- (4-tert-butoxycarbonylpiperazin-) acid 1-yl) -1-propoxy) quinoline-4-carboxylic (10), 5.38 mg (39.8 µmol) of HOBt and 10 µL of DIPEA in 50 µL of DMF. After 15 minutes, (S) -1- (2-aminoacetyl) pyrrolidine-2-carbonitrile (29.9 µmol) of 4-methylbenzenesulfonate in 50 µL of DMF is added. The reaction is quenched with 850 µL of water and purified by HPLC. Freeze-drying provides 12.88 mg (19.4 µmol; 97%) of the title compound.
[0200] [0200] 1H NMR (500 MHz, DMSO-d6) 9.04 (d, J = 5.5 Hz, 1H), 8.24 (d, J = 9.6 Hz, 1H), 8.10 ( d, J = 5.5 Hz, 1H), 7.89 (d, J = 2.3 Hz, 1H), 7.85 (dd, J = 9.6, 2.3 Hz, 1H), 4, 84 (t, J = 6 Hz, 1 H), 4.46–4.36 (m, 4H), 4.26 (d, J = 12.0 Hz, 2H), 3.83 (m, 1H) , 3.67 (m, 3H), 3.47 (t, J = 7.7 Hz, 2H), 3.27 (br, 2H), 3.11 (t, J = 11.5 Hz), 2 , 37 (m, 4H), 2.22 (m, 2H), 1.46 (s, 9H); 13C NMR (125 MHz, DMSO-d6) 168.6, 168.0, 159.4, 155.5, 147.7, 141.8, 135.1, 128.2, 127.5, 123.1 , 120.0, 119.1, 104.7, 82.9, 66.0, 54.3, 51.5, 47.0, 46.3, 42.3, 29.4, 27.4, 24 , 7, 23.1; LC-MS Rt 11.81 minutes, m / z 551.2736 [M + H] +
[0201] [0201] 13.2 mg (22.4 µmol; 75%) were obtained following the previous protocol.
[0202] [0202] LC-MS Rt 11.84 minutes, m / z 605.2610 [M + H] + N- (2- (2-CYAN-4,4-DIFLUOROPIRROLIDIN-1-IL) -2-OXOETYL) -6- (3- (4-BOC-PIPERAZIN- 1-IL) PROPIL-1- (METHIL) AMINO) QUINOLIN-4-CARBOXAMIDE
[0203] [0203] 1.17 mg (1.95 µmol; 92%) were obtained following the previous protocol.
[0204] [0204] LC-MS Rt 12.66 minutes, m / z 600.3057 [M + H] + FAPI-02 (2)
[0205] [0205] 4.85 mg (8.80 mmol) of (S) -N- (2- (2-cyanopyrrolidin-1-yl) -2-oxoethyl) -6- (3- (4-tert-butoxycarbonyl- piperazin-1-yl) -1-propoxy) quinoline-4-carboxamide (11) are dissolved in 1 ml of acetonitrile and 4.2 mg (22.0 µmol) of 4-methylbenzenesulfonic acid monohydrate are added. The reaction is stirred at 45 ° C overnight, before the volatiles are removed under reduced pressure. The residue is taken up in 190 µL of dimethylformamide and 10 µL (7.3 mg; 72 µmol) of triethylamine before adding 6.77 mg (12.9 mmol) of DOTA-p-nitrophenol ester. The reaction mixture is diluted with 1 ml of water and purified by HPLC after stirring for two hours. 5.04 mg (6.02 µmol; 68%) are obtained after freeze-drying.
[0206] [0206] 1H NMR (600 MHz, D2O) 9.02, 8.23, 8.07, 7.87, 7.83, 4.85, 4.45, 4.41, 4.40, 4, 39, 3.83, 3.67, 3.50, 3.49, 2.40, 2.38, 2.36, 2.26, 2.22; 2.16; 13C NMR (150 MHz, D2O) 167.9, 159.1, 147.2, 141.8, 135.4, 127.9, 127.2, 119.8, 119.0, 104.5, 65 , 8, 54.1, 46.8, 46.1, 42.1, 29.2, 24.5, 23.0: LC-MS Rt 8.37 minutes, m / z 837.3872 [M + H] + FAPI-04
[0207] [0207] 3.97 mg (4.55 µmol; 57%) were obtained following the previous protocol.
[0208] [0208] LC-MS Rt 8.80 minutes, m / z 873.3664 [M + H] + FAPI-42
[0209] [0209] 1.91 mg (2.47 µmol; 88%) was obtained following the previous protocol.
[0210] [0210] LC-MS Rt 9.37 minutes, m / z 386.6807 [M + 2H] 2+
[0211] [0211] 39.21 mg (44.3 µmol; 85%) were obtained following the previous protocol.
[0212] [0212] LC-MS Rt 9.03 minutes, m / z 443.7196 [M + 2H] 2+ FAPI-19
[0213] [0213] 1.09 mg (1.86 µmol) of (S) -N- (2- (2-cyano-4,4-difluoropyrrolidin-1-yl) -2-oxoethyl) -6- (3- ( 4-tert-butoxycarbonylpiperazin-1) -yl) -1- propoxy) quinoline-4-carboxamide were deprotected by Boc by the method applied to FAPI-02 and reacted with 2.74 mg (5.91 µmol) of bis ((1 - (2- (tert-butoxy) -2-oxoethyl) -1H-imidazol-2-yl) methyl) glycine, which were pre-activated with 2.13 mg (5.62 µmol) of HBTU and 2.50 µL (1.85 mg; 14.3 µmol) of DIPEA. After purification by HPLC and removal of the solvent, the residue was treated with 200 µL of 2.5% trifluoromethanesulfonic acid in acetonitrile / trifluoroacetic acid 1: 1. After precipitation with diethyl ether and purification by HPLC, 1.06 mg (1.29 µmol; 70%) of the title compound was obtained.
[0214] [0214] LC-MS Rt 8.91 minutes, m / z 820.2933 [M + H] +
[0215] [0215] 1.00 µL (0.74 mg; 5.73 µmol) of DIPEA was added to a solution of 0.95 mg (1.16 µmol) of FAPI-19, 0.42 mg (3.14 µmol) ) of HOBt and 1.10 mg (2.89 µmol) of HBTU in 50 µL of DMF. After 10 minutes, 2.30 mg (5.34 µmol) of H-Asn (Trt) -OtBu was added and reacted for 120 minutes. The tert-butyl protecting groups were removed by 2.5% TfOH in TFA / acetonitrile 8: 2. After HPLC purification and freeze-drying, 0.79 mg (0.75 µmol; 65%) of the title compound was obtained.
[0216] [0216] LC-MS Rt 9.23 minutes, m / z 524.7100 [M + 2H] 2+ FAPI-34
[0217] [0217] 1.01 mg (0.87 µmol; 52%) were obtained following the previous protocol.
[0218] [0218] LC-MS Rt 8.87 minutes, m / z 583.6988 [M + 2H] 2+ FAPI-60
[0219] [0219] 3.91 mg (6.66 µmol) of (S) -N- (2- (2-cyano-4,4-difluoropyrrolidin-1-yl) -2-oxoethyl) -6- (3- ( 4-tert-butoxycarbonylpiperazin-1) -yl) -1- propoxy) quinoline-4-carboxamide was deprotected for 30 minutes by 50 µL of acetonitrile and 100 µL of trifluoroacetic acid. After solvent evaporation and washing with diethyl ether, a 10-minute pre-incubated mixture of 8.02 mg (9.27 µmol) of acetyl-Cys (Trt) -Gly-Cys (Trt) -Gly-OH, 4, 31 mg (31.9 µmol) of HOBt and 4.47 mg (11.8 µmol) of HBTU in 150 µL of dimethylformamide and 2.50 µL (1.85 mg; 14.3 µmol) of DIPEA was added to the residue and reacted for 120 minutes. 4.66 mg (3.49 µmol; 52%) of the title compound protected with S-trityl were obtained after purification by HPLC and freeze-drying.
[0220] [0220] 3.36 mg (2.52 µmol) of the compound protected with trityl was dissolved in 50 µL of acetonitrile. 3 µL of triethylsilane and 100 µL of trifluoroacetic acid were added and reacted for 30 minutes.
[0221] [0221] LC-MS Rt 10.26 minutes, m / z 871.2703 [M + Na] + FAPI-69
[0222] [0222] 0.59 mg (0.60 µmol; 39%) were obtained following the previous protocol.
[0223] [0223] LC-MS Rt 10.25 minutes, m / z 991.3490 [M + H] + FAPI-70
[0224] [0224] 0.61 mg (0.54 µmol; 33%) were obtained following the previous protocol.
[0225] [0225] LC-MS Rt 10.14 minutes, m / z 1120.3884 [M + H] + FAPI-71
[0226] [0226] 0.79 mg (0.66 µmol; 34%) were obtained following the previous protocol.
[0227] [0227] LC-MS Rt 10.17 minutes, m / z 596.7075 [M + 2H] 2+ ATTO488-FAPI-02 (14)
[0228] [0228] 0.66 mg (1.20 µmol) of 11 is treated with 1.33 mg (6.96 µmol) of 4-methylbenzenesulfonic acid monohydrate in 250 µL of acetonitrile at 45 ° C for 4 hours. After removing the solvent, the residue is dissolved in 95 µL of dimethylformamide and 5 µL (3.65 mg; 36.1 µmol) of triethylamine. 0.54 mg (0.55 µmol) of Atto 488 NHS-ester in 25 µL of DMSO was added. After 60 minutes, 0.49 mg (0.43 µmol; 78%) of the title compound was isolated by HPLC and freeze-drying.
[0229] [0229] LC-MS Rt 10.19 minutes, m / z 1022.2706 [M] + FAPI-73
[0230] [0230] 10.95 mg (18.7 µmol) of (S) -N- (2- (2-cyano-4,4-difluoropyrrolidin-1-yl) -2-oxoethyl) -6- (3- ( 4-tert-butoxycarbonylpiperazin-1-yl) -1-
[0231] [0231] LC-MS Rt 9.37 minutes, m / z 649.2892 [M-CF3CO2] + FAPI-72
[0232] [0232] 9.80 mg (12.6 µmol; 70%) were obtained following the previous protocol.
[0233] [0233] LC-MS Rt 9.28 minutes, m / z 662.3237 [M-CF3CO2] + GENERAL CONNECTION OF FMOC-AMINO ACIDS PROTECTED BY SIDE CHAIN (S) -N- (2- (2-CYAN-4,4- DIFLUOROPIRROLIDIN-1-IL) -2-OXOETHYL) -6- (3- (4- (Γ, Γ-DI-TERC- BUTYL) -L-CARBOXI-GLUTAMYLPIPERAZIN-1-IL) -1-PROPOXI) QUINOLIN-4 -CARBOXAMIDA
[0234] [0234] 14.04 mg (23.9 µmol) of (S) -N- (2- (2-cyano-4,4-difluoro pyrrolidin-1-yl) -2-oxoethyl) -6- (3- (1-tert-butoxycarbonyl-piperidin) -4-yl) -1-propoxy) quinoline-4-carboxamide were dissolved in 50 µL of acetonitrile and 100 µL of trifluoroacetic acid. After 10 minutes, the volatiles were removed; the residue was washed with diethyl ether. A 14.95 mg (28.4 µmol) solution of
[0235] [0235] LC-MS Rt 12.85 minutes, m / z 772.3643 [M + H] + FAPI-75
[0236] [0236] 3.37 mg (4.37 µmol) of (S) -N- (2- (2-cyano-4,4-difluoropyrrolidin-1-yl) -2-oxoethyl) -6- (3- ( 4- (γ, γ-di-tert-butyl) -L-carboxyglutamyl piperazin-1-yl) -1-propoxy) quinoline-4-carboxamide and 4.52 mg (10.7 µmol) of NOTE-p-nitrophenol were dissolved in 100 µL of dimethylformamide and 10.0 µL (7.30 mg 72.3 µmol) of triethylamine. After HPLC purification and freeze-drying, the intermediate compound was deprotected by a 60-minute incubation in a solution of 50 µL of acetonitrile, 100 µL of trifluoroacetic acid, 2.5 µL of triisopropylsilane and 2.5 µL of water. 2.62 mg (2.77 µmol; 63%) was obtained after purification by HPLC and freeze-drying.
[0237] [0237] LC-MS Rt 9.38 minutes, m / z 945.3668 [M + H] + PRECURSOR FAPI-77
[0238] [0238] 3.23 mg (3.06 µmol; 73%) were obtained following the general protocol for modifying the active ester. Note: The tert-butyl protecting groups were removed after radiofluorination, HPLC purification and solvent evaporation by treatment with pure TFA at 95 ° C for 3 minutes, followed by treatment with SPE.
[0239] [0239] LC-MS Rt 16.02 minutes, m / z 1219.5858 [M + H] + ACID 2- (2- (4,7,10-TRIS (2- (TERC-BUTOXI) -2-OXOETYL) - 1,4,7,10- TETRAAZACICLODODECAN-1-IL) ACETOXI) ACETIC
[0240] [0240] 28.99 mg (50.6 µmol) of tris-tBu-DOTA, 90.65 (278 µmol) of cesium carbonate and 10.28 µL (15.0 mg; 65.5 µmol) of 2- benzyl bromoacetate were suspended in 300 µL of dimethylformamide and stirred for 2 hours. The product was isolated by HPLC, freeze-dried and dissolved in 25 ml of 10% acetic acid in methanol. 50 mg of 10% Pd / C and hydrogen (ambient pressure) were added. After 2 hours. The solvents were removed and the title compound isolated by HPLC. After freeze-drying, 25.19 mg (39.9 µmol; 79%) of the title compound were obtained.
[0241] [0241] LC-MS Rt 14.14 minutes, m / z 631.4784 [M + H] + TBU-FAPI-79
[0242] [0242] 2.00 mg (3.41 µmol) of (S) -N- (2- (2-cyano-4,4-difluoropyrrolidin-1-yl) -2-oxoethyl) -6- (3- ( 1-tert-butoxycarbonyl-piperidin-4-yl) -1- propoxy) quinoline-4-carboxamide were dissolved in 50 µL of acetonitrile and 100 µL of trifluoroacetic acid. After 10 minutes, the volatiles were removed;
[0243] [0243] LC-MS Rt 12.98 minutes, m / z 1099.7481 [M + H] + FAPI-79
[0244] [0244] 2.26 mg (2.06 µmol) of tBu-FAPI-79 was dissolved in 25 µL of acetonitrile and 100 µL of trifluoroacetic acid and stirred at 35 ° C for 30 minutes. After evaporation of the solvents, the product was isolated by HPLC.
[0245] [0245] LC-MS Rt 8.84 minutes, m / z 466.2737 [M + 2H] 2+ COMPOUND ANALYSIS
[0246] [0246] Reverse phase high performance liquid chromatography (RP-HPLC) was performed using linear gradients of acetonitrile in water (0-100% acetonitrile in 5 minutes; 0.1% TFA; 2 ml flow rate / min) on a Chromolith Performance RP-18e column (100 × 3 mm; Merck KGaA Darmstadt, Germany). UV absorbance was detected at 214 nm. An additional y detector was used for the HPLC analysis of radioactive compounds. The HPLC-MS characterization was performed on an ESI mass spectrometer (Exactive, Thermo Fisher Scientific, Waltham, MA, USA) connected to an Agilent 1200 HPLC system with a Hypersil Gold C18 1.9 μm column (200 ×
[0247] [0247] The radioiodine (I-125) was purchased from Hartmann Analytik (Göttingen, Germany); radioactive lutetium (Lu-177) was obtained from ITG (München, Germany); radioactive gallium (Ga-68) was eluted from a Ge-68 / Ga-68 generator purchased from Themba Labs (Somerset West, South Africa). The Tc-99m was eluted from a Mo-99 / Tc-99m generator (Curium Pharma, Berlin, Germany). Cu-64 was supplied by UKT Tübingen (Tübingen, Germany). Sm-153 was supplied by DSD Pharma (Purkersdorf, Austria). The Pb-203 was supplied by Lantheus (N. Billerica MA, USA). F-18-FDG and F-18 fluoride were supplied by ZAG Zyklotron AG (Eggenstein, Germany). The CRS kit for tricarbonyl was obtained from the Paul Scherrer Institut (Villingen-PSI, Switzerland).
[0248] [0248] For iodination, 10 µL of the FAPI-01 organotin precursor (1 µmol / ml in ethanol) was diluted with 10 µL of 1 M HCl and 10 µL of water before adding 1-20 MBq of iodine-125 in 0.05 M NaOH.
[0249] [0249] Cu-64, Lu-177 and Pb-203 labeling of DOTA compounds was performed by adding 5 MBq of the radioactive nuclide to 100 µL of a 10 µM solution of the individual precursor in 0.1 M NaOAc (pH 5 ) and incubation at 95 ° C for 10 minutes. The solution is used directly in in vitro experiments or diluted with 0.9% NaCl (Braun, Melsungen, Germany) in the case of biodistribution studies. For imaging studies in mice (scintigraphy, PET), the radioactive tracker was processed by solid phase extraction (sep-pak light C18, Waters).
[0250] [0250] TC (I) staining was preceded by the addition of 1 mL of Tc-99m-pertechnetate in 0.9% saline to a CRS Kit and incubation for 20 minutes. After cooling to room temperature, a mixture of 25.0 µL of the precursor (1 mM in water), 150 µL of phosphate buffer (0.4 M, pH 7.4) and 240 µL of hydrochloric acid (1 , 0 M) and the final mixture adjusted to pH 5, if necessary. The reaction was carried out at 95 ° C for 20 minutes and processed by solid phase extraction (sep-pak light C18, Waters).
[0251] [0251] TC (V) staining was preceded by the incubation of 30 µL of SnCl2 solution containing 200 mM glucoheptonate with 200 µL of Tc-99m-pertechnetate in 0.9% saline solution for 10 minutes at room temperature. 5.00 µL of the precursor (1 mM in water) and 3.75 µL of sodium hydroxide solution (0.1 M in water) were added and the final mixture was reacted at 95 ° C for 20 minutes. For imaging studies in mice (scintigraphy), the radioactive tracker was processed by solid phase extraction (sep-pak light C18, Waters).
[0252] [0252] Ga-68 staining for animal studies was carried out by incubating 255 µl of eluate generator (0.6 M HCl; approximately 230 MBq) with a mixture of 1 nmol of DOTA precursor, 1 µL of acid ascorbic 20% in water and 72 µL of NaOAc (2.5 M) at 95 ° C for 10 minutes. The remaining free radioactivity was removed by dilution with 2 ml of water, extraction in solid phase (sep-pak light C18, Waters), washing with 2 ml of water and eluting the product with 1 ml of water / ethanol 1: 1. The obtained solution was evaporated to dryness under reduced pressure and the residue was taken up in 0.9% NaCl (Braun).
[0253] [0253] For the formation of AlF-NOTE complexes, F-18 fluoride was retained in a Sep-Pak QMA with a Waters light cartridge (46 mg adsorbent; preconditioned with 0.5 M NaOAc, pH 3.9 ), washed with water and eluted with 500 µL of 0.1 M NaOAc (pH 3.9). For animal studies, 150 µL of the eluate was pre-incubated with 2 µL of a solution of AlCl3 (10 mM in water) and 50 µL of DMSO. After 5 minutes, the mixture was added to 40 nmol of NOTE precursor (10 µL of a 4 mM solution in water) and 1 µL of 20% ascorbic acid in water. The solution was reacted at 95 ° C for 15 minutes. The product was isolated by HPLC (0-20% acetonitrile in 10 minutes), solvent free and taken up in 0.9% saline solution before injection.
[0254] [0254] For the formation of 6-fluoronicotinamides, fluoride F-18 was retained in a Sep-Pak QMA with a Waters light cartridge (46 mg of adsorbent; preconditioned with 0.5 M KHCO3), washed with water, dried and eluted with a mixture of 7.50 mg (19.9 µmol) of cryptofix 222, 1.99 mg (1.99 µmol) of KHCO3 in 450 µL of acetonitrile and 50 µL of water. After removing the solvent, the residue was dried by azeotropic distillation with 3x1 ml of acetonitrile. The residue was taken up in 100 µL of 1: 1 tert-butanol / acetonitrile and added to 1 mg (about 1.3 µmol) of a trimethylpyridin-2-amine precursor. The solution was reacted at 75 ° C for 10 minutes. The product was isolated by HPLC (0-30% acetonitrile in 10 minutes), solvent-free and taken up in 0.9% saline before injection.
[0255] [0255] Alternatively, 6-fluoronicotinamides were synthesized by retaining F-18 fluoride in a Sep-Pak QMA with a Waters light cartridge (46 mg adsorbent; preconditioned with 0.5 M KHCO3), washed with acetonitrile, dried and eluted with 0.5 mg (about 0.4 - 0.6 µmol) of the precursor of FAPI (protected) in 0.5 ml of methanol. The solvent was removed in vacuo and the residue was taken up in 100 µL of acetonitrile / tert-butanol 1: 4. After 20 minutes at 70 ° C, the reaction mixture was diluted with water and the protected intermediates processed by solid phase extraction (sep-pak light C18, Waters). The solvents were removed and 200 µL of trifluoroacetic acid was added to the residue. The mixture was heated to 95 ° C for 3 minutes, dried in vacuo and diluted with water before the product was isolated by HPLC, which was carried out directly with the diluted reaction mixture in the case of compounds without protecting groups. The products were released from solvents and taken up in 0.9% saline before injection, in the case of animal studies.
[0256] [0256] To determine stability in human serum, radiolabeled compounds (approximately 2.5 MBq for I-125 or 15 MBq for Lu-177) were purified (HPLC or solid phase extraction) and released from the solvent. The residues were taken up in 250 µL of human serum (Sigma-Aldrich) and incubated at 37 ° C. The samples were precipitated with 30 µL of acetonitrile and analyzed by HPLC (0-30% acetonitrile in 10 minutes).
[0257] [0257] In vitro binding studies were performed using human tumor cell lines BxPC3, Capan-2, MCF-7 (purchased from Sigma Aldrich Chemie GmbH) and SK-LMS-1 (purchased from ATCC), as well as FAP cell lines stably transfected HT-1080-FAP, HEK-muFAP and the cell line expressing CD26 HEK-CD26 (obtained from Stefan Bauer, NCT Heidelberg). All cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum at 37 ° C / 5% carbon dioxide. For fluorescence internalization experiments, cells were seeded in coverslips and stained with FAPI-02-Atto488 and DAPI for staining the cell nucleus. The images were obtained using a confocal laser scanning microscope using a 63x oil immersion objective. Radioligand binding studies were performed using HT-1080-FAP cells. The radiolabeled compound was added to the cell culture and incubated for different time intervals, ranging from 10 minutes to 24 hours. Competition experiments were performed by simultaneous exposure to unlabeled compound (10-5 M to 10-9 M) and radiolabeled compound for 60 minutes. For efflux experiments, the radioactive medium was removed after incubation for 60 minutes and replaced with non-radioactive medium for intervals ranging from 1 to 24 hours. For internalization experiments, the surface-bound activity was removed by incubating the cells with 1 M glycine-HCl buffer for 10 minutes. Radioactivity was measured using a γ counter, normalized to 1 million cells and calculated as a percentage of applied dose (% ID). CELL COLORING AND MICROSCOPY
[0258] [0258] For internalization experiments, HT-1080-FAP and HEK muFAP cells were seeded in uncoated coverslips in a 24-well plate and cultured in culture medium containing 10% fetal calf serum until a final confluence of approximately 80-90%. The medium was removed and the cells were washed with 0.5 ml of PBS pH 7.4 for 2 times.
[0259] [0259] For radioligand binding studies, cells were seeded in 6-well plates and cultured for 48 hours until a final confluence of approximately 80-90% (1.2 - 2 million cells / well). The medium was replaced by 1 mL of fresh medium without fetal calf serum. The radiolabeled compound was added to the cell culture and incubated for different time intervals, ranging from 10 minutes to 24 hours. Competition experiments were performed by simultaneous exposure to unlabeled compound (10-5 M to 10-9 M) and radiolabeled compound for 60 minutes. For efflux experiments, the radioactive medium was removed after incubation for 60 minutes and replaced with non-radioactive medium for intervals ranging from 1 to 24 hours. In all experiments, the cells were washed with 1 ml of phosphate buffered saline pH 7.4 twice and subsequently lysed with 1.4 ml of lysis buffer (0.3 M NaOH, 0 SDS, two%). Radioactivity was determined in a γ counter (Cobra II, Packard), normalized to 1 million cells and calculated as a percentage of the applied dose (% ID). Each experiment was performed 3 times and 3 repetitions were acquired per independent experiment.
[0260] [0260] For internalization experiments, the cells were incubated with the radiolabeled compound for 60 minutes at 37 ° C and 4 ° C. Cell uptake was terminated by removing the medium from the cells and washing 2 times with 1 ml of PBS. Subsequently, the cells were incubated with 1 mL of glycine-HCl (1 M in PBS, pH 2.2) for 10 minutes at room temperature to remove surface-bound activity. The cells were washed with 2 ml of ice-cold PBS and lysed with 1.4 ml of lysis buffer to determine the internalized fraction. For cells incubated at 4 ° C, all washing and elution steps were performed using ice-cold buffers. Radioactivity was measured using a γ counter, normalized to 1 million cells and calculated as a percentage of the applied dose (% ID).
[0261] [0261] In order to analyze the binding properties of FAPI-01 to its target protein, radioligand binding assays were performed using different cancer cell lines and cell lines transfected with human and murine FAP, as well as the CD26 membrane protein closely related, also known as DPPIV. Both murine FAP and CD26 show high homology with human FAP-α (muFAP: 90% identity and 94% similarity at the amino acid level; CD26: 52% identity and 71% similarity with high structural similarity) (Kelly T., Drug Resist Updat, 2005).
[0262] [0262] As shown in Figure 1A, FAPI-01 shows no significant binding to FAP-negative cancer cell lines, while targeting cells that express human and murine FAP-α with high affinity (IC50 human FAP-α = 39 , 4 nM). In addition, no substantial binding was observed to cells expressing CD26 (0.05 ± 0.01%), proving that FAPI-01 is selectively targeting FAP-α. This is of particular importance, as CD26 is highly expressed in a variety of normal tissues, including the kidneys, liver and small intestine. In order to avoid a high background signal due to the nonspecific binding to CD26, the high selectivity of the ligand to FAP-α is of great advantage, resulting in excellent image quality.
[0263] [0263] Cell-based internalization assays demonstrate rapid uptake of FAPI-01 in cells (Figure 1B). After 10 minutes of incubation, 95% of the total bound fraction is located intracellularly (total 19.70 ± 0.28%). Over 4 hours, only a marginal decrease in activity is observed (total 17.00 ± 0.40%, of which 94% are internalized).
[0264] [0264] Iodine-labeled compounds generally show time-dependent enzymatic deiodination. This was also observed for FAPI-01, resulting in low intracellular radioactivity of this compound after longer incubation times (3.25 ± 0.29% after 24 hours). Deiodination can be minimized by reducing deiodinase activity after lowering the temperature to 4 ° C, resulting in an increased radioactivity of 26.66 ± 1.59% after 24 hours.
[0265] [0265] To avoid the rapid loss of activity of FAPI-01 due to enzymatic deiodination, the non-halogen derivative FAPI-02 was designed in which the FAP-binding fraction is chemically linked to the DOTA chelator. In addition to the resulting improved stability, this modification offers the possibility to easily incorporate diagnostic or therapeutic radioactive nuclides, allowing FAPI-02 to be used as a teranotic compound. Similar to its iodinated analog, FAPI-02 binds specifically to cells expressing human and murine FAP-α (IC50 human FAP-α = 21 nM) without addressing CD26 (% ID = 0.13 ± 0.01%; Figure 1A). THE
[0266] [0266] The robust internalization of FAPI-02 in cells expressing human and murine FAP-α was confirmed by fluorescence scanning laser microscopy. For this purpose, HT-1080-FAP and HEK-muFAP cells were stained with a fluorescently labeled FAPI-02 derivative (FAPI-02-Atto488) for 1 to 2 hours. As shown in Figure 1D, the compound is completely internalized and accumulates within cells that express FAP-α, while no uptake is detectable in FAP-α negative HEK-CD26 cells. FAPI DERIVATIVES PROJECT WITH CONNECTION PROPERTIES AND IMPROVED PHARMACOKINETICS
[0267] [0267] Other variants of FAPI-02 were designed to increase the retention time of the tumor, aiming at the development of a target agent of teranotic FAP. The FAPI-03 to FAPI-15 variants were characterized in relation to target binding, internalization rate and target specificity. The results are shown in Figure 2.
[0268] [0268] All experiments were carried out in accordance with German animal protection laws and in accordance with the regulations of the
[0269] [0269] For in vivo experiments, 8 week old BALB / c nu / nu mice (Charles River) were inoculated subcutaneously in the right trunk with 5x106 with HT-1080-FAP, Capan-2 or SK-LMS-1 cells, respectively. When the tumor size reached approximately 1 cm 3, the radiolabeled compound was injected through the tail vein (~ 10 MBq for small animal PET imaging; ~ 1 MBq for organ distribution). PET imaging was performed up to 140 minutes after intravenous injection of 1 MBq of Ga-68-labeled compound per mouse using the Inveon PET (Siemens) small animal PET scanner. The images were iteratively reconstructed using the 3D-OSEM + MAP method and were converted into standardized capture value images (SUV). Quantification was performed using an ROI technique and expressed as an average SUV. For the distribution of organs of the Lu-177-labeled compound (approximately 10 MBq per mouse), the animals (n = 3 for each instant) were sacrificed after the indicated moments (from 30 minutes to 24 hours). The distributed radioactivity was measured in all dissected organs and in the blood using a γ counter (Cobra Autogamma, Packard). Values are expressed as a percentage of injected dose per gram of tissue (% ID / g).
[0270] [0270] For pharmacokinetic modeling, the transport constant K1 and the speed constants k2 - k4 were calculated using a two-tissue compartment model implemented in the PMOD software [4], taking into account the vascular fraction (vB) that is associated to the volume of blood exchanging with tissue in a VOI. The rate constants that describe compartmental flows include k1 (binding to the receptor), k2 (detachment), as well as k3 (internalization) and k4 (efflux) in tumor tissue. In this model, the fractional volume of distribution (DV = K1 / k2) is the proportion of the region of interest in which the water marked with 15O is distributed. THE FAPI VARIATORS ACCUMULATE IN HUMAN XENOUNTS EXPRESSING FAP, AS WELL AS IN XENOUNTS WITH NO EXPRESSION OF FAP BY RECRUITMENT AND ACTIVATION OF MOUSE FIBROBLASTES.
[0271] [0271] The tumor accumulation of FAPI-02 and -04 was assessed by PET imaging of small mice animals having positive and negative FAP negative human tumor cell xenografts. In both cases, the radioactive tracker is rapidly enriched within the tumor and is maintained for at least 140 minutes (Figure 3A, C, E, G). At the same time, FAPI-02 and -04 show insignificant and low nonspecific binding and are rapidly eliminated from the blood predominantly through the kidneys and bladder, resulting in a low background and beneficial tumor to organ reasons. The simultaneous administration of a non-competing compound resulted in a complete absence of radioactivity in the tumor, demonstrating the specificity of the radioactive tracker for its target protein (Figure 4). Interestingly, a high tumor uptake of FAPI-02 was observed in mice having tumor cell lines positive for FAP-α (HT-1080-FAP) and also negative for FAP-α (Capan-2) due to the recruitment and activation of fibroblasts of activated mice. The pharmacokinetic characteristics of the radioactive tracker, calculated from PET data, using a two-tissue compartment model according to Burger et al., Nucl Med, 1997, are shown in Table 6.
[0272] [0272] Table 6. Pharmacokinetic characteristics of 68Ga-FAPI-02, calculated from dynamic PET data, using a two-tissue compartment model according to Burger et al., Nucl Med, 1997.
[0273] [0273] These observations were confirmed using 177Lu-FAPI-02 and -04 in a biodistribution study, proving a rapid accumulation of tumors in FAP-α positive and negative human tumors with very low activity in all other organs (values of uptake, see Table 7), resulting in beneficial tumor to organ ratios (Figure 5D-F). Similar results were obtained for 177Lu-FAPI-04 in mice with HT-1080-FAP tumor. Compared to FAPI-02, FAPI-04 shows greater tumor uptake, especially after 24 hours (Figure 5C). A calculation of the area under the curve (AUC) is shown in Table 8. FAPI-02 FAPI-02 FAPI-04 (Capan-2) (HT-1080-FAP) (HT-1080-FAP) Blood 0.83 ± 0.127 1.20 ± 0.178 1.70 ± 0.206 Brain 0.05 ± 0.010 0.06 ± 0.006 0.08 ± 0.010 Heart 0.37 ± 0.031 0.56 ± 0.085 0.80 ± 0.089 Intestines 0.30 ± 0.064 0, 37 ± 0.046 0.66 ± 0.196 Kidneys 1.45 ± 0.106 1.60 ± 0.075 2.28 ± 0.477 Liver 0.36 ± 0.015 0.45 ± 0.074 0.73 ± 0.118 Lungs 0.72 ± 0.021 1.02 ± 0.152 1.50 ± 0.151 Muscle 0.94 ± 0.168 1.17 ± 0.332 0.92 ± 0.020 Spleen 0.25 ± 0.015 0.38 ± 0.051 0.48 ± 0.072 Tumor 3.82 ± 0.390 4.51 ± 0.816 4 , 89 ± 0.817
[0274] [0274] Table 7. Quantification of biodistribution data 1 hour after intravenous administration of Lu-177-labeled FAPI-02 and -04 in nude Balb / c mice having a tumor; n = 3; values reported as mean% of ID / g ± SD. % ID / g 1h 4h 24h AUC FAPI-02 4.5 ± 0.82 4.0 ± 0.56 1.12 ± 0.13 64.0 FAPI-04 4.9 ± 0.82 5.4 ± 1.51 3.0 ± 0.23 99.4 FAPI-05 6.0 ± 0.90 5.8 ± 0.60 2.8 ± 0.40 103.3 FAPI-10 3.2 ± 0.72 2.9 ± 0.12 1.1 ± 0.04 49.3 FAPI-13 6.3 ± 0.57 8.7 ± 0.77 4.8 ± 1.71 157.5 FAPI-15 3.4 ± 1.13 4.6 ± 0.32 1.1 ± 0.25 68.0
[0275] [0275] Table 8. Tumor uptake of selected FAPI derivatives in nude mice having HT-1080-FAP tumor, n = 3. Values are reported as mean ID / g ± SD).
[0276] [0276] The diagnostic imaging of more than 100 patients was performed under the conditions of the updated Helsinki declaration, § 37 (Interventions not proven in clinical practice) and in accordance with German Pharmaceutical Law §13 (2b) for medical reasons, using 68Ga -FAPI-02 or - 04, which was applied intravenously (20 nmol, 122-336 MBq), 10 minutes, 1 and 3 hours after administration of the scanner. The variation in the activity of the injected radioactive tracker is due to the short half-life of 68Ga and the variable elution efficiencies obtained during the lifetime of the 68Ge / 68Ga generator. FDG imaging of a patient was performed 1 hour after intravenous injection of 358 MBq of 18F-FDG. PET / CT scans were performed with a PET / CT Biograph mCT Flow ™ scanner (Siemens Medical Solution) using the following parameters: slice thickness of 5 mm, increment of 3-4 mm, soft tissue reconstruction core, dose of caution.
[0277] [0277] Diagnostic PET / CT scans were performed 1 hour after intravenous administration of 68Ga-FAPI-02 and -04 in patients with breast, lung, pancreas, HNO, small intestine and metastatic ovarian cancer. In all patients, a robust accumulation of the crawler was observed in the primary tumor, as well as in lymph node and bone metastases with maximum SUV values of 48.0. On the other hand, the uptake of the tracker in normal tissue was very low (Figures 6-14). Radioactivity was rapidly eliminated from the bloodstream and excreted predominantly by the kidneys, resulting in high contrast images. Comparative imaging in a patient with locally advanced pulmonary adenocarcinoma revealed an obvious advantage of FAPI-02 compared to the commonly used PET tracker 18F-FDG. As shown in Figure 9, FAPI-02 shows greater uptake with less background activity, leading to greater contrast with better visibility of metastatic lesions. In contrast to FDG, which accumulates highly in cells with high glucose consumption, for example, the brain, FAPI-02 selectively targets the tissues where FAP-α is expressed. Comparative imaging in a patient with prostate cancer revealed an obvious advantage of FAPI-04 compared to the commonly used PET trackers 68Ga-DOTATOC and 68Ga-PSMA, allowing the detection of minor tumor lesions with reduced accumulation of kidney trackers (Figure 14). DISCUSSION
[0278] [0278] Reliable diagnosis of primary tumors, metastatic lesions and affected lymph nodes is of utmost importance to allow effective and adequate therapeutic planning, including tumor staging and treatment choice. To that end, imaging techniques represent indispensable tools for the evaluation of many types of cancer. Due to its high diagnostic accuracy and the possibility of evaluating anatomical and physiological details, combined PET / CT is the method of choice for modern tumor diagnosis. Unlike non-invasive imaging techniques, such as MRT or isolated CT, combined PET / CT, however, requires the use of radioactive scanners with high affinity to target structures with improved expression in tumors compared to normal tissues. An ideal tracker must specifically bind to your target protein to ensure reliable differentiation of cancerous and healthy tissues, as well as low background signals, resulting in high contrast images. Affinity and specificity become even more important if the radioactive tracker represents a teranotic compound, that is, it offers the possibility of being loaded with diagnostic or therapeutic nuclides, which facilitates and improves targeted and personalized treatment. In relation to a potential application of the tracker for therapeutic purposes, the high specificity of the target guarantees reduced side effects, which is especially important for the protection of radiation sensitive tissues, such as bone marrow, reproductive and digestive organs.
[0279] [0279] With this in mind, the inventors have developed a theranostic tracker targeting cancer-associated fibroblasts, which form a major component of the tumor's stroma. They are known to play a critical role in tumor growth, migration and progression and are genetically more stable than cancer cells, so they are less susceptible to developing resistance to therapy.
[0280] [0280] Based on a small molecule enzyme inhibitor, with high affinity for its target protein, we developed the radioactive trackers FAPI-01 to FAPI-73 by focused chemical modification. All compounds show specific binding to human and murine FAP-α with rapid and almost complete internalization without addressing the closely related CD26 / DPP4 protein. Since the iodinated molecules undergo enzymatic deiodination with free iodine efflux, longer incubation times result in low intracellular radioactivity. For this reason, FAPI-02 and subsequent compounds were designed with the FAP-binding portion being chemically linked to the DOTA chelator. This results in a set of teranostic compounds with favorable pharmacokinetic and biochemical properties, of which FAPI-02, FAPI-04, FAPI-46, FAPI-34, FAPI- 42, FAPI-52, FAPI-69, FAPI-70, FAPI -71, FAPI-72 and FAPI-73 represent the most favored ligands. FAPI-02 and FAPI-04 are eliminated significantly more slowly than FAPI-01, with retention of 12% (FAPI-02) and 49% (FAPI-04) of the radioactivity originally accumulated after 24 hours (FAPI-01 1 , 1%) with the other favored compounds having an even stronger bond (figure 16). They internalize rapidly in cells that express FAP-α and show high rates of tumor uptake in mice with tumor and patients with metastatic epithelial carcinomas. On the other hand, there is no accumulation in normal tissue and rapid clearance of the blood system, resulting in high contrast images. Robust internalization in human and murine cells expressing FAP-α was confirmed by confocal microscopy using fluorescence-labeled FAPI-02. In contrast to the first generation FAP antibody F19, which has a high affinity for its target protein without being internalized, FAPI-02 shows complete intracellular uptake after 1 hour of incubation. The internalization mechanism after binding to FAP was studied by Fischer et al. using FAP antibody fragments (Fabs) and an anti-mouse DyLight 549 antibody in SK-Mel-187 cells. The incubation at 37 ° C led to the internalization of the FAP-antibody complexes. As in our small molecule, the internalization process occurred quickly with an almost complete internalization. The colocalization of the Fabs with a marker for early endosomes was observed after 20 minutes and with a marker for late endosomes and lysosomes after 40 minutes. The internalization of Fab-mediated FAP-α was suppressed by a dinamine-dependent endocytosis inhibitor, indicating that endocytosis occurs by a dinamine-dependent mechanism.
[0281] [0281] FAPI-02 and -04 are rapidly eliminated from the body by renal clearance without being retained in the renal parenchyma. Unlike 18F-FDG, which accumulates highly in cells with high glucose consumption, including inflammatory tissue or in the brain, FAPI-02 is selectively enriched in tissues where its target protein is expressed. This opens new perspectives for the detection of malignant lesions in these regions. In addition, it has also been shown that FAP-α is expressed by myofibroblast-like rheumatoid synoviocytes in patients with rheumatoid arthritis and osteoarthritis, atherosclerosis, fibrosis, as well as in ischemic heart tissue after myocardial infarction. These observations suggest the application of FAPI-02 and -04 as image trackers for other indications.
[0282] [0282] The limiting factor for detecting tumor lesions is the degree of expression of FAP-α within the tumor. This depends largely on the number of activated fibroblasts, that is, the percentage of stromal content and / or the number of FAP-α molecules per fibroblast that can be determined by the microenvironment. As the growth of tumors that exceeds a size of 1 to 2 mm essentially requires the formation of a support stroma, the visualization of small lesions in the 3-5 mm range should be possible using FAPI-PET / CT.
[0283] [0283] As with any other targeted approach, FAPI derivatives only achieve optimal results in tissues with sufficiently high FAP-α expression, which is known to be quite heterogeneous in different types of cancer and patients. In addition to breast, colon and pancreatic cancer, which are excellent candidates for FAPI imaging, further analysis needs to explore whether other tumor entities, such as lung cancer, head and neck cancer, ovarian cancer or hepatomas, represent favorable targets.
[0284] [0284] In addition, FAP-α expression has been demonstrated in wound healing and fibrotic tissue, which should be remembered when interpreting radiological findings. These facts emphasize the need to adequately assess which patients are likely to benefit from potential FAPI therapy. Given the ability to use diagnostic or therapeutic nuclides, FAPI-02 and -04 allow simple stratification of the appropriate patient cohort. In any case, it is already clear that the two FAPI trackers represent ideal candidates for the development of a targeted radiopharmaceutical. Due to their high affinity for target, rapid internalization of the tumor and rapid clearance of the body, they are already ideally suited for tumor imaging.
[0285] [0285] All in vitro and in vivo experiments, as well as the clinical evaluation of FAPI derivatives, were performed as described above and according to Loktev et al.1 and Lindner et al.2 A preliminary dosimetry estimate for FAPI- 02 and FAPI-04 was based on two patients examined within 0.2 hours, 1 hour and 3 hours after the injection of the scanner, using the QDOSE dosimetry software. Additional PET / CT scans of tumor patients were acquired 1 hour after injection of FAPI-02 (n = 25) or FAPI-04 (n = 25); for 6 patients, an intra-individual related FDG scan (also acquired 1 hour p.i.) was available. For normal 16-organ tissue, a spherical VOI of 2 cm was placed in the parenchyma; for tumor lesions, a segmented VOI threshold was used to quantify medium SUV / max3. IN VITRO CHARACTERIZATION OF DOTA-FAPI DERIVATIVES
[0286] [0286] To assess the rate of target binding and internalization of DOTA-FAPI derivatives compared to FAPI-04, Lu-177-labeled compounds were incubated with HT-1080 cells that express FAP for 1, 4 and 24 hours, respectively (figure 16). The membrane-bound fraction was removed by acid elution using glycine-HCl, pH 2.2, followed by alkaline cell lysis to determine the internalized fraction. As shown in Figure 16, all derivatives demonstrate greater cell binding compared to FAPI-04 with binding values of up to 500% of the main compound after 1 hour of incubation (up to 750% after 4 hours).
[0287] [0287] To assess target affinity and specificity, competitive binding assays were performed using increasing concentrations of unlabeled as a competitor to the Lu-177-tagged compound
[0288] [0288] Table 9. IC 50 values of selected FAPI derivatives, as determined by competitive binding assays. DISTRIBUTION OF ORGANS FROM DOTA-FAPI DERIVATIVES IN MICE OWN TUMOR
[0289] [0289] For analysis of the pharmacokinetic profile, as well as in vivo tumor uptake, Lu-labeled DOTA-FAPI derivatives were administered intravenously to mice with HT-1080-FAP tumor. The organ distribution of the radiolabeled compounds was determined ex vivo in the blood, healthy tissues and the tumor. As shown in Figure 19, most compounds demonstrate higher rates of tumor uptake compared to FAPI-02 and FAPI-04, notably 24 hours after administration. Due to increased lipophilicity, some of the radioactive trackers show higher blood activity, in addition to increased kidney retention. The determination of tumor-blood ratios also reveals a clear advantage of the compounds FAPI-21 and FAPI-46, which demonstrate ratios significantly higher than FAPI-04 at all times examined (Figure 20). IMAGIOLOGY IN SMALL ANIMALS OF DOTA-FAPI DERIVATIVES IN MICE WITH TUMOR
[0290] [0290] Based on these findings, small animal PET imaging was performed using Ga-68-labeled DOTA-FAPI derivatives up to 140 minutes after intravenous administration of radioactive scanners in mice having HT-1080-FAP tumor.
[0291] [0291] Very similar to the literature values for F-18- FDG, Ga-68-DOTATATE or Ga-68-PSMA-11, an exam with 200 MBq of Ga-68-FAPI-02 and -04 corresponds to a dose equivalent of approximately 3-4 mSv. After rapid clearance through the kidneys, normal organs show low uptake of the crawler, with only minimal changes between 10 minutes and 3 hours p.i. In FAPI-02, tumor uptake from 1 hour to 3 hours p.i. decreases by 75%, while tumor retention is slightly prolonged with FAPI-04 (50% washout). At 1 pm p.i. both FAPI trackers perform equally (Figure 23). In comparison to FDG, tumor uptake is almost equal (maximum average SUV of FDG 7.41; maximum SUV FAPI-02 7.37; n.s.); fundus uptake in the brain (11.01 vs 0.32), liver (2.77 vs 1.69) and oral / pharyngeal mucosa (4.88 vs 2.57) is significantly lower with FAPI-02; other organs were not significantly different between FDG and FAPI-02 (Figure 24). For detailed information and results, see Giesel et al.3 which is incorporated by reference into this document.
[0292] [0292] In addition to rapid uptake of Ga-68-labeled FAPI-04 in different types of cancer, including breast, pancreas, ovarian and HNO tumors, the accumulation of trackers has also been demonstrated in carcinomatous peritonitis (Figure 25A), in addition to inflammatory malignancies, such as myocarditis (Figure 25B) and arthrosis (Figure 25C). These results indicate a potential application of FAPIs labeled with Ga-68 for the detection of non-cancerous malignancies that are characterized by a process of chronic inflammation involving recruitment of activated fibroblasts.
[0293] [0293] As shown in Figure 26, robust accumulation of Ga-68-labeled FAPI-21 was observed in different cancers, including ovarian, rectal and mucoepidermoid carcinoma. Similar tumor uptake was demonstrated for Ga-68-labeled FAPI-46, which rapidly accumulated in colorectal and colorectal carcinoma, lung cancer and solitary fibrous sarcoma (Figure 27). After PET / CT examination using Ga-68-labeled FAPI-46, a first therapeutic approach using the Sm-153-labeled radioactive scanner was performed on two cancer patients. As shown in Figure 28, the robust tumor accumulation of the crawler is detectable up to 20 hours after administration.
[0294] [0294] FAPI-46-PET / CT imaging of three lung cancer patients with idiopathic pulmonary fibrosis revealed a clear difference in accumulation of trackers in cancerous versus fibrotic lesions. As shown in Figure 30, tumor uptake of Ga-68-labeled FAPI-46 was significantly higher in two patients (A, B), but slightly lower in one patient (C), compared to the activity measured in fibrotic tissue. The patient shown in Figure C suffered from exacerbated pulmonary fibrosis compared to the two non-exacerbated cases. Therefore, the tracker is possibly useful for differentiating between fibrosis patients with poor prognosis and patients with good prognosis.
[0295] [0295] To allow the use of alternative radioactive nuclides, a series of FAPI derivatives have been designed and characterized in relation to the target affinity, specificity and pharmacokinetics. In some of these compounds, the original DOTA chelator has been replaced by different chelating portions, which are ideally suited for the incorporation of Tc-99m (FAPI-19, - 27, -28, -29, -33, -34, - 43 , -44, -45, -60, -61, -62). The in vitro FAP affinity and biodistribution in HT-1080-FAP xenografted mice are shown as an example for FAPI-19 and FAPI-34. Both compounds demonstrate robust binding to human FAP in vitro (IC50 FAPI-19: 6.4 nM). In contrast to FAPI-19, which shows insufficient tumor uptake in vivo, as well as rapid accumulation in the liver due to a renal shift towards liver elimination, FAPI-34 is continuously enriched in the tumor and demonstrates significantly less hepatic uptake (Figures 31, 32). A first diagnostic application of Tc-99m-labeled FAPI-34 in a patient with pancreatic cancer with liver metastases shows a stable tumor accumulation of the crawler up to 4 hours after administration.
[0296] [0296] FAPI derivatives radiolabelled with Pb-203 (FAPI-04, -32, -46 and FAPI-04tcmc) show cell binding comparable to HT-
[0297] [0297] To enable radioactive labeling using Cu-64, the derivatives NOTE FAPI-42 and FAPI-52 were developed and characterized in relation to the affinity, specificity and pharmacokinetics of the target. As shown in Figure 37, both trackers show robust binding to HT-1080-FAP cells for up to 24 hours of incubation with similar IC 50 values in the lower nanomolar range (Figure 37A, B). However, FAPI-42 is eliminated significantly more slowly than FAPI-52, resulting in an estimated in vitro half-life of 12 hours (Figure 37C). These results are confirmed by imaging of small animals of HT-1080-FAP xenografted mice. As shown in Figure 38, both compounds demonstrate robust tumor uptake, as well as rapid clearance of the blood stream in vivo. Notably, renal excretion of FAPI-42 occurs significantly more rapidly compared to FAPI-52, while its tumor activity remains slightly greater for 2 to 24 hours after administration.
[0298] [0298] The derivatives Nota FAPI-42 and FAPI-52 were implanted for the formation of aluminum fluoride complexes to allow images with F-18. As shown in Figure 39, both compounds demonstrate rapid tumor uptake in imaging of small xenografted mice HT-1080-FAP. Although both compounds are mainly excreted via the kidney, biliary elimination is also observed. Although renal excretion is faster for FAPI-52, greater tumor accumulation, greater tumor retention and lower proportion of the bile duct are in favor of FAPI-42. REFERENCES
[0299] [0299] 1 Loktev, A. et al. A new method for tumor imaging by targeting cancer associated fibroblasts. Journal of nuclear medicine: official publication, Society of Nuclear Medicine, doi: 10.2967 / jnumed.118.210435 (2018).
[0300] [0300] 2 Lindner, T. et al. Development of quinoline based theranostic ligands for the targeting of fibroblast activation protein. Journal of nuclear medicine: official publication, Society of Nuclear Medicine, doi: 10.2967 / jnumed.118.210443 (2018).
[0301] [0301] 3 Giesel, F. et al. FAPI-PET / CT: biodistribution and preliminary dosimetry estimate of two DOTA-containing FAP-targeting agents in patients with various cancers. Journal of nuclear medicine: official publication, Society of Nuclear Medicine, doi: 10.2967 / jnumed.118.215913 (2018).
[0302] [0302] In order to selectively target FAP positive brain tumors, initial experiments were carried out on mice having a tumor using the U87MG human glioblastoma xenograft model. The tumor accumulation and the distribution of radiolabelled FAPI-02 and -04 organs were analyzed by PET imaging of small animals, as well as in a biodistribution study. As shown in Figures 40 and 41, both FAPI-02 and -04 demonstrate rapid tumor uptake and insignificantly low activity in healthy organs and blood. CLINICAL DATA
[0303] [0303] According to the 2016 WHO classification, gliomas are subdivided into WHO grade I-IV wild-type HDI gliomas and WHO grade II-IV mutant HDI gliomas. The most frequent grade IV gliomas of the WHO are glioblastomas.
[0304] [0304] Clinical PET imaging was performed on 18 patients with glioma (5 mutant HDI gliomas, 13 wild-type HDI glioblastomas; see Table 10). As shown in Figures 42-44, wild-type HDI glioblastomas and grade III / IV mutant HDI gliomas, but not grade II, showed high uptake of the crawler. In glioblastomas, points with greater uptake of projection were observed in areas of increased contrast. CONCLUSION
[0305] [0305] Increased uptake of trackers in HDI wild-type glioblastomas and high-grade mutant HDI astrocytomas, but not diffuse astrocytomas, may allow a non-invasive distinction between low-grade HDI mutants and high-grade gliomas and be useful for follow-up studies. The heterogeneous uptake of trackers in glioblastomas can be useful in planning the biopsy.
[0306] [0306] For reuptake experiments, 177Lu-labeled FAPI-04 and -46 (5 MBq / nmol in DMEM) were added to HT-1080-FAP cells and incubated for 60 minutes at 4 and 37 ° C, respectively. The radioactive medium was removed and the cells were washed twice with phosphate buffered saline (PBS) pH 7.4. Subsequently, non-radioactive medium with and without unmarked FAPI (1 µM) was added at intervals ranging from 10 minutes to 6 hours. The cells were washed twice with PBS pH 7.4. To remove surface-bound activity, cells were incubated with glycine-HCl (1 M in PBS, pH 2.2) for 10 minutes at room temperature. After washing twice with ice-cold PBS, cells were lysed with 1.4 ml of lysis buffer (0.3 M NaOH, 0.2% SDS) to determine the internalized fraction. For cells incubated at 4 ° C, all washing and elution steps were performed using ice-cold buffers. Radioactivity was measured using a γ counter (Packard Cobra II), normalized to 1 million cells and calculated as a percentage of the applied dose (% AD; see Figure 47). ENZYMATIC INHIBITION TEST
[0307] [0307] To determine the potential inhibitory effects of FAPI-04 on the enzymatic activity of FAP, enzyme inhibition assays were performed using the recombinant human FAP protein (1 pmol / well) in a 48-well plate. After incubating FAPI-04 or Talabostat (0 - 1000 nM / well) with human FAP for 30 minutes at 37 ° C, the fluorogenic FAP Z-GP-AMC substrate was added at a final concentration of 0 - 200 µM / well and incubated for 60 minutes at 37 ° C. The enzyme activity of FAP was determined by measuring the fluorescence intensity of the AMC reaction product at 360/460 nm using the SpectraMax M2 Plate Reader (Molecular Devices, San José, USA) (see Figure 46).
[0308] [0308] For biodistribution experiments, 8 week old BALB / c nu / nu mice (Charles River) were inoculated subcutaneously in the right trunk with 5 million HT-1080-FAP cells,
[0309] [0309] All in vitro and in vivo experiments, as well as the clinical evaluation of FAPI derivatives, were performed as described in the initial document and according to Loktev et al. 1 and Lindner et al. two
[0310] [0310] All experiments were carried out in a manner similar to FAPI-42 (labeled with AlF-18) or FAPI-72 (labeled with nicotinamide F-18). Compound IC50 (nM) Compound IC50 (nM) FAPI-72 2.4 FAPI-74 9.2 FAPI-73 5.4 FAPI-75 2.9
[0311] [0311] Table 11. EC50 values of selected FAPI derivatives, as determined by competitive binding assays. DETERMINATION OF DETERMINATION OF BLOOD MIXTURE
[0312] [0312] To estimate the compound's clearance rate, half-lives were calculated by an assumed two-phase exponential decay from the average SUV values (0.375-60 min) of the heart as a representation of the blood mixture. All selected compounds were cleaned very quickly with half-lives below 10 minutes. The calculated plateau values, which were highest for FAPI-13, -21, -36 marked with Ga-68 and FAPI-74 marked with AIF-18, theoretically correspond to a higher fraction of the compound that is not cleaned due to binding unspecific or remaining in circulation (Table 12). As an example for rapid clearance, the activity / time curves for FAPI-04 and -46 between 0 and 15 min are shown in Figure 53. Compound Clearance of the Blood Plateau Value (SUV) mixture (T1 / 2 [min ]) FAPI-04 7.1 0.21 FAPI-13 5.5 0.27 FAPI-21 5.1 0.31 FAPI-36 5.0 0.58 FAPI-46 5.3 0.19 FAPI-74 2.4 0.32
[0313] [0313] Table 12. Half-life of the blood mixture and the hypothetical plateau value of selected FAPI derivatives, calculated from the average SUV values by an assumed two-phase exponential decay.
[0314] [0314] Based on these findings, PET imaging of small animals was performed using F-18-labeled NOTE derivatives and F-18 nicotinamide-tagged FAPI derivatives up to 140 minutes after intravenous administration of radioactive scanners in mice having HT tumor -1080-FAP. Derivatives F-18-nicotinamide FAPI-72, -73 and -77 showed an unfavorable accumulation in the liver, as well as biliary excretion, while FAPI-78 was excreted renally, but did not show tumor uptake. In the case of NOTE derivatives marked with AlF-18, FAPI-74 and -75, high target specificity and rapid clearance were observed, resulting in high contrast images, which allow excellent visualization of FAP positive tumors (Figure 50).
[0315] [0315] For analysis of the pharmacokinetic profile, as well as in vivo tumor uptake, FAPI-75 labeled with AlF-18 was administered intravenously to mice having a HT-1080-FAP tumor. The organ distribution of the radiolabeled compound was determined ex vivo in the blood, healthy tissues and the tumor. As shown in Figure 51, the compounds demonstrate high tumor uptake, although compared to DOTA derivatives labeled with Ga-68, greater accumulation in healthy tissue is observed, while the performance in PET imaging was the same. ITEMS
[0316] [0316] The following items represent preferred embodiments of the present invention.
[0317] [0317] 1. A compound of Formula (I) (I), in which Q, R, U, V, W, Y, Z are individually present or absent, provided that at least three of Q, R, U, V , W, Y, Z are present; Q, R, U, V, W, Y, Z are independently selected from the group consisting of O, CH2, NR4, C = O, C = S, C = NR4, HCR4 and R4CR4,
[0318] [0318] 2. The compound of item 1, in which (i) Q, R, U are CH2 and are individually present or absent; V is CH2, C = O, C = S or C = NR4; W is NR4; Y is HCR4; and Z is C = O, C = S or C = NR4; and / or (ii) Q and R are absent; U is CH2 and is present or absent; R1 and R2 are selected independently from the group consisting of -H and halo; R3 is selected from the group consisting of -H, -CN and - B (OH) 2; R4 is selected from the group consisting of -H and -C1-6alkyl, wherein -C1-6alkyl is optionally substituted with 1 to 3 substituents selected from -OH.
[0319] [0319] 3. The compound of item 1 or 2, in which it is selected from the group consisting of,,, and, optionally, further comprising 1 or 2 heteroatoms selected from O, N and S.
[0320] [0320] 4. The compound of any of the previous items, in which it is selected from the group consisting of,,,,,,,,,,,,,,,,, and.
[0321] [0321] 5. The compound of any of the previous items, where R5 and R6 are H; R7 is, where D is absent; A is O, S, CH2, NH, NCH3; E is C1-6 alkyl or, where m is 1, 2 or 3; A and E, together, form a group selected from:,,,,; B is NR4-C1-6 alkyl or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10-membered N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S, preferably further comprising 1 or 2 nitrogen atoms, preferably wherein the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C1-6 alkyl, aryl, C1-6 aralkyl.
[0322] [0322] 6. The compound of any of the previous items, in which (i) the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle:
[0323] [0323] 7. The compound of any of the previous items, where Q, R, U are absent; V is C = O; W is NH; Y is CH2; Z is C = O; R1 and R2 are selected independently from the group consisting of -H and halo; R3 is -CN; R5 and R6 are H; R7 is, where D is absent; A is O, S, CH2, NH, NCH3; E is C1-6 alkyl or, where m is 1, 2 or 3; or A and E together form a group selected from:,,,,; B is NH-C 1-6 alkyl,,, or; optionally, B is replaced with C1-3 alkyl; and is .
[0324] [0324] 8. The compound of any of the preceding items, where C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl, and / or in which C1-6 aralkyl is selected from the group consisting of benzyl, phenyl-ethyl, phenyl-propyl and phenyl-butyl.
[0325] [0325] 9. The compound of any of the previous items, where R8 is a radioactive portion, where the radioactive portion is a fluorescent isotope, a radioisotope, a radioactive drug or combinations thereof, preferably where the radioactive portion is selected from the group consisting of alpha radiation emitting isotopes, beta radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescence emitting isotopes, such as 11C, 18F, 51Cr, 67Ga, 68Ga, 111In, 99mTc, 186Re, 188Re, 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, 197Hg, 211At, 151Eu, 153Eu, 169Eu, 201Tl, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au, 225Ac, 227Th and 199Ag, preferably 18F, 64Cu, 68Ga, 90Y, 99mTc, 153Sm, 177Lu, 188Re.
[0326] [0326] 10. The compound of any one of items 1 to 8, where R8 is a fluorescent dye selected from the group consisting of the following classes of fluorescent dyes: xanthenes, acridines, oxazins, kinins, stirila dyes, coumarins , porphins, ligand-metal complexes, fluorescent proteins, nanocrystals, perylenes, boro-dipyrometenes and phthalocyanines, as well as conjugates and combinations of these dye classes.
[0327] [0327] 11. The compound of any one of items 1 to 8, wherein R8 is a chelating agent that forms a complex with divalent or trivalent metal cations, preferably in which the chelating agent is selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N, N ', N, N'-tetraacetic acid (DOTA), ethylene diaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTE ), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N, N ', N', N ”-pentaacetic acid (DTPA), bis- (carboxymethylimidazole) glycine and 6-hydrazinopyridine-3-carboxylic acid (HYNIC).
[0328] [0328] 12. The compound of any one of items 1 to 8, wherein R8 is a contrast agent that comprises or consists of a paramagnetic agent, preferably, wherein the paramagnetic agent comprises or consists of paramagnetic nanoparticles.
[0329] [0329] 13. Pharmaceutical composition comprising or consisting of at least one compound according to any one of items 1 to 12; and, optionally, a pharmaceutically acceptable carrier and / or excipient.
[0330] [0330] 14. Composed of any one of items 1 to 12 or the pharmaceutical composition of claim 13, for use in the diagnosis or treatment of a disease characterized by overexpression of fibroblast-activating protein (FAP) in an animal or a human, preferably in which the disease characterized by overexpression of fibroblast activating protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorder, preferably in which the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, cancer of hypopharynx, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, renal clear cell carcinoma, neuroendocrine tumor, osteomalacia oncogenic, sarcoma, CUP (hidden primary carcinoma), thymus carcinoma, desmoid tumors, glioma, astrocytoma, cervical carcinoma and prostate cancer.
[0331] [0331] 15. Kit comprising or consisting of the compound of any one of items 1 to 12 or the pharmaceutical composition of claim 13 and instructions for the diagnosis or treatment of a disease.

权利要求:
Claims (15)
[1]
1. COMPOUND, characterized by being of formula (I) (I), in which Q, R, U, V, W, Y, Z are individually present or absent, provided that at least three of Q, R, U, V , W, Y, Z are present; Q, R, U, V, W, Y, Z are selected independently from the group consisting of O, CH2, NR4, C = O, C = S, C = NR4, HCR4 and R4CR4, with the proviso that two O's are not directly adjacent to each other; R1 and R2 are independently selected from the group consisting of -H, -OH, halo, C1-6 alkyl, -O-C1-6 alkyl, S-C1-6 alkyl; R3 is selected from the group consisting of -H, -CN, - B (OH) 2, -C (O) -alkyl, -C (O) -aryl-, -C = CC (O) -aryl, -C = CS (O) 2-aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2 and 5-tetrazolyl; R4 is selected from the group consisting of -H, -C1-6 alkyl, -O-C1-6 alkyl, -S-C1-6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl and -C1 alkyl -6, each of said - C1-6 alkyl being optionally substituted with 1 to 3 substituents selected from -OH, oxo, halo and optionally connected to Q, R, U, V, W, Y or Z; R5 is selected from the group consisting of -H, halo and C1-6 alkyl; R6 and R7 are selected independently from the group consisting of –H,
and, as long as R6 and R7 are not at the same time H, where L is a ligand,
where D, A, E and B are individually present or absent,
preferably where at least A, E and B are present, where when present:
D is a ligand;
A is selected from the group consisting of NR4, O, S and
CH2;
And it is selected from the group consisting of C1-6 alkyl,
,,
and ; where i is 1, 2 or 3;
where j is 1, 2 or 3;
where k is 1, 2 or 3;
where m is 1, 2 or 3;
B is selected from the group consisting of S, NR4, NR4-O,
NR4-C1-6 alkyl, NR4-C1-6 alkyl-NR4 and an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10 members N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S ,
preferably further comprising 1 or 2 nitrogen atoms, preferably where NR4-C1-6-alkyl-NR4 and the N-containing heterocycle are substituted with 1 to 3 substituents selected from the group consisting of C1-6-alkyl, aryl, aralkyl C1-6; and R8 is selected from the group consisting of radioactive moiety, chelating agent, fluorescent dye, a contrast agent and combinations thereof; is a 1-naphthyl moiety or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10 membered N, where there are 2 ring atoms between the atom of N and X; said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from O, N and S; and X is a C atom; or a tautomer, racemate, hydrate, solvate or pharmaceutically acceptable salt thereof.
[2]
2. COMPOUND, according to claim 1, characterized in that (i) Q, R, U are CH2 and are individually present or absent; V is CH2, C = O, C = S or C = NR4; W is NR4; Y is HCR4; and Z is C = O, C = S or C = NR4; and / or (ii) Q and R are absent; U be CH2 and be present or absent; R1 and R2 are selected independently from the group consisting of -H and halo; R3 is selected from the group consisting of -H, -CN and - B (OH) 2; R4 is selected from the group consisting of -H and -C1-6alkyl, where -C1-6alkyl is optionally substituted with 1 to 3 substituents selected from -OH.
[3]
COMPOSITE according to any one of claims 1 to 2, characterized in that it is selected from the group consisting of,,, and, optionally, further comprising 1 or 2 heteroatoms selected from O, N and S.
[4]
COMPOSITE according to any one of claims 1 to 3, characterized in that it is selected from the group consisting of,,,,,,,,,,,,,
, , , , , and .
[5]
COMPOSITE according to any one of claims 1 to 4, characterized in that R5 and R6 are H; R7 be, where D is absent; A is O; E is C1-6 alkyl or, where m is 1, 2 or 3; B is NR4-C1-6 alkyl or an aromatic or non-aromatic mono- or bicyclic heterocycle containing 5 to 10-membered N, preferably further comprising 1 or 2 heteroatoms selected from O, N and S, preferably further comprising 1 or 2 nitrogen atoms, preferably where the N-containing heterocycle is substituted with 1 to 3 substituents selected from the group consisting of C1-6 alkyl, aryl, C1-6 aralkyl.
[6]
6. A COMPOUND according to any one of claims 1 to 5, characterized in that (i) the N-containing heterocycle comprised in B is an aromatic or non-aromatic monocyclic heterocycle: wherein the heterocycle optionally further comprises 1 or 2 heteroatoms selected from O, N and S, also comprises,
optionally, 1 nitrogen;
it is connected to position 1, 2 or 3, preferably in position 2; l is 1 or 2; and / or
(ii) the heterocycle containing N comprised in B is selected from the group consisting of:
,,,,
,,,,
, , , and
, where if the N-containing heterocycle comprised in B is
, the heterocycle also optionally comprises 1 or 2 heteroatoms selected from O, N and S, it also comprises,
optionally, 1 nitrogen, optionally comprises one or more side chains (for example, derived from amino acids);
it is connected to position 1, 2 or 3, preferably in position 2; o is 1 or 2,
preferably, if the N-containing heterocycle comprised in B is
, the N-containing heterocycle comprised in B is, or; more preferably, if the N-containing heterocycle comprised in B is, the N-containing heterocycle comprised in B is or.
[7]
COMPOUND according to any one of claims 1 to 6, characterized in that Q, R, U are absent; V is C = O; W is NH; Y is CH2; Z is C = O; R1 and R2 are selected independently from the group consisting of -H and halo; R3 is -CN; R5 and R6 are H; R7 be, where D is absent; A is O;
E is C1-6 alkyl or, where m is 1, 2 or 3; B is NH-C 1-6 alkyl,,, or; and is.
[8]
8. COMPOUND according to any one of claims 1 to 7, characterized in that C1-6 alkyl is selected from the group consisting of methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, tert-butyl, pentyl and hexyl, and / or in which C1-6 aralkyl is selected from the group consisting of benzyl, phenyl-ethyl, phenyl-propyl and phenyl-butyl.
[9]
COMPOUND according to any one of claims 1 to 8, characterized in that R8 is a radioactive portion, wherein the radioactive portion is a fluorescent isotope, a radioisotope, a radioactive drug or combinations thereof, preferably in which the portion radioactive is selected from the group consisting of alpha radiation emitting isotopes, beta radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescence emitting isotopes, such as 18F, 51Cr , 67Ga, 68Ga, 111In, 99mTc, 186Re, 188Re, 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, 197Hg, 211At, 151Eu, 153Eu, 169Eu, 201Tl, 203Pb, 212Pb, 64Cu, 67Cu, 188Re, 186Re, 198Au, 225Ac, 227Th and 199Ag.
[10]
10. COMPOUND according to any one of claims 1 to 8, characterized in that R8 is a fluorescent dye selected from the group consisting of the following classes of fluorescent dyes: xanthenes, acridines, oxazins, kinins, stirila dyes, coumarins, porphins, ligand-metal complexes, fluorescent proteins, nanocrystals, perylenes, boron-dipyromethenes and phthalocyanines, as well as conjugates and combinations of these dye classes.
[11]
COMPOSITE according to any one of claims 1 to 8, characterized in that R8 is a chelating agent that forms a complex with divalent or trivalent metal cations, preferably in which the chelating agent is selected from the group consisting of acid 1,4,7,10-tetraazacyclododecane-N, N ', N, N'-tetraacetic (DOTA), ethylene diaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTE) , triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N, N ', N', N ”-pentaacetic acid (DTPA), bis- (carboxymethylimidazole) glycine and 6-hydrazinopyridine-3-carboxylic acid (HYNIC).
[12]
12. COMPOUND according to any one of claims 1 to 8, characterized in that R8 is a contrast agent that comprises or consists of a paramagnetic agent, preferably, wherein the paramagnetic agent comprises or consists of paramagnetic nanoparticles.
[13]
13. PHARMACEUTICAL COMPOSITION, characterized in that it comprises or consists of at least one compound, as defined in any one of claims 1 to 12, and, optionally, a pharmaceutically acceptable vehicle and / or excipient.
[14]
COMPOSITE according to any one of claims 1 to 12, or pharmaceutical composition according to claim 13, characterized in that it is for use in the diagnosis or treatment of a disease that overexpresses fibroblast activating protein (FAP) in an animal or a human being, preferably in which the disease that overexpresses fibroblast activating protein (FAP) is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorder, preferably in which the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer , hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocellular carcinoma, renal clear cell carcinoma, neuroendocrine tumor, the oncogenic steomalacia, sarcoma, CUP (occult primary carcinoma), carcinoma of the thymus, desmoid tumors, glioma, astrocytoma, cervical carcinoma and prostate cancer.
[15]
15. KIT, characterized in that it comprises or consists of the compound, as defined in any one of claims 1 to 12, or the pharmaceutical composition, as defined in claim 13, and instructions for the diagnosis of a disease.

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法律状态:
2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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