Fibroblast-activating protein ligands for targeted delivery applications

Abstract

The present invention relates to ligands of fibroblast activation protein (FAP) for the active delivery of various payloads (e.g., cytotoxic drugs, radionuclides, fluorophores, proteins, and immunomodulators) at sites of disease. In particular, the present invention relates to the development of FAP ligands for targeting applications, especially diagnostic and / or therapeutic or surgical methods associated with diseases or disorders such as cancer, inflammation, or another disease characterized by overexpression of FAP.

Description

[Technical Field]

[0001] This invention relates to ligands for fibroblast-activating protein (FAP) for the active delivery of various payloads (e.g., cytotoxic drugs, radionuclides, fluorophores, proteins, and immunomodulators) at disease sites. In particular, this invention relates to the development of FAP ligands for targeted applications, especially for diagnostic and / or therapeutic or surgical methods related to diseases or disorders such as cancer, inflammation, or other diseases characterized by FAP overexpression. [Background technology]

[0002] Chemotherapy remains widely used for the treatment of cancer patients and other diseases. Conventional anti-cancer chemotherapy agents act on the fundamental mechanisms of cell survival and cannot distinguish between healthy and malignant cells. Furthermore, these drugs do not accumulate effectively at the site of disease when administered systemically. Nonspecific mechanisms of action and ineffective localization at the tumor site are major causes of the unsustainable side effects and insufficient therapeutic efficacy of conventional chemotherapy.

[0003] The development of targeted drugs that can selectively localize to the site of disease after systemic administration is highly desirable. Strategies for generating such drugs are represented by the chemical conjugation of therapeutic payloads, such as cytotoxic drugs or radionuclides, to disease marker-specific ligands. Disease-specific monoclonal antibodies, peptides, and small ligands have been considered as ligands of choice for the development of targeted drug products. The use of small ligands for targeted applications has several advantages compared to larger molecules such as peptides and antibodies: faster and more effective tumor penetration, lower immunogenicity, and lower manufacturing costs.

[0004] Small organic ligands specific to prostate-specific membrane antigens, folate receptors, and carbonic anhydrase IX have shown excellent in vivo distribution profiles in preclinical models of cancer and in patients. These ligands have been conjugated to cytotoxic drugs and radionuclides to generate small molecule-drug conjugates and small molecule-radioconjugate products (SMDCs and SMRCs) for cancer treatment. 177-Lutetium-PSMA-617 represents an example of a late-stage SMRC currently being investigated in a Phase 3 clinical trial (VISION trial) for the treatment of patients with metastatic castration-resistant prostate cancer (mCRPC).

[0005] Fibroblast-activating protein (FAP) is a membrane-bound gelatinase that promotes tumor growth and progression and is overexpressed in cancer-associated fibroblasts. Due to its low expression in normal organs, FAP represents an ideal target for the development of targeted SMDCs and SMRCs.

[0006] WO2019154886 and WO2019154859 describe heterocyclic compounds as fibroblast-activating protein-alpha inhibitors used to treat different cancer types. WO2019118932 describes substituted N-containing cyclic compounds as fibroblast-activating protein-alpha inhibitors used to treat different pathological conditions. WO2019083990 describes fibroblast-activating protein-alpha (FAP-alpha) compounds for imaging and radiotherapy targeting as FAP-alpha inhibitors used to image diseases associated with FAP-alpha and to treat proliferative disorders, noting that the 4-isoquinolinoyl and 8-quinolinoyl derivatives described therein are characterized by very low FAP affinity. WO2013107820 describes substituted pyrrolidine derivatives used in the treatment of proliferative disorders such as cancer and diseases manifested by tissue remodeling, or chronic inflammation such as osteoarthritis. WO2005087235 describes pyrrolidine derivatives as dipeptidyl peptidase IV inhibitors for treating type II diabetes. WO2018111989 describes fibroblast-activating protein (FAP) inhibitors, divalent linkers, and conjugates containing, for example, near-infrared (NIR) dyes, which are useful for eliminating cancer-associated fibroblasts, imaging cell populations in vitro, and treating cancer.

[0007] Tsutsumi et al. (J Med Chem 1994) described the preparation of a series of α-ketoheterocyclic compounds and their in vitro prolyl endopeptidase (PEP) inhibitory activity. Hu et al. (Bioorg Med Chem Lett 2005) described the structure-activity relationships of various N-alkylGly-boro-Pro derivatives against FAP and two other dipeptidyl peptidases. Edosada et al. (J Biol Chem 2006) described the dipeptide substrate specificity of FAP and the development of Ac-Gly-BoroPro FAP selective inhibitors. Gilmore et al. (Biochem Biophys Res Commun 2006) described the design, synthesis, and kinetic studies of a series of dipeptide proline diphenyl phosphonates against DPP-IV and FAP. Tran et al. (Bioorg Med Chem Lett 2007) described the structure-activity relationships of various N-acyl-Gly-, N-acyl-Sar-, and N-blocked-boroPro derivatives against FAP. Tsai et al. (J Med Chem 2010) described structure-activity relationship studies that resulted in numerous FAP inhibitors with excellent selectivity against DPP-IV, DPP-II, DPP8, and DPP9. Ryabtsova et al. (Bioorg Med Chem Lett 2012) described the synthesis and evaluation of the FAP inhibitory properties of a series of N-acylated glycyl-(2-cyano)pyrrolidines. Poplawski et al. (J Med Chem 2013) described N-(pyridine-4-carbonyl)-D-Ala-boroPro as a potent and selective FAP inhibitor. Jansen et al. (ACS Med Chem Lett 2013) described an FAP inhibitor based on the N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. Jansen et al. (Med Chem Commun 2014) described the structure-activity relationship of an FAP inhibitor based on the linagliptin scaffold. Jansen et al. (Med Chem Commun 2014) described a xanthine-based FAP inhibitor with low micromolar potency.Jansen et al. (J Med Chem 2014) described the structure-activity relationship of FAP inhibitors based on the N-4-quinolinoyl-Gly-(2S)-cyanoPro scaffold. Jackson et al. (Neoplasia 2015) described the development of pseudopeptide inhibitors of FAP. Meletta et al. (Molecules 2015) described the use of borate-based FAP inhibitors as non-invasive imaging tracers of atherosclerotic plaques. Dvorakova et al. (J Med Chem 2017) described the preparation of polymer conjugates containing FAP-specific inhibitors for targeted applications. Loktev et al. (J Nucl Med 2018) described the development of iodized and DOTA-coupling radioactive tracers based on FAP-specific enzyme inhibitors. Lindner et al. (J Nucl Med 2018) described the modification and optimization of FAP inhibitors for use as diagnostic and therapeutic tracers. Giesel et al. (J Nucl Med 2019) described the clinical imaging performance of a quinoline-based PET tracer that acts as an FAP inhibitor. [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to address the problem of providing improved binders (ligands) for fibroblast-activating protein (FAP) suitable for targeted applications. The binder should be suitable for targeted delivery of payloads, such as therapeutic or diagnostic agents, to sites that suffer from or are at risk of disease or impairment characterized by FAP inhibition and / or FAP overexpression. Preferably, the binder should form a stable complex with FAP and exhibit increased affinity, increased inhibitory activity, a slower rate of dissociation from the complex, and / or extended retention at the disease site. [Means for solving the problem]

[0009] The inventors have discovered a novel organic ligand for fibroblast-activating protein (FAP) suitable for targeted applications. The compound according to the present invention (also referred to as ligand or binder) contains a small binding site A having the following structure:

[0010] [ka]

[0011] The compound according to the present invention is of the following general formula I,

[0012] [ka]

[0013] It can be represented by its individual diastereoisomers, its hydrate, its solvate, its crystalline form, its individual tautomers, or pharmaceutically acceptable salts thereof, where A is the bonding portion; B is the portion containing a chain of atoms covalently bonded or covalently attaching portions A and C; and C is the payload portion.

[0014] The present invention further provides pharmaceutical compositions comprising the aforementioned compound and pharmaceutically acceptable excipients. The present invention provides the compound or pharmaceutical composition for use in a method of treating a human or animal body by surgery or therapy, or in a diagnostic method performed on a human or animal body; and further provides a method of treating a human or animal body by surgery or therapy, or in a diagnostic method performed on a human or animal body, comprising administering a therapeutically or diagnostically effective amount of the compound or pharmaceutical composition to a subject in need.

[0015] The present invention provides for use in methods of treating or preventing a disease or disorder in a person suffering from or at risk of such disease or disorder; and further provides a method of treating or preventing a disease or disorder, comprising administering a therapeutically or diagnostically effective amount of the compound or pharmaceutical composition to a person suffering from or at risk of such disease or disorder.

[0016] The present invention provides for use in a guided surgical method performed on a subject suffering from or at risk of a disease or disorder; and further provides a guided surgical method comprising administering a therapeutically or diagnostically effective amount of the compound or pharmaceutical composition to a subject suffering from or at risk of a disease or disorder.

[0017] The present invention provides for use in a method of diagnosing a disease or disorder performed on the body of a human or animal and involving nuclear medicine imaging techniques such as positron emission tomography (PET); and further provides a method of diagnosing a disease or disorder, performed on the body of a human or animal and involving nuclear medicine imaging techniques such as positron emission tomography (PET), comprising administering a therapeutically or diagnostically effective amount of the compound or pharmaceutical composition to a subject in need.

[0018] The present invention provides a compound or pharmaceutical composition for use in a method of targeted delivery of a therapeutic or diagnostic agent to a subject suffering from or at risk of a disease or disorder; and further provides a method of targeted delivery of a therapeutically or diagnostically effective amount of the compound or pharmaceutical composition to a subject suffering from or at risk of a disease or disorder.

[0019] Preferably, the aforementioned diseases or disorders are characterized by overexpression of FAP and are independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders. Preferably, cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multidrug-resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cell carcinoma, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumors, tumor-induced osteomalacia, sarcoma, CUP (unknown primary carcinoma), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer, and prostate cancer. More preferably, the diseases or disorders are selected from melanoma and renal cell carcinoma. [Brief explanation of the drawing]

[0020] [Figure 1] This figure shows the chemical structures and LC / MS profiles of compounds (A)ESV6-fluo(26) and (B)Haberkorn-fluo(23). [Figure 2] This figure shows the quality control of recombinant hFAP: A) SDS-PAGE; B) Size exclusion chromatography (Superdex 200 Increase 10 / 300 GL). [Figure 3] This figure shows the co-elution PD-10 experiment with compounds ESV6-fluo(26) and Haberkorn-fluo(23) and hFAP. A stable complex is formed between hFAP and the small ligands ESV6-fluo and Haberkorn-fluo. [Figure 4] This figure shows the affinity determination of small organic ligands for human fibroblast-activating protein (hFAP) by fluorescence polarization. ESV6-fluo(26) exhibits a higher affinity (0.78 nM KD) for hFAP compared to the previously described ligand, Haberkorn-fluo(23) (0.89 nM KD). [Figure 5]This figure shows hFAP inhibition experiments in the presence of small organic ligands. ESV6 ligand (P3) exhibits a lower IC50 (20.2 nM) compared to the previously described ligand, Haberkorn ligand (H6) (24.6 nM). [Figure 6] This figure shows the dissociation rate measurements of ESV6-fluo (26) and Haberkorn-fluo (23) from hFAP. ESV6-fluo dissociates at a slower rate (regression coefficient = -0.093564) compared to Haberkorn-fluo (regression coefficient = -0.075112). [Figure 7-1] This figure shows the evaluation of the targeting performance of the IRDye 750 conjugate in near-infrared fluorescence imaging of BALB / C nu / nu mice carrying SK-MEL-187 melanoma xenografts after intravenous administration (dose of 150 nmol / kg). (A) Images of live animals at various time points (5 minutes, 20 minutes, and 1 hour after injection). (B) Ex vivo organ images at 2 hours are shown. The compound ESV6-IRDye750(18), a derivative of the high-affinity FAP ligand "ESV6," exhibits higher tumor-to-liver, tumor-to-kidney, and tumor-to-intestinal uptake ratios compared to HABERKORN-IRDye750(17). QCOOH-IRDye750(16) (untargeted control) did not localize to SK-MEL-187 lesions in vivo. [Figure 7-2] This figure shows the evaluation of the targeting performance of the IRDye 750 conjugate in near-infrared fluorescence imaging of BALB / C nu / nu mice carrying SK-MEL-187 melanoma xenografts after intravenous administration (dose of 150 nmol / kg). (A) Images of live animals at various time points (5 minutes, 20 minutes, and 1 hour after injection). (B) Ex vivo organ images at 2 hours are shown. The compound ESV6-IRDye750(18), a derivative of the high-affinity FAP ligand "ESV6," exhibits higher tumor-to-liver, tumor-to-kidney, and tumor-to-intestinal uptake ratios compared to HABERKORN-IRDye750(17). QCOOH-IRDye750(16) (untargeted control) did not localize to SK-MEL-187 lesions in vivo. [Figure 8-1] (A) This figure shows the evaluation of the therapeutic activity of ESV6-ValCit-MMAE (21) and HABERKORN-ValCit-MMAE (20) in mice carrying SK-MEL-187 tumors. Data points represent mean tumor volume ± SEM (n=3 per group). Arrows indicate IV infection with different treatments. ESV6-ValCit-MMAE, a drug conjugate derivative of the high-affinity FAP ligand "ESV6", exhibits a more potent antitumor effect compared to HABERKORN-ValCit-MMAE. (B) This figure shows the tolerability of different treatments as determined by the evaluation of the change (%) in body weight of animals during the experiment. ESV6-ValCit-MMAE exhibits lower acute toxicity compared to HABERKORN-ValCit-MMAE. [Figure 8-2] (A) This figure shows the evaluation of the therapeutic activity of ESV6-ValCit-MMAE (21) and HABERKORN-ValCit-MMAE (20) in mice carrying SK-MEL-187 tumors. Data points represent mean tumor volume ± SEM (n=3 per group). Arrows indicate IV infection with different treatments. ESV6-ValCit-MMAE, a drug conjugate derivative of the high-affinity FAP ligand "ESV6", exhibits a more potent antitumor effect compared to HABERKORN-ValCit-MMAE. (B) This figure shows the tolerability of different treatments as determined by the evaluation of the change (%) in body weight of animals during the experiment. ESV6-ValCit-MMAE exhibits lower acute toxicity compared to HABERKORN-ValCit-MMAE. [Figure 9] This figure shows hFAP inhibition experiments in the presence of different small organic ligands. Compound P4 from Example 2 exhibits a lower IC50 (16.83 nM, higher inhibition) compared to compound 24 (33.46 nM, lower inhibition). [Figure 10]This figure shows the affinity determination of small organic ligands for human and mouse fibroblast-activating proteins by fluorescence polarization (FP). (A) Conjugate 15 shows a higher affinity (KD=0.68nM) for hFAP compared to conjugate 25 (KD=1.02nM). (B) Conjugate 15 shows a higher affinity (KD=11.61nM) for mFAP compared to conjugate 25 (KD=30.94nM). Conjugate 15 exhibits superior binding properties for hFAP and better cross-reactivity to mouse antigens compared to conjugate 25. (C) This figure shows the structures of conjugates 15 and 25. [Figure 11] This figure shows a co-elution PD-10 experiment with small molecule ligand conjugate 15 with hFAP(A) and mFAP(B). A stable complex is formed between both proteins and small ligand conjugate 15, enabling the co-elution of the two molecules together. [Figure 12]This figure shows the evaluation of the selective accumulation of conjugate 15 (10 nM) on SK-RC-52.hFAP, HT-1080.hFAP, and wild-type tumor cells via confocal microscopy and FACS analysis. (A) Images of SK-RC-52.hFAP incubated with the compound at different time points (t=0 and 1 hour) show accumulation of conjugate 15 on the cell membrane. (B) Images of SK-RC-52 wild-type after incubation with the compound show no accumulation on the cell membrane (negative control). (C) FACS analysis of SK-RC-52 wild-type (dark gray peak) and SK-RC-52.hFAP (light gray peak) shows FAP-specific cell binding of conjugate 15 (10 nM). (D) Images of HT-1080.hFAP incubated with the compound at different time points (t=0 and 1 hour) show accumulation of conjugate 15 on the cell membrane and inside the cytosol. (E) Images of HT-1080 wild-type after incubation with the compound show no accumulation on the cell membrane or in the cytosol (negative control). (F) FACS analysis of HT-1080 wild-type (dark gray peak) and HT-1080.hFAP (light gray peak) shows FAP-specific cell binding of conjugate 15 (10 nM). [Figure 13-1] This figure shows the evaluation of the targeting performance of conjugate 15 in BALB / C nu / nu mice carrying SK-RC-52.hFAP renal cell carcinoma xenografts after intravenous administration (40 nmol). Ex vivo organ images taken one hour after administration are shown. The compound exhibits high tumor-versus-organ selectivity, rapidly and homogeneously localizing to the in vivo tumor site one hour after intravenous injection. [Figure 13-2] This figure shows the evaluation of the targeting performance of conjugate 15 in BALB / C nu / nu mice carrying SK-RC-52.hFAP renal cell carcinoma xenografts after intravenous administration (40 nmol). Ex vivo organ images taken one hour after administration are shown. The compound exhibits high tumor-versus-organ selectivity, rapidly and homogeneously localizing to the in vivo tumor site one hour after intravenous injection. [Figure 14]This figure shows the radioactive HPLC profiles of radioactive compounds. (A) This figure shows the radioactive HPLC profile of conjugate 9 after labeling with 177Lu (rt11 min). (B) This figure shows the radioactive HPLC profile of free 177Lu (2 min). After radiolabeling, conjugate 9 appears as a single peak in >99% of the conversion. [Figure 15] This figure shows the in vivo distribution experiment of conjugate 9 (which contains a 177Lu radioactive payload) in BALB / C nu / nu cells containing the SK-RC-52.hFAP renal cell carcinoma xenograft. (A) This figure shows the %ID / g ratio analysis in tumors, healthy organs, and tumor-versus-organs at different time points (10 minutes, 1 hour, 3 hours, and 6 hours) after intravenous administration of conjugate 9 (dose = 50 nmol / kg; 0.5~2 MBq). (B) This figure shows the %ID / g ratio analysis in tumors, healthy organs, and tumor-versus-organs 3 hours after intravenous administration of 177Lu conjugate 9 at different doses (125 nmol / kg, 250 nmol / kg, 500 nmol / kg, and 1000 nmol / kg; 0.5~2 MBq). A dose-dependent response may be observed, and target saturation may be reached between 250 nmol / kg and 500 nmol / kg. (C) This figure shows the %ID / g and tumor-to-organ ratio analysis in tumors and healthy organs 3 hours after intravenous administration of 177Lu solution (negative control; 1 MBq). [Figure 16] This figure shows the evaluation of the targeting performance of IRDye 750 conjugate 18 in near-infrared fluorescence imaging of BALB / C nu / nu mice containing xenografts of SK-MEL-187 (right flank) and SK-RC-52.hFAP (left flank) after intravenous administration (dose of 150 nmol / kg). (A) Images of live animals before injection (t=0) and 30 minutes after intravenous injection. (B) Ex vivo organ images at 60 minutes are shown. Compound ESV6-IRDye750(18) accumulated in both SK-RC-52.hFAP and SK-MEL-187 tumors, showing higher accumulation in SK-RC-52.hFAP tumors compared to SK-MEL-187 due to higher FAP expression. [Figure 17]This figure shows hFAP inhibition experiments in the presence of different small organic ligands. Conjugate 28 exhibits lower FAP inhibitory properties compared to Example 2, P4. Conjugate 29, including the L-alanine structural unit between the cyanopyrrolidine headpiece and the pyridine ring, does not inhibit FAP proteolytic activity at the concentrations tested in the assay. [Figure 18] This figure shows the evaluation of the targeting performance of IRDye 750 conjugate 18 in near-infrared fluorescence imaging of BALB / C nu / nu mice containing HT-1080.hFAP and SK-RC-52.wt xenografts after intravenous administration (dose of 150 nmol / kg). Ex vivo organ images at 1 hour are shown. Compound ESV6-IRDye750(18) selectively accumulates in HT-1080.hFAP tumors exhibiting FAP expression, but does not accumulate in SK-RC-52.wt. [Figure 19A] (A) This figure shows the evaluation of the targeting performance of conjugate 30 in BALB / C nu / nu mice containing xenografts of SK-RC-52.hFAP (right flank) and SK-RC-52.wt (left flank) after intravenous administration (40 nmol). Ex vivo organ images taken 1 hour after administration are shown. The compound exhibits excellent tumor-versus-organ selectivity, rapidly, homogeneously, and selectively localizing in vivo to tumors expressing FAP 1 hour after intravenous injection. [Figure 19B] (B) This figure shows the structure of ESV6-Alexa Fluor 488(30). [Figure 20A] (A) This figure shows the evaluation of the targeting performance of conjugate 30 in BALB / C nu / nu mice containing xenografts of HT-1080.hFAP (right flank) and SK-RC-52.wt (left flank) after intravenous administration (40 nmol). Ex vivo organ images taken 1 hour after administration are shown. The compound exhibits excellent tumor-versus-organ selectivity, rapidly, homogeneously, and selectively localizing in vivo to tumors expressing FAP 1 hour after intravenous injection. [Figure 20B] (B) This figure shows the structure of ESV6-Alexa Fluor 488(30). [Figure 21] (A) This figure shows the assessment of the therapeutic activity of ESV6-ValCit-MMAE(21) ​​and QCOOH-ValCit-MMAE(19) in SK-RC-52.hFAP tumor-carrying mice. Data points represent mean tumor volume ± SEM (n=4 per group). The compounds were administered intravenously (tail vein injection) for 6 consecutive days, starting on day 8. ESV6-ValCit-MMAE(21), a drug conjugate derivative of the high-affinity FAP ligand "ESV6," exhibits a more potent antitumor effect compared to QCOOH-ValCit-MMAE(19), the untargeted version of the molecule. (B) This figure shows the tolerability of different treatments as determined by the assessment of the change (%) in body weight of the animals during the experiment. (C) This figure shows the structures of ESV6-ValCit-MMAE(21) ​​and QCOOH-ValCit-MMAE(19). [Figure 22] (A) This figure shows the therapeutic activity of ESV6-ValCit-MMAE(21), L19-IL2, and their combinations in mice carrying SK-RC-52.hFAP tumors. Data points represent mean tumor volume ± SEM (n=4 per group). ESV6-ValCit-MMAE was administered intravenously (tail vein injection) on days 8, 10, and 12. L19-IL2 was administered intravenously (tail vein injection) on days 9, 11, and 13. ESV6-ValCit-MMAE combined with L19-IL2 showed a very potent antitumor effect (4 / 4 complete tumor regression) compared to L19-2 alone. (B) This figure shows the tolerance of different treatments as determined by the evaluation of the change (%) in body weight of the animals during the experiment. [Figure 23]This figure shows the quantitative in vivo distribution of the small molecule-drug conjugate ESV6-ValCit-MMAE(21) ​​in BALB / C nu / nu mice carrying SK-RC-52.hFAP on the right flank and SK-RC-52.wt on the left flank. The compound selectively accumulates in FAP-positive SK-RC-52 tumors (i.e., 18% ID / g at the tumor site 6 hours after intravenous administration). In contrast, ESV6-ValCit-MMAE does not accumulate in FAP-negative SK-RC-52 wild-type tumors. Conjugate uptake in healthy organs is negligible (lower than 1% ID / g). [Figure 24] This figure shows the stability study of conjugate 27 (which contains a 69Ga payload) in mouse serum. HPLC and LC / MS profiles of the treated sample at time 0 and 6 hours after incubation show a single peak with the correct mass (expected mass: 1028.30; MS(ES+)m / z 514.3(M+2H)). [Figure 25] This figure shows the structure, chromatographic profile, and LC / MS analysis of Conjugate 15. MS(ES+)m / z 1348.36(M+1H)+. [Figure 26] This figure shows the structure, chromatographic profile, and LC / MS analysis of ESV6-ValCit-MMAE(21). MS(ES+)m / z 1118.05(M+2H)2+. [Figure 27] This figure shows the structure, chromatographic profile, and LC / MS analysis of ESV6-DOTAGA(8). MS(ES+)m / z 960.39(M+H)+. [Figure 28] This figure shows the structure, chromatographic profile, and LC / MS analysis of P4 in Example 2. MS(ES+)m / z 460.21(M+H)+. [Modes for carrying out the invention]

[0021] The inventors have identified a small molecule binder for fibroblast-activating protein (FAP) suitable for targeted applications. The binder according to the present invention provides high inhibition of FAP, high affinity for FAP, and / or is suitable for targeted delivery of payloads, such as therapeutic or diagnostic agents, to sites affected by or at risk of disease or disorder characterized by FAP overexpression. The binder according to the present invention forms a stable complex with FAP, exhibiting increased affinity, increased inhibitory activity, slower rate of dissociation from the complex, and / or extended retention at the disease site. The binder according to the present invention may further have increased tumor-to-liver, tumor-to-kidney, and / or tumor-to-intestinal uptake ratios; stronger antitumor effects (e.g., measured by mean tumor volume increase); and / or lower toxicity (e.g., determined by assessment of change in body weight (%)). The binder according to the present invention may further have high or improved affinity for human and mouse fibroblast-activating proteins and / or cross-reactivity to mouse antigens. The binder according to the present invention preferably achieves FAP-specific cell binding; selective accumulation of FAP on the cell membrane; and selective accumulation of FAP inside the cytosol. More preferably, the binder according to the present invention can rapidly and homogeneously localize to the in vivo tumor site with high tumor-versus-organ selectivity, particularly for melanoma and / or renal cell carcinoma. Radioactive payload (e.g., 177 The binder according to the present invention, comprising Lu, preferably achieves a dose-dependent response in which the target saturation is reached between 250 nmol / kg and 500 nmol / kg and / or is maintained for up to 12 hours after intravenous administration, more preferably from 1 to 9 hours, and even more preferably from 3 to 6 hours.

[0022] As described above, the present invention provides a compound, its individual diastereoisomers, its hydrate, its solvate, its crystalline form, its individual tautomers, or pharmaceutically acceptable salts thereof, wherein the compound comprises a portion A having the following structure:

[0023] [ka]

[0024] As explained above, the compound according to the present invention can be represented by formula I:

[0025] [ka]

[0026] Therefore, B is a portion containing a covalent bond or a chain of atoms that covalently attaches A to C; C may be an atom, molecule or particle, and / or a therapeutic or diagnostic agent.

[0027] Therefore, the compound according to the present invention may include a portion having the following structure:

[0028] [ka]

[0029] In the formula, B is the part that contains a covalent bond or a chain of covalently bonded atoms. Part A Without intending to link them by any theory, these remarkable technical effects are intended to be related to the special structure of the small bond A, where the quinoline ring is substituted at position 8 by a nitrogen-containing group such as an amino or amide group:

[0030] [ka]

[0031] It has been previously shown that higher target protein affinity of a compound results in longer tumor retention in vivo (Wichert et al., Nature Chemistry 7, 241-249 (2015)). The compounds of the present invention also have increased affinity, a slower dissociation rate with respect to FAP compared to prior art compounds, and therefore are thought to have extended retention at the disease site at therapeutically or diagnostically relevant levels, preferably beyond 1 hour, more preferably beyond 6 hours, after injection. Preferably, the highest enrichment is achieved at 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after injection; and / or enrichment at the disease site is maintained at therapeutically or diagnostically relevant levels over a period of 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after injection, or at least during that period, more preferably beyond 6 hours.

[0032] Preferably, the connecting portion A has the following structure A 1 ;more preferably the following structure A 2 The formula has the following characteristics, where m is 0, 1, 2, 3, 4, or 5, preferably 1:

[0033] [ka]

[0034] Part B Part B is a part comprising a chain of atoms that covalently attaches A to payload C via, for example, one or more covalent bonds. Part B may be a cleavable or incleavable bifunctional or polyfunctional part, which can be used to link one or more payload and / or binder parts to form a targeted conjugate of the present invention. In some embodiments, the structure of the compound independently comprises two or more parts A per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts A; and / or two or more parts C, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts C. Preferably, the structure of the compound comprises two parts A and one part C per molecule; or one part A and two parts C.

[0035] If cleavable linker units are present within portion B, the release mechanism may be identical to that specific to the antibody bound to the cytotoxic payload. In fact, the properties of the binding site are independent in this respect. Therefore, the release is expected to be pH-dependent [Leamon, CP et al. (2006) Bioconjugate Chem., 17, 1226; Casi, G. et al. (2012) J. Am. Chem. Soc., 134, 5887], reductive [Bernardes, GJ et al. (2012) Angew. Chem. Int. Ed. Engl., 51, 941; Yang, J. et al. (2006) Proc. Natl. Acad. Sci. USA, 103, 13872] and enzymatic release [Doronina SO et al. (2008) Bioconjugate Chem, 19, 1960; Sutherland, MSK (2006) J. Biol. Chem, 281, 10540]. In certain settings, if a functional group is present on either the binding site or the payload (e.g., thiol, alcohol), a linker-less connection may be established, thus releasing the intact payload, which significantly simplifies pharmacokinetic analysis.

[0036] Part B can comprise or consist of the units shown in Table 1 below, where the substituents R and R shown in the formula n are, suitably, independently selected from H, halogen, substituted or unsubstituted (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, (hetero)arylalkyl, (hetero)cycloalkyl, (hetero)cycloalkylaryl, heterocyclylalkyl, peptide, oligosaccharide or steroid groups. Preferably, each of R, R1, R2 and R3 is independently selected from H, OH, SH, NH2, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted. Suitably, R and R n are independently selected from H, or C1-C7 alkyl or heteroalkyl. More suitably, R and R n are independently selected from H, methyl or ethyl.

[0037]

Table 1

[0038] Part B, Unit B L and / or Unit B S can suitably include a disulfide linkage as a cleavable bond, since these linkages are stable to hydrolysis while providing appropriate drug release kinetics at the target in vivo and can provide cleavage without trace of the drug moiety including thiol groups.

[0039] Part B, Unit B L and / or Unit B SThe linker may be polar or charged to improve the water solubility of the conjugate. For example, the linker may contain about 1 to about 20, optionally about 2 to about 10, residues of one or more known water-soluble oligomers, such as peptides, oligosaccharides, glycosaminoglycans, polyacrylic acid or its salts, polyethylene glycol, polyhydroxyethyl (meth)acrylate, and polysulfonate. Optionally, the linker may contain a polar or charged peptide moiety comprising, for example, 2 to 10 amino acid residues. The amino acids may refer to any natural or non-natural amino acids. To form the cleavable disulfide linkage with a thiol group on the drug moiety, the peptide linker may optionally contain a free thiol group, preferably an N-terminal cysteine. Any peptide containing L- or D-amino acids may be suitable; particularly suitable peptide linkers of this type are Asp-Arg-Asp-Cys and / or Asp-Lys-Asp-Cys.

[0040] In these and other embodiments, part B, unit B L and / or unit B SThe peptide may include cleavable or incleavable peptide units that are specialized to be selectively enzymatically cleaved from the drug moiety by one or more proteases on the cell surface or extracellular region of target tissue. The amino acid residue chain length of the peptide units may appropriately range from a single amino acid to about eight amino acid residues. A number of specific cleavable peptide sequences suitable for use in the present invention can be designed and optimized for enzymatic cleavage by specific tumor-related enzymes, such as proteases, in their selectivity. Examples of cleavable peptides for use in the present invention include those optimized for proteases MMP-1, 2, or 3, or cathepsins B, C, or D. Particularly suitable are peptides cleavable by cathepsin B. Cathepsin B is a ubiquitous cysteine ​​protease. It is an intracellular enzyme, except in conditions such as metastatic tumors or rheumatoid arthritis. An example of a peptide cleavable by cathepsin B contains the sequence Val-Cit. In any of the above embodiments, part B and, in particular, unit B L The linker further comprises, appropriately, a self-sacrificing moiety that may or may not be present after the linker. Self-sacrificing linkers are also known as electron cascade linkers. These linkers release a drug in an active, preferably free, form by undergoing elimination and fragmentation by enzymatic cleavage of a peptide. The conjugate is stable extracellularly in the absence of an enzyme capable of cleaving the linker. However, upon exposure to a suitable enzyme, the linker is cleaved, inducing a spontaneous self-sacrificing reaction that results in the cleavage of a bond covalently linking the self-sacrificing moiety to the drug, thereby achieving its non-derivativeization or release of the drug in a pharmacologically active form. In these embodiments, the self-sacrificing linker is coupled to the binding moiety via an enzymatically cleavable peptide sequence that provides a substrate for an enzyme that cleaves the amide bond and induces the self-sacrificing reaction. Preferably, the drug moiety is connected to the self-sacrificing moiety of the linker via a chemically reactive functional group protruding from the drug, such as a primary or secondary amine group, a hydroxyl group, a sulfhydryl group, or a carboxyl group.

[0041] An example of a self-sacrificing linker is PABC or PAB (para-aminobenzyloxycarbonyl), which attaches the drug moiety to the binding site in the conjugate (Carl et al. (1981) J.Med.Chem. 24:479~480; Chakravarty et al. (1983) J.Med.Chem. 26:638~644). The amide bond linking the carboxyl terminus of the peptide unit and the para-aminobenzyl of PAB can be substrates and may be cleavable by certain proteases. Aromatic amines become electron-donating, inducing an electron cascade leading to the elimination of the leaving group and the release of the free drug after carbon dioxide elimination (de Groot et al. (2001) Journal of Organic Chemistry 66(26):8815~8830). Further self-sacrificing linkers are described in WO2005 / 082023.

[0042] In other embodiments, the linker includes a glucuronyl group that can be cleaved by glucuronidase present on the cell surface or extracellular region of the target tissue. It has been shown that lysosomal beta-glucuronidase is released extracellularly at high local concentrations in necrotic areas in human cancer and provides a pathway for targeted chemotherapy (Bosslet, K. et al. Cancer Res. 58, 1195-1201 (1998)).

[0043] In any of the above embodiments, part B optionally further comprises a spacer unit. The spacer unit is a unit B which can be linked to the bonding part A via, for example, an amide, amine, or thioether bond. SThis is possible. The spacer unit is, for example, of a length that allows the cleavable peptide sequence to be contacted by a cleavable enzyme (e.g., cathepsin B), and optionally also of a length that allows hydrolysis of the amide bond coupling the cleavable peptide to the self-sacrificing moiety X. The spacer unit can include, for example, repeating units of divalent groups, such as alkylenes, arylenes, heteroarylenes, alkyloxys (e.g., polyethyleneoxys, PEGs, polymethyleneoxys) and alkylaminos (e.g., polyethyleneaminos), or diacid esters as well as amides including succinates, succinamides, diglycolates, malonates and caproamides.

[0044] In any of the embodiments described therein, * represents an attachment point to part A, or an attachment point where the shortest path to part A may contain fewer atoms than the one related to ; represents an attachment point to part C, or where the shortest path to part C may * This represents an attachment point to a portion C containing fewer atoms than those related to the payload portion C. The same applies when a reactive portion L exists rather than a payload portion C. The following notation and all others refer to the attachment point of a particular group or atom (e.g., R) to a further portion:

[0045] [ka]

[0046] If the related structure is a peptide monomer or oligomer, then each * represents an attachment site where the shortest path to part A contains fewer atoms than the path to ; each represents a site where the shortest path to part C is * This represents attachment points containing fewer atoms than those related to R, where n > 1 and each attachment point is R a , R b and R cIf shown on any one of the structures, it can be independently present on one or more peptide monomer units, preferably on the peptide monomer unit furthest distal to the other attachment sites shown in each structure.

[0047] In any of the embodiments described herein, the terms “peptide,” “dipeptide,” “tripeptide,” “tetrapeptide,” etc., refer to peptide monomers or oligomers having a backbone formed by protogenic and / or non-protogenic amino acids. As used herein, the terms “aminoacyl” or “amino acid” generally refer to any protogenic or non-protogenic amino acid. Preferably, in any of the embodiments disclosed herein, the side chain residues of the protogenic or non-protogenic amino acid are R a , R b and R c It is represented by one of the following, and each of these is selected from the list below:

[0048] [ka]

[0049] [ka]

[0050] In the formula, R, R 1 , R 2 and R 3 Each of these is independently selected from H, OH, SH, NH2, halogens, cyano, carboxy, alkyl, cycloalkyl, aryl, and heteroaryl, and each of these is substituted or unsubstituted; Each X is independently selected from NH, NR, S, O, and CH2, preferably NH; Each n and m is an integer, preferably selected independently from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0051] Preferably, in any of the embodiments disclosed herein, the side chain residue of a proteogenic or non-proteogenic amino acid is R a , R b and R c Represented by one of the following: Each of these may be part of a 3-membered, 4-membered, 5-membered, 6-membered, or 7-membered ring. For example, the alpha, beta, and / or gamma positions of the side chains of the proteinogenic or non-proteinogenic amino acids may be part of a ring structure selected from azetidine rings, pyrrolidine rings, and piperidine rings, such as in the following amino acids (proline and hydroxyproline):

[0052] [ka]

[0053] Each of these independently has an unsaturated structure (i.e., each of the R groups a , R b and R c (where geminal H atoms do not exist), for example:

[0054] [ka]

[0055] It is possible. Further preferred non-proteinogenic amino acids can be selected from the following list:

[0056] [ka]

[0057] Particularly preferred embodiments relating to Part B and the compounds according to the present invention are shown in the appended claims. Preferably, B is represented by one of the following general formulas II to V, where:

[0058] [ka]

[0059] Each x is an integer independently selected from the range of 0 to 100, preferably 0 to 50, more preferably 0 to 30, and even more preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; Each y is an integer independently selected from the range of 0 to 30, preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; Each z is an integer independently selected from the range of 0 to 5, preferably selected from 0, 1, 2, 3, and 4; * represents the attachment point to part A; • represents the attachment point to part C, where: (a)B S and / or B L This refers to a group comprising or consisting of a structural unit independently selected from the group consisting of alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkylylene, heteroalkylynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacyl ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxycarbamate, disulfide, vinylene, imine, imidoamide, phosphoramide, sugars, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted; and / or (b)B S and / or B LThis is a group that includes or consists of structural units independently selected from the following group:

[0060] [ka]

[0061] [ka]

[0062] [ka]

[0063] [ka]

[0064] [ka]

[0065] In the formula, R, R 1 , R 2 and R 3 Each of these is independently selected from H, OH, SH, NH2, halogens, cyano, carboxy, alkyl, cycloalkyl, aryl, and heteroaryl, and each of these is substituted or unsubstituted; R 4 and R 5 Each of these is independently selected from alkyl, cycloalkyl, aryl, and heteroaryl groups, and each of these is substituted or unsubstituted; R a , R b and R c Each of these is independently selected from the side chain residues of proteinogenic or non-proteinogenic amino acids, and each of these may be further substituted; Each X is independently selected from NH, NR, S, O, and CH2, preferably NH; Each of n and m is independently selected from integers ranging from 0 to 100, preferably from 0 to 50, more preferably from 0 to 30, even more preferably from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; Here, each * represents an attachment site where the shortest path to part A contains fewer atoms than the path to ; each represents a site where the shortest path to part C is * Represents an attachment point containing fewer atoms than those related to; and / or (c) B L It independently comprises or consists of one or more of the following structural units:

[0066] [ka]

[0067] In the formula, in each of the above structures, n is 1, 2, 3, or 4; each * represents an attachment site where the shortest path to part A contains fewer atoms than the path to ; each represents a site where the shortest path to part C is * This represents attachment points containing fewer atoms than those related to R, where n > 1 and each attachment point is R a , R b and R c If shown on any one of the following, it may independently be present on one or more peptide monomer units, preferably on the peptide monomer unit furthest distal from the other attachment points shown in each structure; and / or (d)B L and B S One or more of the following structures are selected independently:

[0068] [ka]

[0069] During the ceremony, each* represents an attachment site where the shortest path to part A contains fewer atoms than the path to ; each represents a site where the shortest path to part C is * Represents an attachment point containing fewer atoms than those related to; and / or (e) y is 1, 2 or 3; and / or at least one B L It further comprises a cleavable linker group independently selected from the following structures:

[0070] [ka]

[0071] each * represents an attachment site where the shortest path to part A contains fewer atoms than the path to ; each represents a site where the shortest path to part C is * This represents an attachment point containing fewer atoms than those related to the specific site.

[0072] Preferably, B may have the structure defined above and / or the following:

[0073] [ka]

[0074] In the formula, B' s and B'' s Each of the following groups is independently selected:

[0075] [ka]

[0076] Each B L It is independently selected from the following group:

[0077] [ka]

[0078] Each n is 0, 1, 2, 3, 4, or 5; Each m is 0, 1, 2, 3, 4, or 5; Each x' is 0, 1, or 2; Each x'' is 0, 1, or 2; Each y is 0, 1, or 2; z is either 1 or 2. Here, R, R 1 , R 2 , R 3 , R a , R b , R c , X, * And are as defined above.

[0079] More preferably, the compound according to the present invention has a structure represented by one of the following formulas:

[0080] [ka]

[0081] [ka]

[0082] [ka]

[0083] [ka]

[0084] Part C In this invention, part C generally represents a payload which may be any atom (including H), molecule, or particle. Preferably, part C is not a hydrogen atom.

[0085] The payload can be a chelator for radiolabeling. Suitably, no radionuclide is emitted. Chelators are well known to those skilled in the art and include, for example, sulfur colloid, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane,N-(glutamic acid)-N’,N’’,N’’’-triacetic acid (DOTAGA), 1,4,7-triazacyclononane-N,N’,N’’-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA), or any of the preferred chelator structures listed in the appended claims.

[0086] The payload is 223 Ra, 89 Sr, 94m Tc, 99m Tc, 186 Re, 188 Re, 203 Pb, 67 Ga, 68 Ga, 47 Sc, 111 In, 97 Ru, 62 Cu, 64 Cu, 86 Y, 88 Y, 90 Y, 121 Sn, 161 Tb, 153 Sm, 166 Ho, 105 Rh, 177 Lu, 123 I, 124 I, 125 I, 131 I, 18 F, 211 At, 225 Ac, 89 Sr, 225 Ac, 117m Sn and 169 It may be a radioactive group containing or consisting of a radioisotope, including isotopes such as E. Preferably, 18 F and124 Positron emitters such as l, or 99m Tc, 111 In and 123 Gamma emitters such as I are used for diagnostic purposes (e.g., for PET), while, 89 Sr, 131 I and 177 Beta-emitters such as Lu are preferably used for therapeutic applications. 211 At, 225 and 223 Alpha-emitters such as Ra may also be used for therapeutic purposes. In a preferred embodiment, the radioactive isotope is 89 Sr or 223 It is Ra. In a more preferred embodiment, the radioactive isotope is 68 It is Ga.

[0087] The payload may be a chelate of a radioisotope, preferably a chelating agent, preferably a chelate of an isotope listed above together with any of the chelating agents listed above or any of the preferred chelator structures enumerated in the appended claim 9(a); or a group selected from the structures listed in claim 9(c).

[0088] The payload may preferably be a fluorophore group selected from xanthene dyes, acridine dyes, oxazine dyes, cyanine dyes, styryl dyes, coumarin dyes, porphyrin dyes, fluorescent metal-ligand complexes, fluorescent proteins, nanocrystals, perylene dyes, boron-dipyrromethene dyes, and phthalocyanine dyes, and more preferably a fluorophore group selected from the structures listed in claim 9(d).

[0089] The payload may be a cytotoxic agent and / or a cell division inhibitor. Such agents can inhibit or prevent cell function and / or cause cell destruction. Examples of cytotoxic agents include toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including radioisotopes, chemotherapeutic agents, and synthetic analogs and their derivatives. Cytotoxic agents can be selected from the group consisting of auristatin, DNA sulcus binding agents, DNA sulcus alkylating agents, enediynes, requitropsin, duocalmycin, taxanes, puromycin, drastatin, meitansinoids, and vinca alkaloids, or combinations of two or more thereof. Preferred cytotoxic and / or cell division inhibitory payload portions are listed in claim 9(e).

[0090] In one embodiment, the payload may include topoisomerase inhibitors, alkylating agents (e.g., nitrogen mustard; ethyleneimine; alkyl sulfonates; triazenes; piperazine; and nitrosourea), antimetabolites (e.g., mercaptopurine, thioguanine, 5-fluorouracil), antibiotics (e.g., anthracyclines, dactinomycin, bleomycin, adriamycin, mitramycin, dactinomycin), mitotic disruptors (e.g., plant alkaloids - e.g., vincristine and / or microtubule antagonists - e.g., paclitaxel), DNA methylating agents, DNA insertion agents (e.g., For example, the chemotherapeutic agent is selected from the group consisting of carboplatin and / or cisplatin, daunomycin and / or doxorubicin and / or bleomycin and / or thalidomide), DNA synthesis inhibitors, DNA-RNA transcription regulators, enzyme inhibitors, gene regulators, hormone response modifiers, hypoxia-selective cytotoxicities (e.g., tirapazamine), epidermal growth factor inhibitors, antivascular agents (e.g., xanthenone 5,6-dimethylxanthenone-4-acetic acid), radioactive prodrugs (e.g., nitroarylmethyl quaternary (NMQ) salts), or endogenous reducing agents, or combinations of two or more thereof. In some embodiments, the payload (i.e., part C) is not derived from anthracyclines, preferably not from PNU 159682.

[0091] The chemotherapy agents are selected from the group consisting of erlotinib (TARCEVA®), bortezomib (VELCADE®), fulvestrant (FASLODEX®), sutent (SU11248), letrozole (FEMARA®), imatinib mesylate (GLEEVEC®), PTK787 / ZK222584, oxaliplatin (Eloxatin®), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®), lapatinib (GSK572016), ronafarnib (SCH 66336), sorafenib (BAY43-9006), and gefitinib (IRESSA®), AG1478, AG1571 (SU 5271; Sugen), or a combination of two or more of these.

[0092] Chemotherapy agents include alkylating agents—e.g., thiotepa, CYTOXAN® and / or cyclophosphamide; alkyl sulfonates—e.g., busulfan, improsulfan and / or pigosulfan; aziridines—e.g., benzodopa, carbocone, metsuredopa and / or uredopa; ethyleneimines and / or methylamelamamines—e.g., altoretamine, triethylenemelamamine, triethylenephosphoramide, triethylenethiophosphoramide and / or trimethylomelamamine; acetogenins—e.g., bratacin. and / or bratacinone; camptothecin; bryostatin; callistatin; cryptophycin; dorastatin; duocalmycin; eryuterobin; pancratistatin; sarcodicin; spongistatin; nitrogen mustard - e.g., chlorambucil, chlornafadin, clophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide and / or uracil mustard; nitroso Ureas - e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine and / or ranimustine; dinemisine; bisphosphonates - e.g., clodronate; esperamycin; neocarcinostatin chromophore; acrasinomycin, actinomycin, ausuramycin, azaserin, bleomycin, kakutinomycin, carabicin, carminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN ( (Registered Trademark), Doxorubicin - e.g., Morpholino-doxorubicin, Cyanomorpholino-doxorubicin, 2-Pyrrolino-doxorubicin and / or Deoxydoxorubicin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin - e.g., Mitomycin C, Mycophenolic acid, Nogaramycin, Olibomycin, Peplomycin, Potophyllomycin, Puromycin, Queramycin, Rhodolubicin, Streptonigrin, Streptozocin, Tubercidine, Ubenimex, Dinostatin, Zolubicin;Antimetabolites - e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs - e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs - e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs - e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, furoxiuridine; androgens - For example, carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs - for example, aminoglutethimide, mitotane, trilostane; folic acid supplements - for example, folic acid; acegraton; aldofhamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatrexate; defofamine; demecolsin; diaziquan; eflornithine; eriptinium acetate; epoxy Ron; etoglucide; gallium nitrate; hydroxyurea; lentinan; ronidamine; macrocyclic depsipeptides, e.g., meitansine and anthamitosine; mitoglucon; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triadiquan; 2,2',2''-tri Chlorotriethylamine; Trichothecenes - e.g., Beraclin A, Loridine A and / or Anguidin; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside; Cyclophosphamide; Thiotepa; Taxoids - e.g., TAXOL®, Paclitaxel, Abraxane, and / or TAXOTERE®, Doxetaxel; Chlorambucil; GEMZAR®; Gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum analogs - e.g., Cisplatin and Carboplatin; Vinblastine; Platinum; Etoposide; Ifosfamide; Mitoxanthrone; Vincristine; NAVELBINE®, Vinorelbine; Novanthrone; Teniposide;Edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids—e.g., retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, derivatives, or any two or more combinations thereof.

[0093] The payload may be a tubulin-destroying agent, but is not limited to: taxanes—e.g., paclitaxel and docetaxel; vinca alkaloids; discodermorid; eposylone A and B; desoxyepotilone; cryptophycin; crassine A; combretastatin A-4-phosphate; BMS 247550; BMS 184476; BMS 188791; LEP; RPR 109881A; EPO 906; TXD 258; ZD 6126; vinflunin; LU 103793, Dorastatin 10, E7010, T138067 and T900607, Colchicine, Fenstatin, Chalcone, Indanosine, T138067, Oncosidine, Vincristine, Vinblastine, Vinorelbine, Vinflunin, Halichondrin B, Isohomohalichondrin B, ER-86526, Pyronetine, Spongistatin 1, Spiket P, Cryptophycin 1, LU103793 (Sematodin or Semadotin), Rhizoxin, Sarcodicin, Erytherobin, Laurimalidol, VP-16 and D-24851, and pharmaceutically acceptable salts, acids, derivatives, or any two or more combinations of the above.

[0094] The payload may be a DNA insertion material, but is not limited to, the following: acridine, actinomycin, anthracycline, benzothiopyranoindazole, pixantrone, cristonator, brostarisin, CI-958, doxorubicin (adriamycin), actinomycin D, daunorubicin (daunomycin), bleomycin, idarubicin, mitoxantrone, cyclophosphamide, melphalan, mitomycin C, bizeresin, etoposide, mitoxantrone, SN-38, carboplatin, cisplatin, actinomycin D, amsacrin, DACA, pyrazoloacridine, irinotecan and topotecan, as well as pharmaceutically acceptable salts, acids, derivatives, or any two or more combinations of the above.

[0095] The payload may be an anti-estrogen and selective estrogen receptor modulator, including, but not limited to, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone and / or farestone toremifene, as well as pharmaceutically acceptable salts, acids, derivatives, or any two or more combinations thereof. The payload may be an aromatase inhibitor, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, AROMASIN®, exemestane, formestan, fadrozol, RIVISOR®, borozol, FEMARA®, letrozole, and ARIMIDESK® and / or anastrozole, as well as pharmaceutically acceptable salts, acids, derivatives, or any two or more combinations thereof.

[0096] The payload may be an antiandrogen, such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin and / or troxacitabine, as well as pharmaceutically acceptable salts, acids, derivatives, or any two or more combinations thereof.

[0097] The payload may be a protein or an antibody. Preferably, the payload is a cytokine (e.g., interleukins, e.g., IL2, IL10, IL12, IL15; members of the TNF superfamily; or interferons, e.g., interferon gamma).

[0098] Any payload can be used in unmodified or modified form. A combination of payloads that are partially unmodified and partially modified may be used. For example, the payload may be chemically modified. One form of chemical modification is the derivatization of the carbonyl group—for example, an aldehyde.

[0099] In a preferred embodiment, portion C is auristatin (i.e., having a structure derived from a member of the auristatin compound family) or an auristatin derivative. More preferably, portion C has a structure according to the following formula:

[0100] [ka]

[0101] During the ceremony: R 1d These are independently H or C1-C6 alkyl; preferably H or CH3; R 2d These are independently C1-C6 alkyl groups; preferably CH3 or iPr; R 3d These are independently H or C1-C6 alkyl; preferably H or CH3; R 4d These are independently H, C1-C6 alkyl, COO(C1-C6 alkyl), CON(H or C1-C6 alkyl), C3-C 10 Aryl or C3~C 10 Heteroaryl; preferably H, CH3, COOH, COOCH3, or thiazolyl; R 5dThese are independently H, OH, and C1-C6 alkyl groups; preferably H or OH; R 6d These are, independently, C3~C 10 Aryl or C3~C 10 Heteroaryl; preferably a phenyl or pyridyl substituted by optional choice.

[0102] More preferably, portion C is derived from MMAE or MMAF. In a preferred embodiment, part C has a structure according to the following formula:

[0103] [ka]

[0104] During the ceremony: n is 0, 1, 2, 3, 4, or 5; preferably 1; R 1e These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 2e These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; Each R 3e These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 4e These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; X is O, NH, or S; preferably O.

[0105] In a preferred embodiment, part C has a structure according to the following formula:

[0106] [ka]

[0107] During the ceremony: n is 0, 1, 2, 3, 4, or 5; preferably 1. R 1f These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 2f These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; R 3f These are independently H, COOH, aryl-COOH, or heteroaryl-COOH; preferably COOH; X is O, NH, or S; preferably O.

[0108] Particularly preferred embodiments relating to part C and the compounds according to the present invention are shown in the appended claims. Preferred compounds are those having structures according to Table 2 or 3, their individual diastereoisomers, hydrates, solvates, crystalline forms, individual tautomers, or pharmaceutically acceptable salts thereof. Further aspects In one embodiment, disclosed herein are compounds of the general formula I as defined above, individual diastereoisomers thereof, hydrates thereof, solvates thereof, crystalline forms thereof, individual tautomers thereof, or pharmaceutically acceptable salts thereof, where: A is a bonding portion having the structure defined above; B is a portion comprising a covalent bond or a chain of atoms covalently attaching portions A and C; and C is a payload portion.

[0109] In one further embodiment, B is represented by any of the general formulas II to V defined above, where each B S Each B independently represents a spacer base; Lrepresents, independently, a cleavable or non-cleavable linker group; each x is independently selected from the range of 0 to 100, preferably 0 to 50, more preferably 0 to 30, even more preferably an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; each y is independently selected from the range of 0 to 30, preferably an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; each z is independently selected from the range of 0 to 5, preferably an integer selected from 0, 1, 2, 3 and 4; * represents an attachment point to part A; · represents an attachment point to part C.

[0110] In a further aspect according to any of the preceding aspects, the linking part has the structure A as defined above 1 and has. In a further aspect according to any of the preceding aspects, B S and / or B L is a group comprising or consisting of a structural unit independently selected from the group consisting of alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalkenylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxycarbamate, disulfide, vinylene, imine, imidoamide, phosphoramide, saccharides, phosphate ester, phosphoramide, carbamate, dipeptide, tripeptide, tetrapeptide, each of which is substituted or unsubstituted.

[0111] In a further aspect according to any of the preceding aspects, BS and / or B L is a group as defined in appended claim 5(b). In a preferred embodiment according to any of the preceding aspects, one or more B L is as defined in appended claim 5(c). In a preferred embodiment according to any of the preceding aspects, B L and B S one or more of are as defined in appended claim 5(d). In a preferred embodiment according to any of the preceding aspects, y is 1, 2 or 3; and / or at least one B L is as defined in appended claim 5(e). In a preferred embodiment according to any of the preceding aspects, B is as defined in appended claim 6.

[0112] In one further aspect according to any of the preceding aspects, the compound is as defined in appended claim 7. In one further aspect according to any of the preceding aspects, moiety C is as defined in appended claims 8 and / or 9.

[0113] In one further aspect according to any of the preceding aspects, the compound has a structure selected from: Conjugate 1; Conjugate 2; Conjugate 3; Conjugate 4; Conjugate 5; Conjugate 6; Conjugate 7; Conjugate 8; Conjugate 9; Conjugate 10; Conjugate 11; Conjugate 12; Conjugate 13; Conjugate 14; Conjugate 15; and ESV6 - fluo.

[0114] Disclosed are pharmaceutical compositions comprising a compound according to any of the preceding embodiments and a pharmaceutically acceptable excipient. Such pharmaceutical compositions are disclosed for use in (a) a method of treating or performing a diagnostic procedure on a human or animal body by surgery or treatment; or (b) a method of treating or preventing a disease or disorder in a subject suffering from or at risk of disease or disorder; or (c) a guided surgical procedure performed on a subject suffering from or at risk of disease or disorder; or (d) a method of diagnosing a disease or disorder performed on a human or animal body with nuclear medicine imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT); or (e) a method of targeted delivery of a therapeutic or diagnostic agent to a subject suffering from or at risk of disease or disorder, wherein in each of the preceding (b) to (e), the disease The disease or disorder is selected independently from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders, and preferably, cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multidrug-resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cell carcinoma, bladder cancer, cholangiocarcinoma, clear cell renal cancer, neuroendocrine tumors, tumor-induced osteomalacia, sarcoma, CUP (unknown primary carcinoma), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, and prostate cancer; preferably, the compound has an extended retention at the disease site at a therapeutically or diagnostically relevant level, preferably more than 1 hour, more preferably more than 6 hours, after injection. treatment The compounds described herein may be used to treat diseases. Treatment may be therapeutic and / or prophylactic, and the aim is to prevent, reduce or halt unwanted physiological changes or impairments. Treatment may extend survival compared to expected survival without treatment.

[0115] The disease treated by the compound may be any disease that could benefit from the treatment. This includes chronic and acute disorders or diseases, including conditions that make a person susceptible to the disorder.

[0116] The terms "cancer" and "cancerous" are used in their broadest sense to refer to a physiological condition in mammals typically characterized by dysregulation of cell growth. A tumor contains one or more cancerous cells.

[0117] When treating cancer, the observed therapeutic effects may include a reduction in the number of cancer cells; a reduction in tumor size; inhibition or delay of cancer cell invasion into peripheral organs; inhibition of tumor growth; and / or alleviation of one or more cancer-related symptoms.

[0118] In animal models, efficacy can be determined by physical measurements of the tumor during treatment and / or by determining partial and complete remission of the cancer. For cancer treatment, efficacy can be measured, for example, by determining the time to disease progression (TTP) and / or the response rate (RR).

[0119] Particularly preferred embodiments of treatment methods related to the present invention are shown in the appended claims. Disclosed herein are, for example, methods of treating the body of a human or animal by surgical or therapeutic means, or diagnostic methods performed on the body of a human or animal, the methods comprising the step of administering a therapeutically or diagnostically effective amount of the compound or pharmaceutical composition described herein to a subject in need. More specifically, disclosed herein are methods of treating or prophylactically treating a subject suffering from or at risk of disease or disorder; or methods of guided surgical treatment performed on a subject suffering from or at risk of disease or disorder; diagnostic methods of disease or disorder, for example, performed on the body of a human or animal and / or involving nuclear medicine imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT); and methods of targeted delivery of therapeutic or diagnostic agents to a subject suffering from or at risk of disease or disorder. In the aforementioned method, the disease or disorder can be independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders. Preferably, cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multidrug-resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal cancer, neuroendocrine tumors, tumor-induced osteomalacia, sarcoma, CUP (unknown primary carcinoma), thymic carcinoma, desmoid tumor, glioma, astrocytoma, cervical cancer, skin cancer, kidney cancer, and prostate cancer. When used in the method disclosed herein, the compound has an extended retention at the disease site at a therapeutically or diagnostically relevant level, preferably more than 1 hour, more preferably more than 6 hours, after injection. Pharmaceutical composition The compounds described herein may be in the form of pharmaceutical compositions that may be used for human or animal use in human and veterinary medicine, and typically comprise one or more pharmaceutically acceptable diluents, carriers, or excipients. Acceptable carriers or diluents for therapeutic use are well known in pharmaceutical technology and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaro ed. 1985). The choice of pharmaceutically acceptable carrier, excipient, or diluent may be made in relation to the intended route of administration and standard pharmaceutical practices. Pharmaceutical compositions may contain any suitable binders, lubricants, suspending agents, coatings, or solubilizers as carriers, excipients, or diluents—or in addition to them.

[0120] Preservatives, stabilizers, colorants, and even flavorings may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0121] Different compositions / formulation requirements depend on different delivery systems. For example, a pharmaceutical composition can be formulated to be administered using a minipump, or to be administered via a mucosal route, such as a nasal spray or inhalation aerosol or an orally administered solution, or to be administered parenterally, where the composition is formulated in an injectable form for delivery, for example, via an intravenous, intramuscular, or subcutaneous route. Alternatively, formulations can be designed to be administered via multiple routes.

[0122] If a drug is administered mucosally via the gastrointestinal mucosa, it should be able to remain stable during its movement through the gastrointestinal tract; for example, it should be resistant to proteolysis, stable in acidic pH, and resistant to the detergent effect of bile.

[0123] Where appropriate, a pharmaceutical composition may be administered topically by inhalation, in the form of suppositories or pessaries, topically in the form of lotions, solutions, creams, ointments or powders, by use as a skin patch, in the form of tablets containing excipients such as starch or lactose, or in capsules or oval formulations, either alone or in mixtures with excipients, or orally in the form of elixirs, solutions or suspensions containing flavoring agents or coloring agents. Alternatively, a pharmaceutical composition may be administered parenterally, for example, by intravenous, intramuscular or subcutaneous injection. For parenteral administration, the composition may be best used in the form of a sterile aqueous solution that may contain other substances, such as sufficient salts or monosaccharides, to make the solution isotonic with blood. For buccal or sublingual administration, the composition may be administered in the form of tablets or lozenges that can be formulated in a conventional manner.

[0124] The compounds of the present invention can be administered in the form of pharmaceutically acceptable salts or activated salts. Pharmaceutically acceptable salts are well known to those skilled in the art, and include, for example, those described by Berge et al. in J. Pharm. Sci., 66, 1-19 (1977). Examples of salts, though not limited to them, include sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, hydrogen tartrates, ascorbic acid, succinates, maleates, gentisinates, fumarates, glucons, glucarates, sugars, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0125] Routes of administration (delivery) may include, but are not limited to, one or more of the following: oral (e.g., as tablets, capsules, or orally ingestible solutions), topical, mucosal (e.g., as nasal spray or inhaled aerosol), nasal, parenteral (e.g., by injectable form), gastrointestinal, intrathecal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, vaginal, lateral ventricle, intracerebral, subcutaneous, ophthalmic (including intravitreous or anterior chamber), percutaneous, rectal, buccal, vaginal, epidural, and sublingual.

[0126] Typically, a physician determines the actual dosage that is most appropriate for the individual patient. The specific dose level and frequency for any particular patient can vary and depend on a variety of factors, including the activity of the particular compound used, its metabolic stability and duration of action, age, weight, overall health, sex, diet, mode and timing of administration, elimination rate, drug combination, severity of the particular condition, and the individual being treated.

[0127] The formulations can be packaged in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as water, for administration. Immediate injection solutions and suspensions are prepared from the types of sterile powders, granules, and tablets described above. Exemplary unit-dose formulations contain a daily dose or a unit sub-daily dose of the active ingredient, or an appropriate fraction thereof. precursor compound In one aspect of the present invention, what is disclosed herein is a compound, its individual diastereoisomers, its hydrates, its solvates, its crystalline forms, its individual tautomers or its salts, wherein the compound (precursor compound) comprises part A and a reactive moiety L capable of reacting with a conjugation partner to form a covalent bond. Upon conjugation (i.e., upon reaction to form a covalent bond), the former precursor compound attaches the former conjugation partner to a payload moiety C in sequence. The conjugation partner can be an atom, a molecule, a particle, a therapeutic agent and / or a diagnostic agent. Preferably, the conjugation partner is a therapeutic agent and / or a diagnostic agent and can correspond to the payload moiety already described in detail above with respect to the conjugate according to the present invention.

[0128] Preferably, the precursor compound comprises a moiety having the following structure:

[0129]

Chemical formula

[0130] In the formula, B is a moiety containing a covalent bond or a chain of covalently bonded atoms. The precursor compound can be represented by the following formula VI:

[0131]

Chemical formula

[0132] In the formula, B is a moiety containing a covalent bond or a chain of atoms that covalently attaches A to L. Part A preferably has structure A 1 or A 2 where m is 0, 1, 2, 3, 4 or 5.

[0133] Part B preferably has the same structure as described in detail above with respect to the conjugate according to the present invention. Part L can preferably react to form linking groups for amides, esters, carbamates, hydrazones, thiazolidines, methylene alkoxycarbamates, disulfides, alkylenes, cycloalkylenes, arylalkylenes, heteroarylalkylenes, heteroalkylenes, heterocycloalkylenes, alkenylenes, cycloalkenylenes, arylalkenylenes, heteroarylalkenylenes, heteroalkenylenes, heterocycloalkenylenes, alkylylenes, heteroalkylylenes, arylenes, heteroarylenes, aminoacyls, oxyalkylenes, aminoalkylenes, diacyl esters, dialkylsiloxanes, amides, thioamides, thioethers, thioesters, esters, carbamates, hydrazones, thiazolidines, methylene alkoxycarbamates, disulfides, vinylenes, imines, imidoamides, phosphoramides, sugars, phosphate esters, phosphoramides, carbamates, dipeptides, tripeptides, or tetrapeptides. As will be understood by those skilled in the art, there are several possibilities for how to provide reactive groups that can react with conjugation partners to form linking groups according to the aforementioned list, and all of these are encompassed by this disclosure.

[0134] Part B may be a cleavable or incleavable bifunctional or polyfunctional part that can be used to link one or more reactive and / or binder parts to form the conjugate precursor of the present invention. In some embodiments, the structure of the compound independently comprises two or more parts A per molecule, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts A; and / or two or more parts L, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 parts L. Preferably, the structure of the compound comprises two parts A and one part L per molecule; or one part A and two parts L.

[0135] Part L is preferably selected from the following: H, NH2, N3, COOH, SH, Hal

[0136] [ka]

[0137] [ka]

[0138] In the formula, each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each m is independently 0, 1, 2, 3, 4, or 5; each Hal is F, Cl, Br, or I; each R 4 The element is independently selected from carboxy, alkyl, cycloalkyl, aryl, and heteroaryl (where each of the above is substituted or unsubstituted), halogen, and cyano.

[0139] Preferred compounds are those having the structures shown in Table 3, their individual diastereoisomers, hydrates, solvates, crystalline forms, individual tautomers, or salts thereof. How to prepare a conjugate In one embodiment of the present invention, disclosed herein is a method for preparing a conjugate, comprising the step of conjugating a precursor compound described above with a conjugation partner. Preferably, the precursor compound is conjugated to a conjugation partner by reacting with the conjugation partner to form a covalent bond. Preferably, the conjugate thus obtained is a conjugate compound described elsewhere herein.

[0140] The conjugation partner may be an atom, molecule, particle, therapeutic agent, and / or diagnostic agent. Preferably, the conjugation partner is a therapeutic agent and / or diagnostic agent and may correspond to the payload portion already described in detail above with respect to the conjugate according to the present invention.

[0141] Preferably, the method further comprises formulating the conjugate as a pharmaceutical composition or a diagnostic composition. The pharmaceutical or diagnostic composition may be for human or animal use in human and veterinary medicine and typically comprises one or more pharmaceutically acceptable diluents, carriers, or excipients. Acceptable carriers or diluents for therapeutic use are well known in pharmaceutical technology and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaro ed. 1985). The choice of carrier, excipient, or diluent may be made in relation to the intended route of administration and standard pharmaceutical practices. The pharmaceutical or diagnostic composition may contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizer as a carrier, excipient, or diluent—or in addition to them. All formulation details and embodiments disclosed above in the section “Pharmaceutical Compositions” also apply hereto in full. General techniques The practice of this invention employs conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology, and pharmacology known to those skilled in the art, unless otherwise indicated. Such techniques are well described in the literature. For example, Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co.; Hardman, JG, Limbird, LE, and Gilman, AG, eds. (2001) The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Weir, DM, and Blackwell, CC, eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, FM et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John See Wiley & Sons; Ream et al. (eds.) (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton, CR., and Graham, A. (eds.) (1997) PCR (Introduction to Biotechniques Series), 2nd edition, Springer Verlag. chemical synthesis The compounds described herein can be prepared by chemical synthesis techniques.

[0142] It is apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during the synthesis of compounds. This can be achieved by the prior art, for example, as described in "Protective Groups in Organic Synthesis" by TW Greene and PGM Wuts, John Wiley and Sons Inc. (1991), and in "Protecting Groups" by PJ Kocienski, Georg Thieme Verlag (1994).

[0143] In some reactions, any stereocenter present can be epimerized under certain conditions, for example, if a base is used in a reaction with a substrate having an optical center containing a base-sensitive group. As is well known in the art, these potential problems can be avoided by selecting the reaction sequence, conditions, reagents, and protection / deprotection regime. definition Antibodies. The term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), benylated antibodies, antibody fragments, and small immunoproteins (SIPs) (see Int. J. Cancer (2002) 102, 75-85). Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Target antigens generally have numerous binding sites, also called epitopes, which are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen can have two or more corresponding antibodies. Antibodies consist of a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen-binding site that immunospecifically binds to the antigen or a portion of the target antigen of interest. The antibody may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA) or any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or a subclass thereof. The antibody may or may not originate from mice, humans, rabbits, or other species.

[0144] Antibody fragment. The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen-binding region or its variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabolic bodies; linear antibodies; dAb, Camelidae V HH Examples include antibodies, and single-domain antibodies, including IgNAR antibodies from cartilaginous fish. Antibodies and their fragments can be replaced by binding molecules based on alternative non-immunoglobulin scaffolds, peptide aptamers, nucleic acid aptamers, structured polypeptides containing polypeptide loops based on non-peptide backbones, innate receptors or their domains.

[0145] Derivatives. Derivatives involve chemical modifications of compounds. Examples of such modifications include the substitution of hydrogen atoms with halo groups, alkyl groups, acyl groups, or amino groups. Modifications can increase or decrease one or more hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der Waals interactions, and / or dipole interactions.

[0146] Analogues. This term encompasses any enantiomers, racemates, and stereoisomers, as well as all pharmaceutically acceptable salts and hydrates of such compounds. Unless otherwise specified, the following definitions apply to chemical terms used in relation to the compounds of the present invention and compositions containing such compounds.

[0147] Alkyl refers to a branched or unbranched saturated hydrocarbyl group. Preferably, an alkyl group contains 1 to 100 carbon atoms, preferably 3 to 30, and more preferably 5 to 25 carbon atoms. Preferably, alkyl refers to methyl, ethyl, propyl, butyl, pentyl, or hexyl groups.

[0148] An alkenyl refers to a branched or unbranched hydrocarbyl group containing one or more carbon-carbon double bonds. Appropriately, an alkenyl group contains 2 to 30 carbon atoms, preferably 5 to about 25 carbon atoms.

[0149] Alkynyl refers to a branched or unbranched hydrocarbyl group containing one or more carbon-carbon triple bonds. Typically, an alkynyl group contains about 3 to about 30 carbon atoms, for example, about 5 to about 25 carbon atoms.

[0150] Halogen refers to fluorine, chlorine, bromine, or iodine, preferably fluorine or chlorine. A cycloalkyl group refers to an alicyclic moiety having, appropriately, three, four, five, six, seven, or eight carbon atoms. The group may be a bridged or polycyclic ring system. More frequently, cycloalkyl groups are monocyclic. This term includes references to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and bicyclo[2.2.2]octyl.

[0151] The term aryl refers to an aromatic carbocyclic system containing, appropriately, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring carbon atoms. The aryl may also be a polycyclic system having two or more rings, at least one of which is aromatic. This term includes references to groups such as phenyl, naphthylfluorenyl, azlenyl, indenyl, and anthryl.

[0152] In this specification, the prefix (hetero) means that one or more carbon atoms of the group may be substituted with nitrogen, oxygen, phosphorus, silicon, or sulfur. Examples of heteroalkyl groups include alkyloxy groups and alkylthio groups. In this specification, heterocycloalkyl groups or heteroaryl groups may have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon, and sulfur. In particular, 3-membered to 10-membered rings or ring systems and even more particularly 5-membered or 6-membered rings may be saturated or unsaturated. For example, oxyranil, azilinil, 1,2-oxathiolanil, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranil, thiopyranil, thiantrenil, isobenzofuranil, benzofuranil, clomenil, 2H-pyrrolyl, pyrrolyl, pyrrolinil, pyrrolidinil, imidazolyl, imidazolidinil, benzimidazolyl, pyrazolyl, pyrazinil, pyrazolidinil, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinil, pyrimidinil, piperidyl, piperazinil, pyridadinil, morpholinil, thiomorpholinil, especially thiomorpholinyl, indolidinil, 1,3-dioxo-1,3-dihydro-isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, coumarill, i Selected from ndazolyl, triazolyl, tetrazolyl, prinyl, 4H-quinolidinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranil, dibenzofuranil, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthilidinyl, quinoxalyl, quinazolinil, quinazolinil, sinnolinyl, pteridinyl, carbazolyl, [beta]-carbolinyl, phenanthiazinyl, acridinyl, perimidinyl, phenanthrolinyl, flazanil, phenazinyl, phenothiazinyl, phenoxadinyl, clomenyl, isochromanil, clomanil, 3,4-dihydro-2H-isoquinolin-1-one, 3,4-dihydro-2H-isoquinolinyl, etc.

[0153] "Substituted" means that one or more hydrogen atoms in the aforementioned part, particularly up to five, and more particularly one, two, or three, are independently replaced by a corresponding number of substituents. The term "optionally substituted," as used herein, includes being substituted or unsubstituted. Naturally, it is understood that substituents are only in positions where they are chemically possible, and those skilled in the art can determine (either experimentally or theoretically) without undue effort whether a particular substitution is possible. For example, an amino or hydroxyl group with free hydrogen may be unstable if it is bonded to a carbon atom by an unsaturated (e.g., olefin) bond. Preferably, the term "substituted" means that one or more hydrogen atoms in the aforementioned part, particularly up to five, and more particularly one, two, or three, are independently replaced by a corresponding number of substituents selected from OH, SH, NH2, halogens, cyano, carboxy, alkyl, cycloalkyl, aryl, and heteroaryl. In addition, the substituents described herein may themselves be substituted by any substituent, subject to the aforementioned limitations on appropriate substitutions recognized by those skilled in the art. Preferably, any of the aforementioned substituents may be further substituted by any of the aforementioned substituents, and each of these may be further substituted by any of the aforementioned substituents.

[0154] Suitable substituents include halogen atoms and halomethyl groups, e.g., CF3 and CCl3; oxygen-containing groups, e.g., oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alcoil, alcoyloxy, aryloxy, aryloyl, and aryloyloxy; nitrogen-containing groups, e.g., amino, alkylamino, dialkylamino, cyano, azide, and nitro; sulfur-containing groups, e.g., thiol, alkylthiol, sulfonyl, and sulfoxide; heterocyclic groups, which may be substituted themselves; alkyl groups, which may be substituted themselves; and aryl groups, which may be substituted themselves, e.g., phenyl and substituted phenyl. Examples of alkyl groups include substituted and unsubstituted benzyl.

[0155] When it is stated that two or more parts are selected "independently" from a list of atoms or groups, this means that the parts may be the same or different. The identity of each part is therefore independent of the identity of one or more other parts.

[0156] For ease of reference, the numbers and structures of some of the compounds disclosed herein are summarized in Tables 2, 3, and 4 below. In case of any doubt, the numbers and structures below shall prevail.

[0157] [Table 2-1]

[0158] [Table 2-2]

[0159] [Table 2-3]

[0160] [Table 2-4]

[0161] Table 2-5

[0162] Table 2-6

[0163] Table 2-7

[0164] Table 2-8

[0165] Table 2-9

[0166] Table 2-10

[0167] Table 2-11

[0168] Table 2-12

[0169] Table 2-13

[0170] Table 2-14

[0171] Table 2-15

[0172] Table 2-16

[0173] Table 2-17

[0174] Table 2-18

[0175] Table 2-19

[0176] Table 2-20

[0177] Table 2-21

[0178] Table 2-22

[0179] Table 2-23

[0180] Table 3-1

[0181] [Table 3-2]

[0182] [Table 3-3]

[0183] [Table 3-4]

[0184] [Table 4-1]

[0185] [Table 4-2]

[0186] [Table 4-3]

[0187] [Table 4-4] [Examples]

[0188] Materials & Methods Overview and Procedures Yield refers to the compound purified by chromatography.

[0189] Proton (1H) nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AV400 (400 MHz) spectrometer. Shifts are shown in ppm using residual solvent as an internal standard. Coupling constants (J) are reported in Hz, and the following abbreviations are used to indicate splitting: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.

[0190] Mass spectroscopy (LC-ESI-MS) spectra were obtained using an Agilent 6100 Series Single Quadrupole MS System combined with an Agilent 1200 Series LC System, employing an InfinityLab Poroshell 120 EC-C18 column, 4.6 mm × 56 mm, for 2 mL. -1 The flow rates were recorded as linear gradients for solvents A and B (A = Millipore water containing 0.1% formic acid [FA], B = MeCN containing 0.1% formic acid [FA]).

[0191] High-resolution mass spectrometry (HRMS) spectra and reversed-phase super-performance liquid chromatography (UPLC) for analysis were obtained on a Waters Xevo G2-XS QTOF coupled to a Waters Acquity UPLC H-Class System with a PDA UV detector, using an ACQUITY UPLC BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 50 mm, for 0.6 mL min. -1 The flow rates were recorded as linear gradients for solvents A and B (A = Millipore water with 0.1% FA, B = MeCN with 0.1% FA).

[0192] Preparative reverse-phase high-pressure liquid chromatography (RP-HPLC) was performed on an Agilent 1200 Series System using a Phenomenex Gemini® 5μm NX-C18 half-portion column, 110Å, 150mm × 10mm, yielding 5 mL of liquid. -1 The test was performed using a linear gradient of solvents A and B (A = Millipore water containing 0.1% trifluoroacetic acid [TFA], B = MeCN containing 0.1% trifluoroacetic acid [TFA]) at the specified flow rates.

[0193] Comparative Example 1: Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-6-(3-(4-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carbonyl)piperazine-1-yl)propoxy)quinoline-4-carboxamide ("HABERKORN-FLUO", 23) Step 1: 6-Hydroxy-4-Quinolinecarboxylic Acid (H1) A solution of 6-methoxyquinoline carboxylic acid (250 mg, 1.08 mmol, 1.0 equivalent) in 48 wt.% HBr in H2O (5 mL) was heated at 130°C for 2 hours, and then concentrated under vacuum to obtain the product as an orange powder (292 mg, 1.08 mmol, quantitative yield). MS(ES) + )m / z 271(M+H) + . Step 2: 1-Boc-4-(3-hydroxypropyl)piperazine(H2) Anhydrous K2CO3 (815 mg, 5.91 mmol, 1.1 equivalent) was added to a solution of 1-Boc-piperazine (1.0 g, 5.37 mmol, 1.0 equivalent) in dry THF (15 mL), followed by 3-bromo-1-propanol (530 μL, 5.91 mmol, 1.1 equivalent). The reaction mixture was stirred at room temperature for 72 hours. Volatile substances were removed under reduced pressure, the residue was diluted with water, extracted with SiO2 (twice), and the combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by flash chromatography (DCM / MeOH at 98:2 to 90:10) to obtain the title compound as a colorless oil (1.2 g, 4.91 mmol, 91% yield). MS(ES) + )m / z 245(M+H) + . Step 3: 3-(4-(tert-butoxycarbonyl)piperazine-1-yl)propyl 6-(3-(4-(tert-butoxycarbonyl)piperazine-1-yl)propoxy)quinoline-4-carboxylate(H3) A stirred solution of H1 (100 mg, 0.37 mmol, 1.0 equivalent), H2 (180 mg, 0.74 mmol, 2.0 equivalents), and triphenylphosphine (193 mg, 0.74 mmol, 2.0 equivalents) in dry THF (25 mL) was treated with diisopropyl azodicarboxylate (DIAD; 145 μL, 0.74 mmol, 2.0 equivalents). The reaction mixture was stirred at room temperature for 1 hour, then concentrated under vacuum, and the title compound was obtained as a white powder by direct purification by flash chromatography (DCM / MeOH at 95:5 to 90:10) (100 mg, 0.156 mmol, 42% yield). MS(ES) + )m / z 642(M+H) + .

[0194] [ka]

[0195] Step 4: 6-(3-(4-tert-butoxycarbonylpiperazine-1-yl)propoxy)quinoline-4-carboxylic acid (H4) A stirring solution of H3 (100 mg, 0.156 mmol, 1.0 equivalent) in THF (5 mL) was mixed with a solution of LiOH (13 mg, 0.312 mmol, 2.0 equivalents) in H2O (2 mL), and the reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with water, extracted with siRNA, washed with NH4Cl ss, dried over Na2SO4, and filtered to obtain the product as a white powder (60 mg, 0.144 mmol, 92% yield). MS(ES) + )m / z 416(M+H) + . Step 5: 1-Piperazine carboxylic acid, 4-[3-[[4-[[[2-[(2S)-2-cyano-4,4-difluoro-1-pyrrolidinyl]-2-oxoethyl]amino]carbonyl]-6-quinolinyl]oxy]propyl]-, 1,1-dimethylethyl ester (H5) To a stirred solution of H4 (15 mg, 0.036 mmol, 1.0 equivalent), HATU (20 mg, 0.054 mmol, 1.5 equivalents), and HOBt (7.3 mg, 0.054 mmol, 1.5 equivalents) in DCM (3 mL), a solution of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile (10 mg, 0.054 mmol, 1.5 equivalents) in DMF (1.0 mL) and DIPEA (25 μL, 0.144 mmol, 4 equivalents) was added, and the reaction mixture was stirred at room temperature for 9 hours. The mixture was evaporated under vacuum, dissolved in DMSO, and purified by RP-HPLC to obtain the product as a white solid (6.0 mg, 0.01 mmol, 28% yield). MS(ES) + )m / z 587(M+H) + . Step 6: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-6-(3-(piperazine-1-yl)propoxy)quinoline-4-carboxamide(H6) A stirred solution of H5 (5.0 mg, 8.0 μmol, 1.0 equivalent) and 4-methylbenzenesulfonic acid monohydrate (6.8 mg, 40 μmol, 5.0 equivalents) in MeCN (3 mL) was heated at 55°C for 2 hours. The mixture was concentrated under vacuum, and the product was used directly in subsequent steps. White powder (8.0 mg, 8.0 μmol, quantitative yield). MS(ES) + )m / z 487(M+H) + . Step 7: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-6-(3-(4-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-carbonyl)piperazine-1-yl)propoxy)quinoline-4-carboxamide ("HABERKORN-FLUO", 23) To a stirred solution of H6 (4.5 mg, 4.0 μmol, 1.0 equivalent) and TEA (1.1 μL, 8.0 μmol, 2.0 equivalents) in DMF (1 mL), NHS-fluorescein (2.8 mg, 6.0 μmol, 1.5 equivalents) was added, and the reaction mixture was stirred at room temperature for 9 hours. The mixture was directly purified by RP-HPLC, yielding the product as an orange powder (0.9 mg, 1.0 μmol, 26% yield). MS(ES) + )m / z 845(M+H) + (See Comparative Example 1B; Figure 1B).

[0196] Example 2: Synthesis of (S)-N1-(4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)-N4-(2-(2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethoxy)ethoxy)ethyl)succinimide ("ESV6-FLUO", 26) Step 1: 5,8-Dioxa-2,11-Diazadodecanoic acid, 12-[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-6-yl)amino]-12-thioxo-,1,1-dimethylethyl ester (P1) To a stirred solution of tert-butyl N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}carbamate (173 μL, 0.731 mmol, 1.5 equivalents) in THF (20 mL), fluorescein-5-isothiocyanate (190 mg, 0.487 mmol, 1.0 equivalent) was added, and the reaction was stirred at room temperature for 12 hours. The mixture was concentrated under vacuum, and the crude product was purified directly by flash chromatography (DCM / siRNA, 9:1 to 8:2) to obtain the compound as an orange powder (200 mg, 0.314 mmol, 64% yield). MS(ES) + )m / z 638(M+H) + . Step 2: Thiourea, N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-N'-(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-5-yl)-(P2) To a stirred solution of P1 (150 mg, 0.235 mmol, 1.0 equivalent) in DCM (10 mL) cooled to 0°C, HCl 4M in Et2O (5 mL) was added. The reaction mixture was stirred, slowly heated to room temperature for 2 hours, then concentrated under vacuum, and after several co-evaporations with Et2O, an orange powder was obtained (135 mg, 0.235 mmol, quantitative yield). MS(ES) + )m / z 538(M+H) + .

[0197] [ka]

[0198] Step 3: (S)-8-amino-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide(P3) Commercially available 8-amino-quinoline-4-carboxylic acid (19.0 mg, 100 μmol, 1.0 equivalent), DIPEA (70.0 μL, 400 μmol, 4.0 equivalents), and HATU (38.0 mg, 100 μmol, 1.0 equivalent) were dissolved in a 1:1 DCM / DMF mixture (2 mL). After 15 minutes, a solution of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitride trifluoroacetate (30.3 mg, 100 μmol, 1.0 equivalent) in DCM was added. The reaction mixture was stirred at room temperature for 1 hour, washed with water, dried over Na2SO4, filtered, and concentrated to obtain a brown crude product as a viscous oil. The residue was purified by flash chromatography (DCM / MeOH at 91:1 to 90:10) to obtain a pure product as a brownish oil (24.8 mg, 68.9 μmol, 69% yield). MS(ES) + )m / z 360(M+H) + . Step 4: (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (P4) Triethylamine (20.8 μL, 150 μmol, 2.0 equivalents) and 4-dimethylaminopyridine (0.91 mg, 10.0 μmol, 0.1 equivalents) were added to a chilled solution (0°C) of P3 (26.8 mg, 70.0 μmol, 1.0 equivalent) in DCM, followed by dropwise addition of succinic anhydride (15.0 mg, 150 μmol, 2.0 equivalents). The reaction mixture was allowed to return to room temperature. After placing the reaction mixture in a preheated 40°C oil bath, complete conversion was observed. The solvent was evaporated, and the residue was purified by RP-HPLC to obtain a pure product as a white powder (9.42 mg, 20.0 μmol, 28% yield). MS(ES) + )m / z 460(M+H) + Figure 28 shows the structure, chromatographic profile, and LC / MS analysis of Example 2, P4. MS(ES+)m / z 460.21(M+H) + . Step 5: (S)-N1-(4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)-N4-(2-(2-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethoxy)ethoxy)ethyl)succinamide ("ESV6-FLUO", 26) P2 (22.0 mg, 40.0 μmol, 2.0 equivalents) and HATU (15.6 mg, 40.0 μmol, 2.0 equivalents) were added to a 1:1 DCM / DMF mixture (2 mL) and DIPEA (7.20 μl, 40.0 μmol, 2.0 equivalents). The resulting mixture was stirred at room temperature for 15 minutes. P4 (9.42 mg, 20.0 μmol, 1.0 equivalent) in DCM was added, and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated, and the residue was purified by preparative RP-HPLC to obtain a pure product as a yellow solid (2.50 mg, 10.0 μmol, 25% yield). MS(ES) + )m / z 979(M+H) + (Figure 1A).

[0199] Further conjugates according to the present invention are listed below. Conjugate 1: (2S,5R,8R,11R)-2-(((1-(6-(((S)-1-(((R)-1-((4-((5R,8S,11R,12S)-11-((R)-sec-butyl)-12-(2-((R)-2-((1S,2S)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazateto Radicyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)-2,5-dioxopyrrolidine-3-yl)thio)methyl)-5,11-bis(carboxymethyl)-16-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-8-(3-guanidinopropyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0200] [ka]

[0201] Conjugate 2: (2S,5R,8R,11R)-8-(4-aminobutyl)-2-(((1-(6-(((S)-1-(((R)-1-(((4-((5R,8S,11R,12S)-11-((R)-sec-butyl)-12-(2-((R)-2-((1S,2S)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa -4,7,10-Triazatetradecyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)-2,5-dioxopyrrolidine-3-yl)thio)methyl)-5,11-bis(carboxymethyl)-16-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0202] [ka]

[0203] Conjugate 3: (2S,5R,8R,11R)-2-(((1-(6-(((S)-1-(((R)-1-((4-((5R,8S,11R,12S)-11-((R)-sec-butyl)-12-(2-((R)-2-((1S,2S)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-trioxa Thetetradecyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-6-oxohexyl)-2,5-dioxopyrrolidine-3-yl)thio)methyl)-5,11-bis(carboxymethyl)-16-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-8-(3-guanidinopropyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0204] [ka]

[0205] Conjugate 4:N 6 -(4-((3-((3-(((2R)-1-(((1 4 R,1 6 R,3 3 R, 2S, 4S, 10E, 12Z, 14S)-8 6 -Chloro-1 4 -Hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2,6-Dioxo-7-Aza-1(6,4)-Oxadinana-3(2,3)-Oxirana-8(1,3)-Benzenacyclotetradecafan-10,12-Dien-4-yl)oxy)-1-Oxopropane-2-yl)(methyl)amino)-3-Oxopropyl)thio)-2,5-Dioxopyrrolidine-1-yl)methyl)cyclohexane-1-carbonyl)-N 2 -(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoyl)-D-aspartyl-D-arginyl-D-aspartyl-D-lysine

[0206] [ka]

[0207] Conjugate 5: (2R,5R,8R,11R)-5,11-bis(carboxymethyl)-16-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-2-((1-(14-((4,7-dimethyl-3,8,11-trioxo-11-((2S,4S)-2,5,12-trihydroxy-7-methoxy-4-(((1S,3R,4aS,9S,9aR,10aS)-9-methoxy-1-methyloctahydro-1H-pyrano[ [4',3':4,5]oxazolo[2,3-c][1,4]oxazin-3-yl)oxy)-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracene-2-yl)-2,9-dioxa-4,7-diazaundecyl)phenyl)carbamoyl)-15-methyl-3,12-dioxo-7,10-dioxa-4,13-diazahexadecyl)-2,5-dioxopyrrolidine-3-yl)thio)-8-(3-guanidinopropyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0208] [ka]

[0209] Conjugate 6: (10R,13R,16R,19R)-1-((3aR,4R,5S,10bR)-4-acetoxy-3a-ethyl-9-((3S,5S,7S,9S)-5-ethyl-5-hydroxy-9-(methoxycarbonyl)-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methano[1]azacycloundecino[5,4-b]indole-9-yl)-5-hydroxy-8-methoxy-6-methyl-3a,3a 1 ,4,5,5a,6,11,12-Octahydro-1H-Indolidino[8,1-cd]carbazol-5-yl)-10-Carboxy-13-(carboxymethyl)-19-(4-((4-((2-((R)-2-Cyanol-4,4-Difluoropyrrolidine-1-yl)-2-Oxoethyl)Carbamoyl)Quinoline-8-yl)amino)-4-Oxobutanamide)-16-(3-Guanidinopropyl)-1,4,12,15,18-Pentaoxo-5-oxa-8,9-Dithia-2,3,11,14,17-Pentaazahenicosan-21-Eucic Acid

[0210] [ka]

[0211] Conjugate 7: 2,2',2''-(10-(2-((2-(3-(((2S,5R,8R,11R)-8-(4-aminobutyl)-2-carboxy-5,11-bis(carboxymethyl)-16-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecyl)thio)-2,5-dioxopyrrolidine-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0212] [ka]

[0213] Conjugate 8: 2,2',2''-(10-(1-carboxy-4-((2-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0214] [ka]

[0215] Conjugate 9: 177-Lutetium-labeled 2,2',2''-(10-(1-carboxy-4-((2-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0216] [ka]

[0217] Conjugate 10: 225-Actinium-labeled 2,2',2''-(10-(1-carboxy-4-((2-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0218] [ka]

[0219] Conjugate 11: 64-copper-labeled 2,2',2''-(10-(1-carboxy-4-((2-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0220] [ka]

[0221] Conjugate 12:68-Gallium-labeled (S)-3-((S)-2-amino-6-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)hexanamide)-4-(((R)-1-carboxy-2-mercaptoethyl)amino)-4-oxobutanoic acid

[0222] [ka]

[0223] Conjugate 13: (S)-3-((S)-2-amino-6-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)hexanamide)-4-(((R)-1-carboxy-2-mercaptoethyl)amino)-4-oxobutanoic acid

[0224] [ka]

[0225] Conjugate 14:99m-technetium-labeled (S)-3-((S)-2-amino-6-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)hexanamide)-4-(((R)-1-carboxy-2-mercaptoethyl)amino)-4-oxobutanoic acid

[0226] [ka]

[0227] Conjugate 15: (2R,5S,8S,11S)-8-(4-aminobutyl)-5,11-bis(carboxymethyl)-16-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-2-(((1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)-2,5-dioxopyrrolidine-3-yl)thio)methyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0228] [ka]

[0229] Example 3: Characterization of the fluorescein-labeled binder "ESV6-FLUO" (26) Human FAP expression Amino acid sequence of polyhistidine-tagged human fibroblast activating protein (hFAP): LRPSRVHNSEENTMRALTLKDILNGTFSYKTFFPNWISGQEYLHQSADNNIVLYNIETGQSYTILSNRTMKSVNASNYGLSPDRQFVYLESDYSKLWRYSYTATYYIYDLSNGEFVRGNELPRPIQYLCWSPVGSKLAYVYQNNIYLKQRPGDPPFQITFNGRENKIFNGIPDWVYEEEMLATKY ALWWSPNGKFLAYAEFNDTDIPVIAYSYYGDEQYPRTINIPYPKAGAKNPVVRIFIIDTTYPAYVGPQEVPVPAMIASSDYYFSWLTWVTDERVCLQWLKRVQNVSVLSICDFREDWQTWDCPKTQEHIEESRTGWAGGFFVSTPVFSYDAISYYKIFSDKDGYKHIHYIKDTVENAIQITSGKW EAINIFRVTQDSLFYSSNEFEEYPGRRNIYRISIGSYPPSKKCVTCHLRKERCQYYTASFSDYAKYYALVCYGPGIPISTLHDGRTDQEIKILEENKELENALKNIQLPKEEIKKLEVDEITLWYKMILPPQFDRSKKYPLLIQVYGGPCSQSVRSVFAVNWISYLASKEGMVIALVDGRGTAFQ GDKLLYAVYRKLGVYEVEDQITAVRKFIEMGFIDEKRIAIWGWSYGGYVSSLALASGTGLFKCGIAVAPVSSWEYYASVYTERFMGLPTKDDNLEHYKNSTVMARAEYFRNVDYLLIHGTADDNVHFQNSAQIAKALVNAQVDFQAMWYSDQNHGLSGLSTNHLYTHMTHFLKQCFSLSDHHHHHH Human fibroblast-activating protein (hFAP; UniProtKB-Q12884(SEPR_HUMAN) amino acids 25-760) was cloned in pCDNA3.1(+) having a secretory sequence at its 5'- and a 6× polyhistidine tag at its 3' end, and expressed in CHO cells by transient gene expression. The transfection mix was constructed as follows: 0.625 μg plasmid DNA, 10 6 For each cell, 2.5 μg of polyethyleneimine (PEI), 4 × 10⁶ 6Cell density in cells / ml. Cells were incubated at 37°C for 6 days in 5% CO2. 22 The cells were incubated at 120 rpm. Cells were collected by centrifugation (4500 rpm, 30 min, 4°C), and 1 mL (dry volume) of complete His-tagged purified resin was added to the filtered supernatant and incubated at 120 rpm for 2 hours at room temperature. The resin was washed with 800 mL of washing buffer (10 mM imidazole, 250 mM PBS / NaCl), and hFAP was eluted with 250 mM imidazole PBS / NaCl. The eluted fraction (1.5 mL) was checked for absorbance at 280 nm using a spectrophotometer. The hFAP-enriched fraction was pooled and dialyzed against HEPES buffer (50 mM HEPES, 100 mM NaCl, pH=7.4).

[0230] Recombinant hFAP samples were analyzed by SDS-PAGE and size exclusion chromatography (see Figure 2: A) SDS-PAGE; B) Size exclusion chromatography (Superdex 200 Increase 10 / 300 GL), and enzyme activity was confirmed in an inhibition assay ("In vitro human FAP enzyme IC5"). 50 (See the section titled "Assays"). Affinity measurement for human FAP using fluorescence polarization (Figure 4) Fluorescence polarization measurements were performed in a 384-well plate (unbound, ps, f-bottom, black, high volume, final volume 30 μL). A stock solution of human FAP (4 μM) was sequentially diluted with buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4) while maintaining a constant final binder concentration at 10 nm. After incubation in the dark at 37°C for 30 minutes, measurements were performed by fluorescence polarization (Figure 4). ESV6-fluo was found to be linked to the previously described ligand, Haberkorn-fluo (0.89 nM K). D ) compared to hFAP(0.78nM K D It exhibits a higher affinity for ). In vitro human FAP enzyme IC50 assay (Figure 5) The enzymatic activity of human FAP against the Z-Gly-Pro-AMC substrate was measured at room temperature on a microtiter plate reader, and fluorescence was monitored at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The reaction mixture consisted of 20 μM substrate, 20 nM human FAP, assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4), and inhibitor (10 -6 from 10 -11 It contained IC in a total volume of 30 μL (within the range of M). 50 The value is defined as the concentration of the inhibitor required to reduce enzyme activity by 50% after a 30-minute pre-incubation using the enzyme at 37°C prior to substrate addition.

[0231] Inhibitor stock solution (200 mM) was prepared in DMSO. Immediately before the start of the experiment, the stock was immersed in assay buffer for 10 minutes. -6 The solution was further diluted with M, and from this, a 1:2 series of dilutions was prepared. All measurements were performed in triplicate form.

[0232] Figure 5 shows the results from hFAP inhibition experiments in the presence of small organic ligands. ESV6 ligand exhibits a lower IC50 (20.2 nM) compared to the previously described ligand, Haberkorn ligand (24.6 nM). Chromatographic co-elution experiment of ligand-protein complex (Figure 3) The PD-10 column was pre-equilibrated with assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4) before loading the complex. Human FAP (2 μM) and fluorescein-labeled binder (6 μM) were incubated at 37°C in the dark for 30 minutes. The mixture was loaded, and the column was flushed with assay buffer. The flow-through was collected in a 96-well plate, and fluorescence was immediately measured on a TECAN microtiter plate reader, monitoring fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Protein content was estimated by measuring absorbance at 280 nm using a Nanodrop 2000 / 2000c spectrophotometer.

[0233] Figure 3 shows the results of co-elution PD-10 experiments with the small molecule ligands ESV6-fluo and Haberkorn-fluo and hFAP. Stable complexes were formed between hFAP and the small ligands ESV6-fluo and Haberkorn-fluo. Dissociation rate measurement (Figure 6) k off Fluorescence polarization measurements were performed in a 384-well plate to determine the values ​​(unbound, ps, f-bottom, black, high volume, final volume 30 μL). Measurements were performed after preliminary incubation of a 2.0 nM fluorescein-labeled binder using human FAP at a constant concentration of 1.0 μM in the dark at 37°C. Dissociation of the fluorescein-labeled compounds was induced by adding a large excess of the corresponding fluorescein-free binder (compound P3 obtained in step 3 of Example 2 and compound H6 obtained in step 6 of Comparative Example 1, each at a final concentration of 20 μM).

[0234] Figure 6 shows the results from dissociation rate measurements of ESV6-fluo and Haberkorn-fluo from hFAP. ESV6-fluo dissociates at a slower rate (regression coefficient = -0.093564) compared to Haberkorn-fluo (regression coefficient = -0.075112).

[0235] The compound of Comparative Example 1 in this application ("HABERKORN-FLUO") can be considered representative of the structure of ligand FAPI-04 described in the prior art disclosures, and in particular in the prior art (WO2019 / 154886 and WO2019 / 154859). The above results show that the compounds according to the present invention not only form stable complexes with hFAP, but also, surprisingly, exhibit a significant increase in affinity (lower K) compared to the prior art. D ), increased inhibitory activity (lower IC 50 ), and also demonstrate slower rates of dissociation.

[0236] Example 4: Comparative experiment in a tumor model PART 1 - Conjugate Preparation Synthesis of tert-butyl(8-aminoquinoline-4-carbonyl)glycinate

[0237] [ka]

[0238] DIPEA (185 μL, 1.063 mmol, 4 equivalents) was added dropwise to a stirred solution of tert-butylglycinate hydrochloride (89 mg, 0.532 mmol, 2.0 equivalents), 8-aminoquinoline-4-carboxylic acid (50 mg, 0.266 mmol, 0.1 equivalent), and HATU (111 mg, 0.292 mmol, 1.1 equivalents) in 300 μL of DMF and 3 mL of DCM. The reaction mixture was stirred at room temperature for 1 hour. The mixture was concentrated under vacuum, and the crude material was directly purified by flash chromatography (100% to 9.5:0.5 DMC / MeOH) to obtain tert-butyl(8-aminoquinoline-4-carbonyl)glycinate as a brown oil (70 mg, 0.232 mmol, 87.5%). MS(ES+)m / z 302.14(M+H) + Synthesis of 4-((4-((2-(tert-butoxy)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid

[0239] [ka]

[0240] 4-dimethylaminopyridine (14 mg, 0.116 mmol, 0.5 equivalents) was added to a stirred solution of tert-butyl(8-aminoquinoline-4-carbonyl)glycinate (70 mg, 0.232 mmol, 1.0 equivalent) and dihydrofuran-2,5-dione (232 mg, 2.324 mmol, 10.0 equivalents) in THF (3 mL). The reaction mixture was heated at 50°C for 6 hours. The mixture was concentrated under vacuum, diluted in DCM, and washed with water. The organic phase was concentrated under vacuum and purified by flash chromatography (DMC / MeOH, 9:1 to 7:3) to obtain the compound as a brown oil (50 mg, 0.125 mmol, 83.3%). MS(ES+)m / z 402.16(M+H) + Synthesis of Cys(STrt)-Asp(OtBu)-Lys(NHBoc)-Asp(OtBu)-NHFmoc on resin

[0241] [ka]

[0242] A commercially available preload of Fmoc-Cys(Trt) (500 mg, 0.415 mmol, RAPP Polymere) on Tentagel resin was expanded in DMF (3 × 5 min × 5 mL), the Fmoc groups were removed with 20% piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL), and the resin was washed with DMF (6 × 1 min × 5 mL). The peptides were extended with Fmoc-Asp(tBu)-OH, Fmoc-Lys(NHBoc)-OH, and Fmoc-Asp(tBu)-OH in the order shown. For this purpose, Fmoc-protected amino acids (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) were dissolved in DMF (5 mL). The mixture was allowed to stand at 0°C for 10 minutes and then reacted with the resin for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the Fmoc group was removed with 20% piperidine in DMF (1 × 1 min × 5 min and 2 × 10 min × 5 mL). Following the deprotection step, a washing step with DMF (6 × 1 min × 5 mL) was performed, followed by coupling with the next amino acid. Synthesis of (2R,5S,8S,11R)-8-(4-aminobutyl)-5,11-bis(carboxymethyl)-16-((4-((carboxymethyl)carbamoyl)quinoline-8-yl)amino)-2-(mercaptomethyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0243] [ka]

[0244] Cys(STrt)-Asp(OtBu)-Lys(NHBoc)-Asp(OtBu)-NHFmoc (50 mg, 0.025 mmol) on a resin was expanded in DMF (3 × 5 min × 5 mL). The peptide was extended with 4-((4-((2-(tert-butoxy)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (20 mg, 0.05 mmol, 2.0 equivalents), HATU (19 mg, 0.05 mmol, 2.0 equivalents), and DIPEA (17 μL, 0.1 mmol, 4.0 equivalents), and allowed to react for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the resin was cleaved by stirring at room temperature for 4 hours with a mixture of TFA (15%), TIS (2.5%), and H2O (2.5%) in DCM. The resin was washed with methanol (2 × 5 mL), and the combined cleavage solution and washing solution were concentrated under vacuum. The crude product was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) and lyophilized to obtain a white solid (2.4 mg, 0.003 mmol, 12%). MS(ES+)m / z 807.45(M+H) + Synthesis of (2R,5S,8S,11S)-8-(4-aminobutyl)-5,11-bis(carboxymethyl)-16-(4-(3-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazine-1-yl)-2-(mercaptomethyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0245] [ka]

[0246] Cys(STrt)-Asp(OtBu)-Lys(NHBoc)-Asp(OtBu)-NHFmoc (60 mg, 0.03 mmol) on a resin was expanded in DMF (3 × 5 min × 5 mL). The peptide was extended with (S)-4-(4-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazine-1-yl)-4-oxobutanoic acid (17.5 mg, 0.03 mmol, 1.0 equivalent), HATU (22 mg, 0.06 mmol, 2.0 equivalents) and DIPEA (200 μL, 1.2 mmol, 40 equivalents), and allowed to react for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the resin was cleaved by stirring at room temperature for 4 hours with a mixture of TFA (15%), TIS (2.5%), and H2O (2.5%) in DCM. The resin was washed with methanol (2 × 5 mL), and the combined cleavage solution and washing solution were concentrated under vacuum. The crude product was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) and lyophilized to obtain a white solid (1 mg, 0.95 μmol, 0.3%). MS(ES+) m / z 1048.39(M+H) + Synthesis of (2R,5S,8S,11S)-8-(4-aminobutyl)-5,11-bis(carboxymethyl)-16-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-2-(mercaptomethyl)-4,7,10,13,16-pentaoxo-3,6,9,12-tetraazahexadecanoic acid

[0247] [ka]

[0248] Cys(STrt)-Asp(OtBu)-Lys(NHBoc)-Asp(OtBu)-NHFmoc (80 mg, 0.04 mmol) on a resin was expanded in DMF (3 × 5 min × 5 mL). The peptide was extended with (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (37 mg, 0.08 mmol, 2 equivalents), HATU (30 mg, 0.08 mmol, 2.0 equivalents), and DIPEA (28 μL, 0.16 mmol, 4.0 equivalents), and allowed to react for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the resin was cleaved by stirring at room temperature for 4 hours with a mixture of TFA (15%), TIS (2.5%), and H2O (2.5%) in DCM. The resin was washed with methanol (2 × 5 mL), and the combined cleavage solution and washing solution were concentrated under vacuum. The crude product was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) and lyophilized to obtain a white solid (1 mg, 0.95 μmol, 0.3%). MS(ES+) m / z 921.29(M+H) + Synthesis of "QCOOH-IRDye750" SH-Cys-Asp-Lys-Asp-QCOOH (140 μg, 0.174 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (800 μL). IRDye750 maleimide (200 μg, 0.174 μmol, 1.0 equivalent) was added as a dry DMF solution (200 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a green solid (0.06 μmol, 40%). MS(ES+)m / z 978(M+2H) 2+

[0249] [ka]

[0250] Conjugate 16: Structure of "QCOOH-IRDye750". Synthesis of "HABERKORN-IRDye750" SH-Cys-Asp-Lys-Asp-HK (181 μg, 0.174 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (800 μL). IRDye750 maleimide (200 μg, 0.174 μmol, 1.0 equivalent) was added as a dry DMF solution (200 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a green solid (0.06 μmol, 40%). MS(ES+) m / z 1099.8 (M+2H) 2+

[0251] [ka]

[0252] Conjugate 17: Structure of "HABERKORN-IRDye750". Synthesis of ESV6-IRDye750 SH-Cys-Asp-Lys-Asp-ESV6 (160 μg, 0.174 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (800 μL). IRDye750 maleimide (200 μg, 0.174 μmol, 1.0 equivalent) was added as a dry DMF solution (200 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a green solid (0.08 μmol, 50%). MS(ES+) m / z 1036.3 (M+2H) 2+

[0253] [ka]

[0254] Structure of Conjugate 18:ESV6-IRDye750. Synthesis of "QCOOH-ValCit-MMAE" SH-Cys-Asp-Lys-Asp-QCOOH (612 μg, 0.760 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (840 μL). MC-ValCit-PAB-MMAE (1000 μg, 0.760 μmol, 1.0 equivalent) was added as a dry DMF solution (160 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid (322 μg, 20%). MS(ES+)m / z 2124.03(M+H) +

[0255] [ka]

[0256] Conjugate 19: The structure of "QCOOH-ValCit-MMAE". Synthesis of "HABERKORN-ValCit-MMAE" SH-Cys-Asp-Lys-Asp-HK (795 μg, 0.760 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (840 μL). MC-ValCit-PAB-MMAE (1000 μg, 0.760 μmol, 1.0 equivalent) was added as a dry DMF solution (160 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid (322 μg, 20%). MS(ES+)m / z 2364.18(M+H) +

[0257] [ka]

[0258] Conjugate 20: The structure of "HABERKORN-ValCit-MMAE". Synthesis of "ESV6-ValCit-MMAE" SH-Cys-Asp-Lys-Asp-ESV6 (700 μg, 0.760 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (840 μL). MC-ValCit-PAB-MMAE (1000 μg, 0.760 μmol, 1.0 equivalent) was added as a dry DMF solution (160 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid (322 μg, 20%). MS(ES+)m / z 2236.07(M+H) +

[0259] [ka]

[0260] Conjugate 21: Structure of "ESV6-ValCit-MMAE". Figure 26 shows the structure, chromatographic profile, and LC / MS analysis of ESV6-ValCit-MMAE(21). MS(ES+)m / z 1118.05(M+2H) 2+ . PART 2 - Animal Experiments Tumor cell preparation After thawing, SK-MEL-187 tumor cells were cultured at 37°C and 5% CO2 in RPMI medium supplemented with fetal bovine serum (10%, FBS) and antibiotic-antifungal agent (1%, AA). For subculturing, when 90% confluence was reached, the cells were detached using 0.05% trypsin-EDTA and re-seeded at a 1:4 dilution. IVIS experiment Tumors transplanted with SK-MEL-187 xenografted were transplanted into female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above and allowed to grow to an average volume of 0.1 mL. Mice carrying subcutaneous SK-MEL-187 tumors were intravenously injected with ESV6-IRDye750, HABERKORN-IRDye750, or QCOOH-IRDye750 (as a 30 μM solution prepared in 150 nmol / Kg sterile PBS, pH 7.4). Mice were anesthetized with isoflurane, and fluorescence images were acquired on an IVIS Spectrum imaging system (Xenogen, exposure 1 s, binning coefficient 8, excitation at 745 nm, emission filter at 800 nm, f / 2, field of view 13.1). Images were taken 5 minutes, 20 minutes, and 1 hour after injection. Food and water were provided without restriction during this period. Mice were subsequently sacrificed by CO2 asphyxiation (at a 2-hour time point). Sections of blood, heart, lungs, kidneys, liver, spleen, stomach, intestines, and SK-MEL-187 tumors were collected and individually imaged using the parameters described above (Figure 7).

[0261] Specifically, Figure 7 shows the evaluation of the targeting performance of the IRDye 750 conjugate in near-infrared fluorescence imaging of BALB / C nu / nu mice carrying SK-MEL-187 melanoma xenografts after intravenous administration (dose of 150 nmol / kg): (A) Images of live animals at various time points (5 minutes, 20 minutes, and 1 hour after injection); (B) Ex vivo organ images at 2 hours. The compound ESV6-IRDye750, a derivative of the high-affinity FAP ligand "ESV6," exhibits higher tumor-to-liver, tumor-to-kidney, and tumor-to-intestinal uptake ratios compared to HABERKORN-IRDye750. QCOOH-IRDye750 (untargeted control) does not localize to SK-MEL-187 lesions in vivo. Treatment experiment Tumors transplanted with SK-MEL-187 xenografted were transplanted into female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above and allowed to grow to an average volume of 0.1 mL. Mice were randomly assigned to three treatment groups. They were injected daily with HABERKORN-ValCit-MMAE (250 nmol / kg) and ESV6-ValCit-MMAE (250 nmol / kg) in a sterile PBS solution containing 1% DMSO for seven consecutive days. Tumors were measured using electronic calipers, and animals were weighed daily. Tumor volume (mm²) 3 The ratio was calculated using the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5 (Figure 8). Prism 6 software (GraphPad Software) was used for data analysis (Bonferroni trial following a standard two-directional ANOVA).

[0262] Specifically, Figure 8 shows the assessment of the therapeutic activity of ESV6-ValCit-MMAE and HABERKORN-ValCit-MMAE in mice carrying SK-MEL-187 tumors. Data points represent mean tumor volume ± SEM (n=3 per group). (A) Arrows indicate IV infection with different treatments. ESV6-ValCit-MMAE, a drug conjugate derivative of the high-affinity FAP ligand "ESV6," exhibits a more potent antitumor effect compared to HABERKORN-ValCit-MMAE. (B) Tolerance of different treatments is shown as determined by the assessment of the change in body weight (%) of the animals during the experiment. ESV6-ValCit-MMAE exhibits lower acute toxicity compared to HABERKORN-ValCit-MMAE.

[0263] Example 5: Preparation of a conjugate for radiolabeling "HABERKORN-DOTA" synthesis (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-6-(3-(piperazine-1-yl)propoxy)quinoline-4-carboxamide (15 mg, 0.030 mol, 1.0 equivalent), HATU (13 mg, 0.039 mmol, 1.1 equivalent), and tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (19 mg, 0.039 mmol, 1.1 equivalent) were dissolved in DCM / MDF (800 μL / 50 μL). DIPEA (18 μL, 0.101 mmol, 3 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature for 1 hour. The crude product was treated overnight with TFA (40%), purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes), and lyophilized to obtain a white solid (4 mg, 15%). MS(ES+)m / z 873.4(M+H) +

[0264] [ka]

[0265] Conjugate 22: The structure of "HABERKORN-DOTA". Synthesis of "ESV6-DOTA" (8) (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (15 mg, 0.032 mmol, 1.0 equivalent) was dissolved in dry DMSO (400 μL). Dicyclohexylcarbodiimide (9 mg, 0.042 mmol, 1.3 equivalents) and N-hydroxysuccinimide (4.5 mg, 0.039 mmol, 1.3 equivalents) were added, and the reaction mixture was stirred overnight at room temperature and protected from light. 100 μL of PBS solution containing 2,2',2”-(10-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (20 mg, 0.039 mmol, 1.2 equivalents) was added, and the reaction mixture was stirred for 2 hours. The crude product was purified by reverse-phase HPLC (0.1% water TFA / 0.1% acetonitrile TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid (2.4 mg, 8%). MS(ES+)m / z 960.39(M+H) +

[0266] [ka]

[0267] Conjugate 8: Structure of "ESV6-DOTA". Figure 27 shows the structure, chromatographic profile, and LC / MS analysis of ESV6-DOTAGA(8). MS(ES+)m / z 960.39(M+H) + .

[0268] Example 6: Comparative experiment between compound P4 and compound 24 PART 1 - Preparation of Compound 24 Synthesis of 7-(phenylamino)quinoline-4-carboxylic acid

[0269] [ka]

[0270] 7-Bromoquinoline-4-carboxylic acid (30 mg, 0.119 mmol, 1.0 equivalent) was added in a pressure vial to a stirred solution of aniline (111 mg, 1.19 mmol, 198 μL, 10.0 equivalents) in toluene (1 mL) and dioxane (500 μL). The solution was degassed for 5 minutes (argon-vacuum cycle), and then BrettPhos Palladacycle (10 mg, 0.0119 mmol, 0.1 equivalent) and potassium tert-butoxide (53 mg, 0.476 mmol, 4.0 equivalents) were added. The reaction mixture was heated at 110°C for 4 hours and checked by LC / MS. The crude product was absorbed onto silica and purified by flash chromatography (DMC / MeOH, 9:1 to 2:8) to obtain the compound as an orange oil (31 mg, 0.119 mmol, 100%). MS(ES+)m / z 265.09(M+H) + Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-7-(phenylamino)quinoline-4-carboxamide (compound 24)

[0271] [ka]

[0272] 7-(phenylamino)quinoline-4-carboxylic acid (31 mg, 0.119 mmol, 1.0 equivalent), (S)-4,4-difluoro-1-glycylpyrrolidine-2-carbonitrile (24 mg, 0.129 mmol, 1.1 equivalent), and HATU (89 mg, 0.234 mmol, 2 equivalents) were added to solution DMF (200 μL) and dichloromethane (1 mL). DIPEA (45 mg, 0.352 mmol, 61 μL, 3 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature for 15 minutes. DCM was evaporated, and the crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes), and lyophilized to obtain a yellow solid (3.5 mg, 0.008 mmol, 6.9%). MS(ES+)m / z 436.15(M+1H) 1+ PART 2 - In Vitro Experiments In vitro inhibition assay for hFAP The enzymatic activity of human FAP against the Z-Gly-Pro-AMC substrate was measured at room temperature on a microtiter plate reader in the presence of different small organic ligands (compound P4 from Example 2; compound 24), and fluorescence was monitored at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The reaction mixture consisted of 20 μM substrate, 20 nM human FAP (constant), assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4), and inhibitor (10 -6 from 10 -11 It contained a series dilution of M (1:2) in a total volume of 20 μL. 50 The value is defined as the concentration of the inhibitor required to reduce enzyme activity by 50% after substrate addition.

[0273] Figure 9 shows that compound P4 from Example 2 exhibits lower IC50 compared to compound 24 (33.46 nM, lower inhibition). 50 This demonstrates (16.83 nM, higher inhibition). Example 7: Synthesis of conjugates 15 and 25 and their characterization PART 1 - Conjugate Preparation Synthesis of Conjugate 15

[0274] [ka]

[0275] SH-Cys-Asp-Lys-Asp-ESV6 (2 mg, 2.171 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (800 μL). Fluorescein-5-maleimide (1.8 mg, 4.343 μmol, 2.0 equivalents) was added as a dry DMSO solution (200 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 after 20 minutes) and lyophilized to obtain a yellow solid (420 nmol, 19.3%). MS(ES+) m / z 1348.36 (M+1H) 1+ Figure 25 shows the structure, chromatographic profile, and LC / MS analysis of conjugate 15. MS(ES+)m / z 1348.36(M+1H) + . Synthesis of Conjugate 25

[0276] [ka]

[0277] SH-Cys-Asp-Lys-Asp-HK (1 mg, 0.954 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (800 μL). Fluorescein-5-maleimide (817 μg, 1.909 μmol, 2.0 equivalents) was added as a dry DMSO solution (200 μL). The reaction mixture was stirred for 3 hours. The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a yellow solid (373 nmol, 39.1%). MS(ES+) m / z 1476.47 (M+1H) 1+ PART 2 - In Vitro Experiments Affinity measurement of human and mouse FAP using fluorescence polarization (FP) Fluorescence polarization experiments were performed in 384-well plates (unbound, ps, f-bottom, black, high volume, final volume 30 μL). Stock solutions of human FAP (4 μM) and mouse FAP (5 μM) were sequentially diluted with buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4) while maintaining a constant final binder concentration at 10 nm. Fluorescence anisotropy was measured on a TECAN microtiter plate reader. Experiments were performed in triplicate, and mean anisotropy values ​​were fitted using prism 7.

[0278] Figure 10A shows that conjugate 15 is connected to conjugate 25(K D Compared to (=1.02nM), hFAP(K D Figure 10B shows that conjugate 15 has a higher affinity for conjugate 25 (K = 0.68 nM). D Compared to mFAP(K = 30.94nM), D It shows higher affinity for (=11.61nM). Conjugate 15 exhibits superior binding properties to hFAP and better cross-reactivity to mouse antigens compared to conjugate 25. Chromatographic co-elution experiments of ligand-protein complexes The PD-10 column was pre-equilibrated with assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4) before loading the complex. Mixtures of different proteins (hFAP=2 μM, mFAP=5 μM) and conjugate 15 (100 nM) were incubated and loaded onto the column. The mixture was flushed using assay buffer. The flow-through was collected in a 96-well plate, and fluorescence intensity was immediately measured on a TECAN microtiter plate reader to monitor fluorescence at the excitation wavelength of 485 nm and the emission wavelength of 535 nm. Protein concentrations were estimated by measuring absorbance at 280 nm using a Nanodrop 2000 / 2000c spectrophotometer.

[0279] Figure 11 shows the results of co-elution PD-10 experiments with small molecule ligand conjugates 15 with hFAP(A) and mFAP(B). A stable complex is formed between both the protein and the small ligand conjugate 15, enabling the co-elution of the two molecules together. PART 3 - Animal Experiments cell culture After thawing, SK-RC-52.hFAP and SK-RC-52 cells were cultured at 37°C and 5% CO2 in RPMI medium supplemented with fetal bovine serum (10%, FBS) and antibiotic-antifungal agent (1%, AA). For subculturing, when 90% confluence was reached, the cells were detached using 0.05% trypsin-EDTA and reseeded at a 1:4 dilution.

[0280] After thawing, HT-1080.hFAP and HT-1080 cells were cultured at 37°C and 5% CO2 in DMEM medium supplemented with fetal bovine serum (10%, FBS) and antibiotic-antifungal agent (1%, AA). For subculturing, when 90% confluence was reached, the cells were detached using 0.05% trypsin-EDTA and reseeded at a 1:4 dilution. Confocal microscopy analysis of SK-RC-52.hFAP, SK-RC-52, HT-1080.hFAP, and HT-1080. SK-RC-52.hFAP and SK-RC-52 cells were placed in RPMI medium supplemented with 10% FCS, AA, and HEPES (10 mM) at a rate of 10 per well. 4 Cells were seeded at a specific cell density in 4-well coverslip chamber plates and grown for 24 hours under standard culture conditions. Hoechst 33342 nuclear dye was used to stain the nuclear structure.

[0281] The culture medium was replaced with fresh medium containing conjugate 15 (100 nM). Randomly selected colonies were imaged on an SP8 confocal microscope equipped with an AOBS (Leica Microsystems) device.

[0282] HT-1080.hFAP and HT-1080 cells were placed in DMEM medium (1 mL) supplemented with 10% FCS, AA, and HEPES (10 mM) at a rate of 10 per well. 4 Cells were seeded at a specific cell density in 4-well coverslip chamber plates and grown for 24 hours under standard culture conditions. Hoechst 33342 nuclear dye was used to stain the nuclear structure.

[0283] The culture medium was replaced with fresh medium containing conjugate 15 (100 nM). Randomly selected colonies were imaged on an SP8 confocal microscope equipped with an AOBS (Leica Microsystems) device.

[0284] Figure 12 shows the evaluation of selective accumulation of conjugate 15 (10 nM) on SK-RC-52.hFAP, HT-1080.hFAP, and wild-type tumor cells via confocal microscopy and FACS analysis. (A) Images of SK-RC-52.hFAP incubated with the compound at different time points (t=0 and 1 hour) show accumulation of conjugate 15 on the cell membrane. (B) Images of SK-RC-52 wild-type after incubation with the compound showed no accumulation on the cell membrane (negative control). (C) FACS analysis of SK-RC-52 wild-type (dark gray peak) and SK-RC-52.hFAP (light gray peak) shows FAP-specific cell binding of conjugate 15 (10 nM). (D) Images of HT-1080.hFAP incubated with the compound at different time points (t=0 and 1 hour) show accumulation of conjugate 15 on the cell membrane and inside the cytosol. (E) Images of HT-1080 wild-type after incubation with compound do not show accumulation on the cell membrane or in the cytosol (negative control). FACS analysis Using Accutase, SK-RC-52.hFAP, SK-RC-52.wt, HT-1080.wt, and HT-1080.hFAP were detached from the culture plate, counted, and 1.5 × 10⁶ units were added to a 1% v / v solution of FCS in PBS pH 7.4. 6Suspended to the final concentration of cells / mL. 3 × 10 5 Aliquots of cells (200 μL) were spun down and resuspended in a solution of conjugate 15 (15 nM) in a 1% v / v solution of FCS in PBS pH 7.4 (200 μL), and incubated on ice for 1 hour. The cells were washed once with 200 μL of a 1% v / v solution of FCS in PBS pH 7.4 (200 μL), spun down, and resuspended in a 1% v / v solution of FCS in PBS pH 7.4 (300 μL), and analyzed on a CytoFLEX cytometer (Beckman Coulter). Raw data were processed with FlowJo 10.4 software.

[0285] The results are shown in Figure 12F: FACS analysis of HT-1080 wild-type (dark gray peak) and HT-1080.hFAP (light gray peak) shows FAP-specific cell binding of conjugate 15 (10 nM). animal research All animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under license number ZH04 / 2018, issued by Veterinaramt des Kantons Zurich. Subcutaneous SK-RC-52.hFAP tumor transplantation SK-RC-52.hFAP cells were grown to 80% confluence and detached with 0.05% trypsin-EDTA. The cells were then placed in HBSS medium in 5 × 10⁻⁶ units. 7 Resuspended to the final concentration of cells / mL. 5 × 10 6 Aliquots of cells (100 μL suspension) were injected subcutaneously into the right flank of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old). Ex vivo experiment Mice carrying subcutaneous SK-RC-52.hFAP tumors were intravenously injected with conjugate 15 (40 nmol in sterile PBS, pH 7.4). The animals were sacrificed by CO2 asphyxiation one hour after intravenous injection, organs and tumors were excised, snap-frozen in OCT medium, and stored at -80°C. Cryostat sections (7 μm) were cut, and the nuclei were stained with Fluorescence Mounting Medium (Dako Omnis, Agilent). Images were obtained using an Axioskop2 mot plus microscope (Zeiss) and analyzed with ImageJ 1.53 software.

[0286] Figure 13 shows the results of evaluating the targeting performance of conjugate 15 in BALB / C nu / nu mice carrying SK-RC-52.hFAP renal cell carcinoma xenografts after intravenous administration (40 nmol). Ex vivo organ images taken one hour after administration are shown. The compound exhibits high tumor-versus-organ selectivity, rapidly and homogeneously localizing to the tumor site in vivo one hour after intravenous injection.

[0287] Example 8: Synthesis and Characterization of Conjugate 9 PART 1 - Conjugate Preparation Synthesis of 177-lutetium-labeled conjugate 9(2,2',2''-(10-(1-carboxy-4-((2-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0288] [ka]

[0289] Conjugate 8 was dissolved in acetate buffer (1M, pH=4) to a final concentration of 1 μg / μL. A stock solution of conjugate 8 (25 μg in 25 μL) was diluted with 250 μL of acetate buffer (1M, pH=4). 177 LuCl3 solution (250 μL, 25 MBq) was added, and the mixture was heated at 95°C for 15 minutes. The labeling mixture was diluted by adding sterile PBS (1975 μL, pH=7.4), and the labeling efficiency was monitored via radiation HPLC (5-10 μL, 0.5-1 MBq, 0.1% TFA in water as solvent A and 0.1% TFA in acetonitrile as solvent B; program: 0-8 min, 20-65% solvent B and flow rate 1 ml / min). Quantitative conversion to conjugate 9 was achieved (radiation labeling efficiency >99%, Figure 14).

[0290] Figure 14A shows, 177 Figure 14B shows the emission HPLC profile (rt11 min) of conjugate 9 after labeling with Lu. 177 The emission HPLC profile of Lu (2 min) is shown. After emission labeling, conjugate 9 appears as a single peak in >99% of the conversions. PART 2 - Animal Experiments Radiolabeling and in vivo distribution experiments of SK-RC-52.hFAP SK-RC-52.hFAP tumor cells were transplanted into female BALB / c nu / nu mice as described in Example 7, and 250 mm 3 The mice were allowed to grow to an average volume for 3 weeks. Mice were randomized (n=4 per group) and intravenously injected with radiolabeled preparations of Conjugate 9 (50, 125, 250, 500 or 1000 nmol / kg; 0.5–2 MBq). Mice were sacrificed by CO2 asphyxiation 10 minutes, 1 hour, 3 hours, and 6 hours after injection, organs were extracted, weighed, and radiometrically measured using a Packard Cobra γ counter. Values ​​are expressed as %ID / g ± SD (Figure 15). Food and water were provided without restriction during the period.

[0291] Figure 15 shows conjugate 9 in BALB / C nu / nu carrying the SK-RC-52.hFAP renal cell carcinoma xenograft (this is, 177 The results of in vivo distribution experiments of Lu (including radioactive payload) are shown. (A) Analysis of %ID / g and tumor-to-organ ratio in tumors and healthy organs at different time points (10 minutes, 1 hour, 3 hours, and 6 hours) after intravenous administration of conjugate 9 (dose = 50 nmol / kg; 0.5~2 MBq). (B) At different doses (125 nmol / kg, 250 nmol / kg, 500 nmol / kg, and 1000 nmol / kg; 0.5~2 MBq) 177 Analysis of %ID / g and tumor-to-organ ratio in tumors and healthy organs 3 hours after intravenous administration of Lu conjugate 9. A dose-dependent response may be observed, and target saturation can be reached between 250 nmol / kg and 500 nmol / kg. (C) 177 Analysis of %ID / g (negative control; 1 MBq) and tumor-to-organ ratio in tumors and healthy organs 3 hours after intravenous administration of Lu solution.

[0292] Example 9: Synthesis of Compounds 27-32 Synthesis of 2,2',2''-(10-(1-carboxy-4-((2-(4-((4-((2-((R)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)ethyl)amino)-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid conjugate 27, labeled with 69-gallium.

[0293] [ka]

[0294] Conjugate 8 was dissolved in acetate buffer (1M, pH=4) to a final concentration of 1 μg / μL. A stock solution of Conjugate 8 (100 μg in 100 μL) was diluted with 250 μL of acetate buffer (1 M, pH=4). GaCl3 solution (183 μg in 183 μL of HCl) was added, and the mixture was heated at 90°C for 15 minutes. The reaction was checked via LC / MS. MS(ES+)m / z 1027.30(M+H) + Synthesis of methyl(6-methoxyquinoline-4-carbonyl)glycinate

[0295] [ka]

[0296] 6-methoxyquinoline-4-carboxylic acid (200 mg, 0.985 mmol, 1.0 equivalent), HBTU (400 mg, 1.03 mmol, 1.05 equivalent), HOBt (167 mg, 1.05 mmol, 1.15 equivalent), and glycine methyl hydrochloride (107 mg, 1.08 mmol, 1.1 equivalent) were dissolved in 5 mL of DMF and stirred at room temperature. DIPEA (613 μL, 4.42 mmol, 4.5 equivalents) was added dropwise, and the reaction was checked by LC / MS until competition occurred. The crude product was directly purified by chromatography (DCM:MeOH, 100:0 to 80:20 at 10 mins) to obtain a pale yellow solid (40 mg, 0.145 mmol, 14.7%). MS(ES+)m / z 275.1(M+1H). Synthesis of (6-methoxyquinoline-4-carbonyl)glycinate

[0297] [ka]

[0298] Methyl(6-methoxyquinoline-4-carbonyl)glycinate (30 mg, 0.109 mmol, 1.0 equivalent) was dissolved in 2 mL of 1 M LiOH solution of THF / H2O (1:1) and stirred at room temperature for 6 hours. Upon completion, the base was quenched with 1 M HCl until a slightly acidic pH was reached, and the crude product was freeze-dried to obtain a white solid (quantitative yield). MS(ES+)m / z 261.08(M+1H) 1+ Synthesis of (S)-N-(2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)-6-methoxyquinoline-4-carboxamide, conjugate 28

[0299] [ka]

[0300] (6-methoxyquinoline-4-carbonyl)glycinate (28 mg, 0.109 mmol, 1.0 equivalent), (S)-pyrrolidine-2-carbonitrile (16 mg, 0.120 mmol, 1.1 equivalent), and HATU (62 mg, 0.164 mmol, 1.5 equivalent) were dissolved in 2 mL of DMF, and the suspension was stirred at room temperature. DIPEA (47 μL, 2.62 mmol, 24 equivalents) was added dropwise, and the reaction was checked by LC / MS until competition occurred. The crude product was directly purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes), and a yellow solid (2 mg, 5.91 μmol, 5.4%) was obtained by lyophilization. MS(ES+)m / z 339.14(M+1H) 1+ Synthesis of tert-butyl((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl)carbamate

[0301] [ka]

[0302] (S)-4,4-difluoropyrrolidine-2-carbonitrile (50 mg, 0.379 mmol, 1 equivalent), (tert-butoxycarbonyl-L-alanine (154 mg, 0.75 mmol, 2.0 equivalents), and HATU (288 mg, 0.75 mmol, 2 equivalents) were dissolved in 4 mL of DMF, and the suspension was stirred at room temperature. DIPEA (335 μL, 1.893 mmol, 5 equivalents) was added dropwise, and the reaction was checked via LC / MS until competition occurred. The DMF was removed under vacuum, and the crude product was diluted in DCM. The organic phase was washed with water and 1 M HCl, and then dried to obtain a white foam (115 mg, 0.379 mmol, quantitative yield). MS(ES+)m / z 304.14(M+1H) 1+ Synthesis of (S)-1-(L-alanyl)-4,4-difluoropyrrolidine-2-carbonitrile

[0303] [ka]

[0304] 115 mg (0.379 mmol, 1 equivalent) of tert-butyl((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl) carbamate was dissolved in 2 mL of DCM, and 203 μL (7 equivalents) of TFA was added dropwise. The reaction mixture was stirred at room temperature and checked by LC / MS until competition occurred. The crude product was diluted in DCM, and the product was extracted with 1 M HCl. The acidic aqueous phase was then quenched with 1 M NaOH, and the product was extracted with DCM and dried to obtain a pale yellow oil (30 mg, 0.147 mmol, 38.7%). MS(ES+)m / z 204.07(M+1H) 1+ Synthesis of tert-butyl((4-(((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridine-2-yl)methyl)carbamate

[0305] [ka]

[0306] (S)-1-(L-alanyl)-4,4-difluoropyrrolidine-2-carbonitrile (30 mg, 0.147 mmol, 1 equivalent), 2-(((tert-butoxycarbonyl)amino)methyl)isonicotinic acid (74 mg, 0.295 mmol, 2.0 equivalents), and HATU (112 mg, 0.295 mmol, 2 equivalents) were dissolved in 1 mL of DMF, and the suspension was stirred at room temperature. DIPEA (102 μL, 0.590 mmol, 4 equivalents) was added dropwise, and the reaction was checked by LC / MS until competition occurred. The DMF was removed under vacuum, and the crude product was diluted in DCM and purified by chromatography (DCM:MeOH, 99:1 to 70:30 at 15 mins) to obtain a yellow solid (15 mg, 0.034 mmol, 19.7%). MS(ES+)m / z 438.19(M+1H) 1+ Synthesis of 2-(aminomethyl)-N-((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl)isonicotinamide

[0307] [ka]

[0308] 15 mg, 0.034 mmol, 1.0 equivalent of tert-butyl((4-(((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridine-2-yl)methyl)carbamate was dissolved in 400 μL of DCM, and 200 μL, 20% by volume of TFA was added dropwise. The reaction mixture was stirred at room temperature and checked by LC / MS until competition occurred. The crude product was dried and freeze-dried in 500 μL of a 1:1 water:acetonitrile solution to obtain a yellow powder (4 mg, 11.86 μmol, 34.8%). MS(ES+)m / z 338.14(M+1H) 1+ Synthesis of 4-(((4-(((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl)carbamoyl)pyridine-2-yl)methyl)amino)-4-oxobutanoic acid conjugate 29

[0309] [ka]

[0310] 2-(aminomethyl)-N-((S)-1-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-1-oxopropan-2-yl)isonicotinamide (4 mg, 11.86 μmol, 1.0 equivalent) was dissolved in 500 μL of THF. DMAP (6 mg, 48 μmol, 4.0 equivalents) and succinic anhydride (3.5 mg, 35.6 μmol, 3.0 equivalents) were added, and the reaction mixture was stirred at room temperature and checked by LC / MS until competition occurred. The crude product was directly purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) and lyophilized to obtain a yellow solid (1.3 mg, 2.97 μmol, 25%). MS(ES+)m / z 438.15(M+1H) 1+ Synthesis of ESV6-Alexa Fluor 488 and conjugate 30

[0311] [ka]

[0312] SH-Cys-Asp-Lys-Asp-ESV6 (293 μg, 0.32 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (300 μL). Alexa Fluor® 488 C5 Maleimide (200 μg, 0.29 μmol, 0.9 equivalents) was added as a dry DMSO solution (200 μL). The reaction mixture was stirred for 3 hours.

[0313] The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 after 20 minutes) and lyophilized to obtain an orange solid (70 nmol, 21.9%). MS(ES+)m / z 1619.39(M+1H) 1+ Synthesis of ESV6-ValCit-PNU 159682 conjugate 31

[0314] [ka]

[0315] SH-Cys-Asp-Lys-Asp-ESV-6 (2 mg, 2.17 μmol, 1.2 equivalents) was dissolved in PBS pH 7.4 (750 μL). MA-PEG4-VC-PAB-DMAE-PNU 159682 (2.5 mg, 1.75 μmol, 1.0 equivalent) was added as a dry DMF solution (250 μL). The reaction mixture was stirred for 3 hours.

[0316] The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid (3 mg, 73%). MS(ES+)m / z 2348.88(M+H) + Synthesis of QCOOH-ValCit-PNU 159682 conjugate 32

[0317] [ka]

[0318] SH-Cys-Asp-Lys-Asp-QCOOH (1.6 mg, 2.17 μmol, 1.2 equivalents) was dissolved in PBS pH 7.4 (750 μL). MA-PEG4-VC-PAB-DMAE-PNU 159682 (2.5 mg, 1.75 μmol, 1.0 equivalent) was added as a dry DMF solution (250 μL). The reaction mixture was stirred for 3 hours.

[0319] The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and freeze-dried to obtain a white solid (2.8 mg, 73%).

[0320] Example 10: Characterization and biological testing of compounds 18, 19, 21, P4, 27, 28, 29, and 30. material and method In vitro inhibition assay for hFAP The enzymatic activity of human FAP against the Z-Gly-Pro-AMC substrate was measured at room temperature using a microtiter plate reader, and fluorescence was monitored at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. The reaction mixture consisted of 20 μM substrate, 20 nM human FAP (constant), assay buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4), and the test compound (10 -6 from 10 -11 It contained a series dilution of M (1:2) in a total volume of 20 μL. 50 The value is defined as the concentration of the inhibitor required to reduce enzyme activity by 50% after substrate addition. cell culture After thawing, SK-MEL-187, SK-RC-52.hFAP, and SK-RC-52.wt cells were cultured at 37°C and 5% CO2 in RPMI medium supplemented with fetal bovine serum (10%, FBS) and antibiotic-antifungal agent (1%, AA). For subculturing, when 90% confluence was reached, the cells were detached using 0.05% trypsin-EDTA and reseeded at a 1:4 dilution.

[0321] After thawing, HT-1080.hFAP and HT-1080.wt cells were cultured at 37°C and 5% CO2 in DMEM medium supplemented with fetal bovine serum (10%, FBS) and antibiotic-antifungal agent (1%, AA). For subculturing, when 90% confluence was reached, the cells were detached using 0.05% trypsin-EDTA and reseeded at a 1:4 dilution. animal research All animal experiments were conducted in accordance with Swiss animal welfare laws and regulations under license number ZH04 / 2018, issued by Veterinaramt des Kantons Zurich. Subcutaneous SK-RC-52.hFAP and HT-1080.hFAP tumor transplantation SK-RC-52.hFAP, HT-1080.hFAP, and SK-RC-52.wt cells were grown to 80% confluence and detached with 0.05% trypsin-EDTA. Cell counts were 5 × 10⁻⁶. 7 The solution was resuspended in HBSS medium at a final concentration of cells / mL. 5 × 10 6 Aliquots of cells (100 μL suspension) were injected subcutaneously into the flanks of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old). SK-MEL-187 cells were grown to 80% confluence and detached with 0.05% trypsin-EDTA. Cells were measured in 10 × 10⁶ units. 7 The cells were resuspended in a 1:1 mixture of HBSS:Matrigel to a final concentration of 5 × 10⁶ cells / mL. 6 Aliquots of cells (200 μL suspension) were injected subcutaneously into the flanks of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old). IVIS experiment In the first experiment, tumors transplanted with HT-1080.hFAP were transplanted into the right flank of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above, and allowed to grow to an average volume of 0.1 mL. Tumors transplanted with SK-RC-52.wt were transplanted into the right flank of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above, and allowed to grow to an average volume of 0.1 mL.

[0322] Mice were intravenously injected with ESV6-IRDye750 (18, 150 nmol / kg, as a 30 μM solution prepared in sterile PBS, pH 7.4). The mice were sacrificed by CO2 asphyxiation (at a 1-hour time point), and fluorescence images of all recovered organs (blood, heart, muscle, lung, kidney, liver, spleen, stomach, intestinal sections, SK-RC-52.wt tumor, and HT-1080.hFAP) were acquired on an IVIS Spectrum imaging system (Xenogen, 1 s exposure, binning coefficient 8, excitation at 745 nm, emission filter at 800 nm, f / 2, field of view 13.1).

[0323] In different experiments, tumors xenografted with SK-MEL-187 were transplanted into the right flank of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above and allowed to grow to an average volume of 0.1 mL. Tumors xenografted with SK-RC-52.hFAP were transplanted into the left flank of female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above and allowed to grow to an average volume of 0.1 mL. ESV6-IRDye750 (18, 150 nmol / kg, as a 30 μM solution prepared in sterile PBS, pH 7.4) was intravenously injected into the mice. Mice were euthanized by CO2 asphyxiation (at a 1-hour time point), and fluorescence images of all recovered organs (blood, heart, muscle, lung, kidney, liver, spleen, stomach, intestinal sections, SK-MEL-187 tumor, and SK-RC-52.hFAP) were acquired on the IVIS Spectrum imaging system (Xenogen, 1s exposure, binning coefficient 8, excitation at 745nm, emission filter at 800nm, f / 2, field of view 13.1). Ex vivo experiment Mice carrying subcutaneous SK-RC-52.hFAP or HT-1080.hFAP tumors on the right flank and SK-RC-52.wt tumors on the left flank were intravenously injected with conjugate 30 (40 nmol in sterile PBS, pH 7.4). Animals were sacrificed by CO2 asphyxiation 1 hour after intravenous injection, organs and tumors were excised, snap-frozen in OCT medium (Thermo Scientific), and stored at -80°C. Cryostat sections (7 μm) were cut, and the nuclei were stained with Fluorescence Mounting Medium (Dako Omnis, Agilent). Images were obtained using an Axioskop2 mot plus microscope (Zeiss) and analyzed with ImageJ 1.53 software. Treatment experiment Tumors transplanted with SK-RC-52.hFAP xenografted were transplanted into female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above, and allowed to grow to an average volume of 0.1 mL.

[0324] Mice were randomly assigned to eight treatment groups of four mice each (five single-drug groups, two combination groups, and a vehicle group). ESV6-ValCit-MMAE (21, 500 nmol / kg) and QCOOH-ValCit-MMAE (19, 500 nmol / kg) were injected as sterile PBS solution in 2% DMSO. L19-IL2 was diluted to 330 μg / mL in a suitable pharmaceutical buffer and administered intravenously at a dose of 2.5 mg / kg.

[0325] Mice in the single-drug group received daily injections of either ESV6-ValCit-MMAE, QCOOH-ValCit-MMAE, or L19-IL2. In the combination group, mice received injections of ESV6-ValCit-MMAE on days 8, 10, and 12 post-tumor transplantation, and injections of L19-IL2 on days 9, 11, and 13 post-tumor transplantation.

[0326] The tumors were measured with electronic calipers, and the animals were weighed daily. Tumor volume (mm³) 3The value was calculated using the formula (long side, mm) × (short side, mm) × (short side, mm) × 0.5. Prism 6 software (GraphPad Software) was used for data analysis (Bonferroni trial followed by standard two-way ANOVA). In-body distribution experiment Tumors transplanted using SK-RC-52.hFAP (right flank) and SK-RC-52.wt (left flank) were transplanted into female thymus-deficient BALB / C nu / nu mice (6-8 weeks old) as described above, and allowed to grow to an average volume of 0.1 mL.

[0327] Mice were injected with ESV6-ValCit-MMAE (21, 250 nmol / kg), and 6 hours after intravenous injection, they were sacrificed by CO2 asphyxiation. Organs and tumors were excised and stored at -80°C. Frozen organs were cut with a scalpel (50 mg) and suspended in 600 μL of 95:5 acetonitrile / water (0.1% TFA). A solution of D8-MMAE in 3:97 acetonitrile / water (0.1% FA; internal standard, 50 μL, 50 nM) was added. The samples were mechanically dissolved using a Tissue Lyser II (Qiagen, 30 Hz shaking, 15 min). Plasma samples (50 μL) were added with a solution of D8-MMAE in 3:97 acetonitrile / water (0.1% FA; internal standard, 50 μL, 50 nM), and the proteins were precipitated by adding 600 μL of 95:5 acetonitrile / water (0.1% TFA). Proteins from both organ and plasma samples were pelletized by centrifugation, and the supernatant was dried in a vacuum centrifuge. The solid residue was resuspended in 1 mL of 3:97 acetonitrile / water (0.1% TFA), and the solution was purified via a first purification step on an HBL Oasis column and a second purification on C18 Macro Spin Columns. The purified and dried eluate was resuspended in 150 μL of 3:97 acetonitrile / water (0.1% FA) and analyzed by MS. All samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using an EASY-nLC 1000 and a fitted Q Exactive Mass Spectrometer (both Thermo Fisher Scientific). Analytes were resolved by performing a linear gradient from 3% to 50% ACN over 60 minutes at a flow rate of 0.3 μL / min using an Acclaim PepMap RSLC C18, 50 μm × 150 mm, 2 μm analytical column (Thermo Fisher Scientific). All buffer solutions contained 0.1% formic acid. MS spectra were recorded in SIM scan mode with a resolution of 70000 and a maximum injection time of 100 ms. MS / MS spectra were recorded with a resolution of 35000 and a maximum injection time of 250 ms.The four SIM windows were placed in the center of the dual and triple-charged ions of ESV6-ValCit-MMAE, 1119.0435 m / z, and 746.3648 m / z, respectively.

[0328] The raw files were then analyzed using Skyline software. The MS1 area of ​​two ions was used for quantification. Mouse serum stability A labeling solution of Conjugate 27 (100 μL, 200 μM) was spiked into 100 μL of mouse serum and incubated at room temperature. Protein precipitation was induced by adding 100 μL of methanol immediately after addition, and again at 10 minutes, 1 hour, 3 hours, and 6 hours. The sample was centrifuged at 16300 rpm for 10 minutes. The supernatant was collected and checked by analytical HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes), and peak identity was determined by LC / MS. result Figure 16 shows the results of evaluating the targeting performance of the IRDye 750 conjugate 18 in near-infrared fluorescence imaging of BALB / C nu / nu mice containing xenografts of SK-MEL-187 (right flank) and SK-RC-52.hFAP (left flank) after intravenous administration (dose of 150 nmol / kg): (A) Images of live animals before injection and 30 minutes after intravenous injection; (B) Ex vivo organ images at 1 hour are shown. Compound ESV6-IRDye750(18) accumulated in both SK-RC-52.hFAP and SK-MEL-187 tumors, showing higher accumulation in SK-RC-52.hFAP tumors compared to SK-MEL-187 due to higher FAP expression.

[0329] Figure 17 shows hFAP inhibition experiments in the presence of different small organic ligands. Conjugate 28 exhibits lower FAP inhibitory properties compared to Example 2, P4. Conjugate 29, including the L-alanine structural unit between the cyanopyrrolidine headpiece and the pyridine ring, does not inhibit FAP proteolytic activity at the concentrations tested in the assay.

[0330] Figure 18 shows the results of evaluating the targeting performance of IRDye 750 conjugate 18 in near-infrared fluorescence imaging of BALB / C nu / nu mice containing HT-1080.hFAP and SK-RC-52.wt xenografts after intravenous administration (dose of 150 nmol / kg). Ex vivo organ images at 1 hour are shown. Compound ESV6-IRDye750(18) selectively accumulates in HT-1080.hFAP tumors exhibiting FAP expression, but does not accumulate in SK-RC-52.wt.

[0331] Figure 19 shows the results of evaluating the targeting performance of conjugate 30 in BALB / C nu / nu mice containing xenografts of SK-RC-52.hFAP (right flank) and SK-RC-52.wt (left flank) after intravenous administration (40 nmol). Ex vivo organ images taken 1 hour after administration are shown. The compound exhibits excellent tumor-versus-organ selectivity, rapidly, homogeneously, and selectively localizing in vivo to tumors expressing FAP 1 hour after intravenous injection. (B) shows the structure of ESV6-Alexa Fluor 488(30).

[0332] Figure 20 shows the results of evaluating the targeting performance of conjugate 30 in BALB / C nu / nu mice containing xenografts of HT-1080.hFAP (right flank) and SK-RC-52.wt (left flank) after intravenous administration (40 nmol). Ex vivo organ images taken 1 hour after administration are shown (A). The compound exhibits excellent tumor-versus-organ selectivity, rapidly, homogeneously, and selectively localizing in vivo in tumors expressing FAP 1 hour after intravenous injection. (B) shows the structure of ESV6-Alexa Fluor 488(30).

[0333] Figure 21 shows the results of the assessment of the therapeutic activity of ESV6-ValCit-MMAE(21) ​​and QCOOH-ValCit-MMAE(19) in SK-RC-52.hFAP tumor-carrying mice (A). Data points represent mean tumor volume ± SEM (n=4 per group). The compounds were administered intravenously (tail vein injection) for 6 consecutive days starting on day 8. ESV6-ValCit-MMAE(21), a drug conjugate derivative of the high-affinity FAP ligand "ESV6," exhibits a more potent antitumor effect compared to QCOOH-ValCit-MMAE(19), the untargeted version of the molecule. (B) shows the tolerability of different treatments as determined by the assessment of the change (%) in body weight of the animals during the experiment. (C) Structures of ESV6-ValCit-MMAE(21) ​​and QCOOH-ValCit-MMAE(19).

[0334] Figure 22 shows the results of the assessment of the therapeutic activity of ESV6-ValCit-MMAE (21), L19-IL2, and combinations thereof in SK-RC-52.hFAP tumor-carrying mice (A). Data points represent mean tumor volume ± SEM (n=4 per group). ESV6-ValCit-MMAE was administered intravenously (tail vein injection) on days 8, 10, and 12. L19-IL2 was administered intravenously (tail vein injection) on days 9, 11, and 13. ESV6-ValCit-MMAE combined with L19-IL2 showed a very potent antitumor effect (4 / 4 complete tumor regression) compared to L19-2 alone. (B) shows the tolerability of different treatments as determined by the assessment of the change (%) in body weight of the animals during the experiment.

[0335] Figure 23 shows the results of quantitative in vivo distribution experiments of the small molecule-drug conjugate ESV6-ValCit-MMAE(21) ​​in BALB / C / nu- / nu mice carrying SK-RC-52.hFAP on the right flank and SK-RC-52.wt on the left flank. The compound selectively accumulates in FAP-positive SK-RC-52 tumors (i.e., 18% ID / g at the tumor site 6 hours after intravenous administration). In contrast, ESV6-ValCit-MMAE does not accumulate in FAP-negative SK-RC-52 wild-type tumors. Uptake of the conjugate in healthy organs is lower (less than 1% ID / g).

[0336] Figure 24 shows conjugate 27 in mouse serum (this is, 69 The results of the stability study (including the Ga payload) are shown. HPLC and LC / MS profiles of the processed sample at time 0 and 6 hours after incubation show a single peak with the correct mass (expected mass: 1028.30; MS(ES+)m / z 514.3(M+2H)).

[0337] Example 11: Synthesis of further conjugates Synthesis of Conjugate 39

[0338] [ka]

[0339] (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (15 mg, 0.032 mmol, 1.0 equivalent) is dissolved in dry DMSO (400 μL). Dicyclohexylcarbodiimide (9 mg, 0.042 mmol, 1.3 equivalents) and N-hydroxysuccinimide (4.5 mg, 0.039 mmol, 1.3 equivalents) are added, and the reaction mixture is stirred overnight at room temperature and protected from light. Add 100 μL of PBS solution containing 2,2'-(7-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (16.2 mg, 0.039 mmol, 1.2 equivalents), and stir the reaction mixture for 2 hours. The crude product is purified by reverse-phase HPLC (0.1% TFA water / 0.1% TFA acetonitrile, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid. MS(ES+)m / z 858.35(M+H) + Synthesis of Conjugate 40

[0340] [ka]

[0341] A solution of Conjugate 39 (1 mL, 150 μM) in sodium acetate buffer (0.1 M, pH 4) was prepared using newly prepared (Al 18 F) 2+ The solution (obtained as previously described in the literature, Cleeren et al., Bioconjugate.Chem.2016) is added to separate vials. The sealed vials are heated at 95°C for 12 minutes. The formation of the complex is confirmed by radiation HPLC and radiation TLC analysis. Synthesis of Conjugate 41

[0342] [ka]

[0343] Dissolve SH-Cys-Asp-Lys-Asp-ESV6 (2 mg, 2.171 μmol, 1.0 equivalent) in PBS pH 7.4 (800 μL). Add maleimide-NOTA (3.0 mg, 4.343 μmol, 2.0 equivalents) as a dry DMSO solution (200 μL). Stir the reaction mixture for 3 hours. Purify the crude material by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and freeze-dry to obtain a yellow solid. MS(ES+)m / z 1345.48(M+1H) 1+ . Synthesis of conjugate 43a

[0344] [ka]

[0345] A commercially available preload of Fmoc-Lys(NeBoc) (300 mg, 0.18 mmol, RAPP Polymere) on Tentagel resin is expanded in DMF (3 × 5 min × 5 mL), the Fmoc groups are removed with 20% piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL), and the resin is washed with DMF (6 × 1 min × 5 mL). The peptide is extended with Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH and (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid in the order shown. For this purpose, Fmoc-protected amino acids (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) are dissolved in DMF (5 mL). The mixture is allowed to stand at 0°C for 10 minutes, and then reacted with the resin for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the Fmoc group is removed with 20% piperidine in DMF (1 × 1 min × 5 min and 2 × 10 min × 5 mL). Following the deprotection step, a washing step using DMF (6 × 1 min × 5 mL) is performed, followed by coupling with the next amino acid. The peptide is cleaved from the resin at room temperature for 1 hour with a mixture of 20% TFA in DCM. The solvent is removed under reduced pressure, the crude product is precipitated in cold diethyl ether, centrifuged, dissolved in water / ACN, purified by HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 5:5 over 15 minutes), and lyophilized to obtain a white solid. The compound is then extracted using 2,3,5,6-tetrafluorophenyl 6-(trimethyl-λ). 4 -Azanyl)nicotinate (2.0 equivalents) is reacted overnight in dry acetonitrile (2 mL). The crude compound is then [ 18 The final compound is obtained by reacting [F]TBAF (2.0 equivalents), TBAHCO3 (2.0 equivalents), and a mixture of tBuOH:MeOH (5:2) at 50°C for 10 minutes. MS(ES+)m / z 967.33(M+1H) 1+ Synthesis of Cys(STrt)-Cys(STrt)-Asp(OtBu)-Lys(NHBoc)-Asp(OtBu)-NHFmoc on resin

[0346] [ka]

[0347] A commercially available preload of Fmoc-Cys(Trt) (500 mg, 0.415 mmol, RAPP Polymere) on Tentagel resin was expanded in DMF (3 × 5 min × 5 mL), the Fmoc group was removed with 20% piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL), and the resin was washed with DMF (6 × 1 min × 5 mL). The peptide was extended with Fmoc-Cys(Trt), Fmoc-Asp(tBu)-OH, Fmoc-Lys(NHBoc)-OH, and Fmoc-Asp(tBu)-OH in the order shown. For this purpose, Fmoc-protected amino acids (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) were dissolved in DMF (5 mL). The mixture was allowed to stand at 0°C for 10 minutes, and then reacted with the resin for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the Fmoc group was removed with 20% piperidine in DMF (1 × 1 min × 5 min and 2 × 10 min × 5 mL). Following the deprotection step, a washing step with DMF (6 × 1 min × 5 mL) was performed, followed by coupling with the next amino acid. Synthesis of (2R,5R,8S,11S,14S)-11-(4-aminobutyl)-8-(carboxymethyl)-14-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanamide)-2,5-bis(mercaptomethyl)-4,7,10,13-tetraoxo-3,6,9,12-tetraazahexadecanedioic acid (SH-Cys-SH-Cys-Asp-Lys-Asp-ESV6, P7)

[0348] [ka]

[0349] Cys(STrt)-Cys(STrt)-Asp(OtBu)-Lys(NHBoc)-Asp(OtBu)-NHFmoc (80 mg, 0.04 mmol) on a resin was expanded in DMF (3 × 5 min × 5 mL). The peptide was extended with (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (37 mg, 0.08 mmol, 2 equivalents), HATU (30 mg, 0.08 mmol, 2.0 equivalents), and DIPEA (28 μL, 0.16 mmol, 4.0 equivalents), and allowed to react for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the resin was cleaved by stirring at room temperature for 4 hours with a mixture of TFA (15%), TIS (2.5%), and H2O (2.5%) in DCM. The resin was washed with methanol (2 × 5 mL), and the combined cleavage solution and washing solution were concentrated under vacuum. The crude product was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) and lyophilized to obtain a white solid (8 mg, 0.95 μmol, 2.4%). MS(ES+) m / z 1024.28(M+H) + Synthesis of ESV6-ValCit-MMAE-bis(44)

[0350] [ka]

[0351] Dissolve SH-Cys-SH-Cys-Asp-Lys-Asp-ESV6 (P7, 1.2 mg, 1.175 μmol, 1.0 equivalent) in PBS pH 7.4 (840 μL). Add MC-ValCit-PAB-MMAE (4.6 mg, 3.525 μmol, 3.0 equivalent) as a dry DMF solution (160 μL). Stir the reaction mixture for 3 hours.

[0352] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a white solid. MS(ES+)m / z 3656.9(M+H) + Synthesis of ESV6_2-ValCit-MMAE-bis(45)

[0353] [ka]

[0354] SH-Cys-Asp-Lys-Asp-ESV6 (P7, 1 mg, 1.09 μmol, 1.0 equivalent) is dissolved in PBS pH 7.4 (840 μL). MC-ValCit-PAB-MMAE (1.4 mg, 1.09 μmol, 1.0 equivalent) and OSu-Glu-ValCit-PAB-MMAE (1.4 mg, 1.09 μmol, 1.0 equivalent) are added as dry DMF solution (160 μL). The reaction mixture is stirred for 3 hours. The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a white solid. MS(ES+)m / z 3456.8(M+H) + Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-8-(hexa-5-inamide)quinoline-4-carboxamide (P8)

[0355] [ka]

[0356] 5-hexic acid (94 mg, 0.84 mmol, 1.5 equivalents) was dissolved in 1.5 mL of DCM and 20 μL of DMF in a 25 mL round-bottom flask. The mixture was cooled to 0°C, and oxalyl chloride (107 mg, 0.84 mmol, 1.5 equivalents) was added dropwise. The ice bath was removed, and the reaction mixture was stirred for 15 minutes. The mixture was then added to the cooled solution of Example 2-P4 in DMF. After 30 minutes, the crude product was diluted with an aqueous solution of NaHCO3, extracted with DCM, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by chromatography (DCM / MeOH, 100:0 to 90:10 in 10 minutes) to obtain a yellow oil (78 mg, 0.267 mmol, 34%). MS(ES+)m / z 454.16(M+1H) 1+ Synthesis of (S)-8-(4-(1-(5-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)butanamide)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide (P9)

[0357] [ka]

[0358] A commercially available preload of amino-PEG2 (300 mg, 0.2 mmol, Novabiochem) on Tentagel resin was expanded in DMF (3 × 5 min × 5 mL). The resin was stretched with 5-azidovaleric acid (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) in DMF (5 mL). The mixture was allowed to stand at 0°C for 10 minutes, and then reacted with the resin for 1 hour with gentle stirring. (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-8-(hexa-5-inamide)quinoline-4-carboxamide (78 mg, 0.17 mmol, 0.86 equivalents), CuI (4 mg, 0.02 mmol, 0.1 equivalent), and TBTA (34 mg, 0.06 mmol, 0.3 equivalents) were dissolved in 5 mL of a 1:1 mixture of DMF / THF.

[0359] The peptide was cleaved from the resin at room temperature for 1 hour in a mixture of 20% TFA in DCM. The solvent was removed under reduced pressure, the crude product was precipitated in cold diethyl ether, centrifuged, dissolved in water / ACN, and purified via HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 5:5 over 15 minutes). Lyophilization yielded a white solid (30 mg, 21%). MS(ES+)m / z 727.8(M+H) + Synthesis of Conjugate 46

[0360] [ka]

[0361] (S)-8-(4-(1-(5-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-5-oxopentyl)-1H-1,2,3-triazole-4-yl)butanamide)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)quinoline-4-carboxamide (1 mg, 1.4 μmol, 1.0 equivalent) was dissolved in THF (200 μL), and dried DIPEA (400 μg, 3.3 μmol, 2.4 equivalents) was added dropwise. FITC isomer I (0.8 mg, 2.1 μmol, 1.5 equivalents) was added as DMSO solution at a concentration of 1 mg in 20 μL. The reaction mixture was stirred for 3 hours, protected from light, and the crude material was directly purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain a yellow powder (1.1 mg, 70.4%). MS(ES+)m / z 1116.4(M+H) + Synthesis of (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-8-(hexa-5-inamide)quinoline-4-carboxamide (P10)

[0362] [ka]

[0363] (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (50 mg, 0.11 mmol, 1 equivalent), propargylamine (7 mg, 0.13 mmol, 1.2 equivalents), and HATU (49 mg, 0.13 mmol, 1.2 equivalents) were dissolved in 2 mL of DCM and 100 μL of DMF. DIPEA (56 mg, 0.44 mmol, 4 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature for 30 minutes. Water was added, and the mixture was separated from the organic layer, then extracted three times with DCM. The crude product was dried over sodium sulfate, filtered, and evaporated. Dark oil was obtained by purifying the crude product via chromatography (DCM / MeOH, 100:0 to 95:5 in 10 minutes) (32 mg, 0.0638 mmol, 58%). MS(ES+)m / z 495.47(M+1H) 1+ Synthesis of Bi-ESV6-peptide (P11)

[0364] [ka]

[0365] A commercially available preload of Fmoc-Cys(Trt) (300 mg, 0.18 mmol, RAPP Polymere) on Tentagel resin was expanded in DMF (3 × 5 min × 5 mL), the Fmoc groups were removed with 20% piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL), and the resin was washed with DMF (6 × 1 min × 5 mL). The peptides were extended with Fmoc-Asp(tBu)-OH, Fmoc-Lys(NHBoc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-N3-Lys, Fmoc-Asp(tBu)-OH and (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid in the order shown. For this purpose, Fmoc-protected amino acids (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) were dissolved in DMF (5 mL). The mixture was allowed to stand at 0°C for 10 minutes, and then reacted with the resin for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the Fmoc group was removed with 20% piperidine in DMF (1 × 1 min × 5 min and 2 × 10 min × 5 mL). Following the deprotection step, a washing step with DMF (6 × 1 min × 5 mL) was performed, followed by coupling with the following amino acids: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-8-(hexa-5-inamide)quinoline-4-carboxamide (174 mg, 0.35 mmol, 2 equivalents), CuI (4 mg, 0.02 mmol, 0.1 equivalent), and TBTA (28 mg, 0.05 mmol, 0.3 equivalents) were dissolved in 5 mL of a 1:1 mixture of DMF / THF. The peptide was cleaved from the resin with a mixture of 20% TFA in DCM at room temperature for 1 hour. The solvent was removed under reduced pressure, the crude product was precipitated in cold diethyl ether, centrifuged, dissolved in water / ACN, purified by HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 5:5 over 15 minutes), and lyophilized to obtain a white solid (18 mg, 6%). MS(ES+)m / z 1687.7(M+H) + Synthesis of Conjugate 47

[0366] [ka]

[0367] Dissolve Bi-ESV6-peptide (P11, 1 mg, 0.59 μmol, 1.0 equivalent) in PBS pH 7.4 (840 μL). Add maleimide-fluorescein (0.76 mg, 1.77 μmol, 3.0 equivalents) as a dry DMF solution (160 μL). Stir the reaction mixture for 3 hours.

[0368] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a yellow solid. MS(ES+)m / z 2114.7(M+H) + Synthesis of Conjugate 48

[0369] [ka]

[0370] Dissolve Bi-ESV6 peptide (P11, 1 mg, 0.59 μmol, 1.0 equivalent) in PBS pH 7.4 (300 μL). Add Alexa Fluor® 488 C5 Maleimide (200 μg, 0.29 μmol, 0.5 equivalent) as a dry DMSO solution (200 μL). Stir the reaction mixture for 3 hours.

[0371] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain an orange solid. MS(ES+)m / z 2385.8(M+1H) 1+ Synthesis of Conjugate 49

[0372] [ka]

[0373] Dissolve Bi-ESV6-peptide (P11, 1 mg, 0.59 μmol, 1.0 equivalent) in PBS pH 7.4 (840 μL). Add MC-ValCit-PAB-MMAE (1 mg, 0.76 μmol, 1.3 equivalents) as a dry DMF solution (160 μL). Stir the reaction mixture for 3 hours.

[0374] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a white solid. MS(ES+)m / z 3003.5(M+H) + Synthesis of Conjugate 50

[0375] [ka]

[0376] Dissolve Bi-ESV6 peptide (P11, 1 mg, 0.59 μmol, 3.3 equivalents) in PBS pH 7.4 (300 μL). Add IRDye750 (200 μg, 0.174 μmol, 1.0 equivalent) as a dry DMSO solution (200 μL). Stir the reaction mixture for 3 hours.

[0377] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain an orange solid. MS(ES+)m / z 2838.0(M+1H) 1+ Synthesis of Conjugate 51

[0378] [ka]

[0379] Dissolve Bi-ESV6 peptide (P11, 1 mg, 0.59 μmol, 1 equivalent) in PBS pH 7.4 (300 μL). Add maleimide-DOTA (465 μg, 0.59 μmol, 1.0 equivalent) as a dry DMSO solution (200 μL). Stir the reaction mixture for 3 hours.

[0380] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain an orange solid. MS(ES+)m / z 2213.9(M+1H) 1+ Synthesis of AlbuTag-ESV6-peptide (P12)

[0381] [ka]

[0382] A commercially available preload of Fmoc-Cys(Trt) (300 mg, 0.18 mmol, RAPP Polymere) on Tentagel resin was expanded in DMF (3 × 5 min × 5 mL), the Fmoc groups were removed with 20% piperidine in DMF (1 × 1 min × 5 mL and 2 × 10 min × 5 mL), and the resin was washed with DMF (6 × 1 min × 5 mL). The peptides were extended with Fmoc-Asp(tBu)-OH, Fmoc-Lys(NHBoc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-N3-Lys, Fmoc-a(tBu)-Asp-OH, Fmoc-Glu(tBu)-OH, and 4-(4-iodophenyl)butanoic acid in the order shown. For this purpose, Fmoc-protected amino acids (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) were dissolved in DMF (5 mL). The mixture was allowed to stand at 0°C for 10 minutes, and then reacted with the resin for 1 hour with gentle stirring. After washing with DMF (6 × 1 min × 5 mL), the Fmoc group was removed with 20% piperidine in DMF (1 × 1 min × 5 min and 2 × 10 min × 5 mL). Following the deprotection step, a washing step using DMF (6 × 1 min × 5 mL) was performed, followed by coupling with the following amino acid: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-8-(hexa-5-inamide)quinoline-4-carboxamide (174 mg, 0.35 mmol, 2 equivalents), CuI (4 mg, 0.02 mmol, 0.1 equivalent), and TBTA (28 mg, 0.05 mmol, 0.3 equivalents) were dissolved in 5 mL of a 1:1 mixture of DMF / THF. The peptides were cleaved from the resin at room temperature for 1 hour in a mixture of 20% TFA in DCM. The solvent was removed under reduced pressure, the crude product was precipitated in cold diethyl ether, centrifuged, dissolved in water / ACN, purified by HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 5:5 over 15 minutes), and lyophilized to obtain a white solid (6 mg, 2%). MS(ES+)m / z 1646.6(M+H) + Synthesis of Conjugate 52

[0383] [ka]

[0384] AlbuTag-ESV6-peptide (P12, 1 mg, 0.61 μmol, 1.0 equivalent) is dissolved in PBS pH 7.4 (840 μL). Maleimide-fluorescein (0.78 mg, 1.82 μmol, 3.0 equivalents) is added as a dry DMF solution (160 μL). The reaction mixture is stirred for 3 hours.

[0385] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a yellow solid. MS(ES+)m / z 2073.6(M+H) + Synthesis of Conjugate 53

[0386] [ka]

[0387] AlbuTag-ESV6 peptide (P12, 1 mg, 0.61 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (300 μL). Alexa Fluor® 488 C5 Maleimide (400 μg, 0.58 μmol, 0.95 equivalents) was added as a dry DMSO solution (200 μL). The reaction mixture was stirred for 3 hours.

[0388] The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 after 20 minutes) and lyophilized to obtain an orange solid (180 nmol, 30%). MS(ES+)m / z 2345.7(M+1H) 1+ Synthesis of Conjugate 54

[0389] [ka]

[0390] Dissolve AlbuTag-ESV6 peptide (P12, 1 mg, 0.61 μmol, 1.0 equivalent) in PBS pH 7.4 (840 μL). Add MC-ValCit-PAB-MMAE (1 mg, 0.76 μmol, 1.25 equivalents) as a dry DMF solution (160 μL). Stir the reaction mixture for 3 hours.

[0391] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a white solid. MS(ES+)m / z 2963.8(M+H) + Synthesis of Conjugate 55

[0392] [ka]

[0393] AlbuTag-ESV6 peptide (P12, 1 mg, 0.61 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (300 μL). IRDye750 (400 μg, 0.384 μmol, 0.58 equivalents) was added as a dry DMSO solution (200 μL). The reaction mixture was stirred for 3 hours.

[0394] The crude material was purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and lyophilized to obtain an orange solid (55 nmol, 9%). MS(ES+)m / z 2797.9(M+1H) 1+ Synthesis of Conjugate 56

[0395] [ka]

[0396] AlbuTag-ESV6 peptide (P12, 1 mg, 0.61 μmol, 1.0 equivalent) is dissolved in PBS pH 7.4 (300 μL). Maleimide-DOTA (480 μg, 0.61 μmol, 1.0 equivalent) is added as a dry DMSO solution (200 μL). The reaction mixture is stirred for 3 hours.

[0397] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain an orange solid. MS(ES+)m / z 2172.7(M+1H) 1+ Synthesis of Bi-ESV6(P13)

[0398] [ka]

[0399] Expand a commercially available 2-chlorotrityl chloride resin (300 mg) in DMF (3 × 5 min × 5 mL). Extend the resin with NHFmoc-azidrolynthetic acid (1 mmol), HBTU (1.0 equivalent), HOBt (1.0 equivalent), and DIPEA (2.0 equivalents) in DMF (5 mL). Allow the mixture to stand at 0°C for 10 minutes, then react with the resin for 1 hour with gentle stirring. Wash the resin with methanol. Extend the resin with (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (1 mmol), HOBt (1.0 equivalent), and DIPEA (2.0 equivalents) in DMF (5 mL). (S)-N-(2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)-8-(hexa-5-inamide)quinoline-4-carboxamide (78 mg, 0.17 mmol, 0.86 equivalents), CuI (4 mg, 0.02 mmol, 0.1 equivalent), and TBTA (34 mg, 0.06 mmol, 0.3 equivalents) are dissolved in 5 mL of a 1:1 mixture of DMF / THF. The peptides are cleaved from the resin at room temperature for 1 hour in a mixture of 50% HFIP in DCM. The solvent is removed under reduced pressure, the crude product is precipitated in cold diethyl ether, centrifuged, dissolved in water / ACN, purified by HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 5:5 over 15 minutes), and lyophilized to obtain a white solid. MS(ES+)m / z 1111.1(M+H) + Synthesis of DOTA-GA-Bi-ESV6(57)

[0400] [ka]

[0401] Dissolve Bi-ESV6 (P13, 45 mg, 40.5 μmol, 1.0 equivalent) in dry DMSO (400 μL). Add dicyclohexylcarbodiimide (10.9 mg, 52.7 μmol, 1.3 equivalents) and N-hydroxysuccinimide (14 mg, 122 μmol, 3 equivalents), and stir the reaction mixture overnight at room temperature, protecting it from light.

[0402] A 100 μL solution of PBS containing 2,2',2”-(10-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (25 mg, 48.6 μmol, 1.2 equivalents) was added, and the reaction mixture was stirred for 2 hours.

[0403] The crude product is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and freeze-dried to obtain a white solid. MS(ES+)m / z 1624.8(M+H) + General procedure for the synthesis of MC-amino acids (OtBu)

[0404] [ka]

[0405] The general procedure described above can be carried out, for example, for AA=glycine, as shown below. 6-Maleimidohexanoic acid (1.0 equivalent) was dissolved in dry CH2Cl2 (1 mL / mmol) under an argon atmosphere, and the solution was cooled to 0°C. EDC·HCl (1.1 equivalents), DIPEA (2.3 equivalents), and the desired H-AA-OtBu·HCl (1.1 equivalents) were subsequently added. The reaction mixture was stirred at room temperature until completion. The mixture was diluted with AcOEt and washed with 1 M aqueous KHSO4 solution, saturated NaHCO3 solution, and brine. The organic phase was dried and concentrated to obtain the desired product as a white powder. General procedure for MC-amino acid synthesis

[0406] [ka]

[0407] The general procedure described above can be carried out, for example, for R, which corresponds to AA = glycine, as shown below. The desired MC-amino acid-OtBu (1.0 equivalent) is dissolved in dry CH2Cl2 (0.3 mL / mmol) under an argon atmosphere. TFA (22.0 equivalents) is added, and the mixture is stirred at room temperature for 2 hours. The solution is concentrated and precipitated with hexane to obtain the product as a white powder. (9H-fluoren-9-yl)methyl(2S)-2-((4-(hydroxymethyl)phenyl)carbamoyl)-1λ 4 Synthesis of pyrrolidine-1-carboxylate

[0408] [ka]

[0409] Fmoc-Pro-OH (312 mg; 0.92 mmol; 1.0 equivalent) and HATU (393 mg; 1.01 mmol; 1.1 equivalent) were dissolved in dry DMF (4 mL) under an argon atmosphere, and the solution was cooled to 0°C. DIPEA (505 μL; 2.76 mmol; 3.0 equivalents) was added dropwise, and the mixture was stirred at the same temperature for 15 minutes. 4-aminobenzyl alcohol (226 mg, 1.84 mmol, 2 equivalents) was added as a solution in dry DMF. The mixture was stirred overnight at room temperature. The reaction mixture was diluted with AcOEt and washed with 1 M KHSO4 aqueous solution, saturated NaHCO3, and brine. The pooled organic phase was dried and concentrated under vacuum. The crude product was purified by chromatography (DCM / MeOH, 99:1 to 95:5 in 10 minutes), yielding a white powder (274 mg; 0.66 mmol; 66% yield). MS(ES+)m / z 443.19(M+1H) 1+ Synthesis of (S)-N-(4-(hydroxymethyl)phenyl)pyrrolidine-2-carboxyamide

[0410] [ka]

[0411] (9H-fluoren-9-yl)methyl(2S)-2-((4-(hydroxymethyl)phenyl)carbamoyl)-1λ 4 -Pyrrolidine-1-carboxylate (274 mg; 0.66 mmol) is dissolved in dry DMF (30 mL) under an argon atmosphere and cooled to 0°C. Piperidine (325 μL; 3.29 mmol) is added and the mixture is stirred at room temperature for 1 hour. The solution is concentrated under high vacuum, dissolved in AcOEt (100 mL), and washed with aqueous NaHCO3 solution and brine. The organic phase is dried and concentrated under vacuum. The crude material is purified by chromatography (99:1 to 90:10 DCM / MeOH with 0.5% TEA) to obtain the product as a brown oil. MS(ES+)m / z 221.12(M+1H) 1+ General procedure for the synthesis of MC-AA-Pro-PAB

[0412] [ka]

[0413] The desired MC-amino acid (1.1 equivalents) is dissolved in dry DMF (0.5 mL / mmol) under an argon atmosphere, and the solution is cooled to 0°C. HATU (1.1 equivalents), (S)-N-(4-(hydroxymethyl)phenyl)pyrrolidine-2-carboxamide (1.0 equivalent), and DIPEA (1.5 equivalents) are then added. The reaction mixture is allowed to slowly reach room temperature and stirred overnight. The mixture is diluted with AcOEt and washed with 1 M KHSO4 and brine. The organic phase is dried, concentrated under vacuum, and the crude product is purified by chromatography (DCM / MeOH 93:7) to obtain the desired MC-AA-Pro-PAB. General procedure for the synthesis of MC-AA-Pro-PAB-PNP

[0414] [ka]

[0415] A solution of 4-nitrophenyl chloroformate (2.2 equivalents) in dry CH2Cl2 (1 mL / mmol) is added under an argon atmosphere to a suspension of the desired MC-AA-Pro-PAB (1.0 equivalent) in dry CH2Cl2 (1 mL / mmol) and pyridine (1.5 equivalents). After completion, the solvent is removed under vacuum, and the crude mixture is filtered over a silica pad that elutes with AcOEt to obtain MC-AA-Pro-PAB-PNP. General procedure for the synthesis of MC-AA-Pro-PAB-MMAE

[0416] [ka]

[0417] Monomethyl auristatin E (MMAE·TFA 1.1 equivalents) is dissolved in dry DMF (200 μL) under a nitrogen atmosphere. MC-AA-Pro-PAB-PNP (1.0 equivalent), HOAt (0.5 equivalents), and DIPEA (5.0 equivalents) are then added. The mixture is stirred at room temperature for 48 hours and concentrated under vacuum. The crude product is diluted 1:1 with water / methanol in 200 μL of the mixture and purified over reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) to obtain the desired product as a white solid. General procedure for the synthesis of ESV6-AA-Pro-MMAE(58)

[0418] [ka]

[0419] Dissolve SH-Cys-Asp-Lys-Asp-ESV6 (P7, 700 μg, 0.760 μmol, 1.0 equivalent) in PBS pH 7.4 (840 μL). Add MC-AA-Pro-PAB-MMAE (1.0 equivalent) as a dry DMF solution (160 μL). Stir the reaction mixture for 3 hours.

[0420] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a white solid.

[0421] The AA, m / z, and yield of the derivatives used are listed in the table below.

[0422] [Table 5]

[0423] General procedure for the synthesis of Py-SS-MMAE

[0424] [ka]

[0425] Monomethyl auristatin E (MMAE·TFA 1.1 equivalents) is dissolved in dry DMF (200 μL) under a nitrogen atmosphere. Various substituted 4-nitrophenyl (2-(pyridine-2-yldisulfanyl)ethyl) carbonate (1.0 equivalent), HOAt (0.5 equivalents), and DIPEA (5.0 equivalents) are then added. The mixture is stirred at room temperature for 48 hours and concentrated under vacuum. The crude product is diluted with 200 μL of the mixture in a 1:1 water / methanol ratio and purified on reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 mins) to obtain the desired product as a white solid. General procedure for the synthesis of ESV6-SS-MMAE(59)

[0426] [ka]

[0427] Dissolve SH-Cys-Asp-Lys-Asp-ESV6 (P7, 700 μg, 0.760 μmol, 1.0 equivalent) in PBS pH 7.4 (840 μL). Add the desired Py-SS-MMAE (1.0 equivalent) as a dry DMF solution (160 μL). Stir the reaction mixture for 3 hours.

[0428] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a white solid.

[0429] The R group used in the derivatives, m / z, and yield are listed in the table below.

[0430] [Table 6]

[0431] Synthesis of PenCys-Asp-Lys-Asp-ESV6 peptide (P14)

[0432] [ka]

[0433] A commercially available 2-chlorotrityl chloride resin (300 mg) is expanded in DMF (3 × 5 min × 5 mL). The resin is extended with Fmoc-PenCys(Trt), Fmoc-Asp(tBu)-OH, Fmoc-Lys(NHBoc)-OH, and Fmoc-Asp(tBu)-OH in the order shown. For this purpose, Fmoc-protected amino acids (2.0 equivalents), HBTU (2.0 equivalents), HOBt (2.0 equivalents), and DIPEA (4.0 equivalents) are dissolved in DMF (5 mL). The resin is extended in DMF (5 mL) with (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (1 mmol), HBTU (2.0 equivalents), and DIPEA (2.0 equivalents). The peptide is cleaved from the resin at room temperature for 1 hour in a mixture of 50% HFIP in DCM. The solvent is removed under reduced pressure, the crude product is precipitated in cold diethyl ether, centrifuged, dissolved in water / ACN, purified by HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 5:5 over 15 mins), and lyophilized to obtain a white solid. MS(ES+)m / z 949.3(M+H) + General procedure for the synthesis of PenESV6-SS-MMAE(60)

[0434] [ka]

[0435] PenCys-Asp-Lys-Asp-ESV6 (700 μg, 0.760 μmol, 1.0 equivalent) was dissolved in PBS pH 7.4 (840 μL). The desired Py-SS-MMAE (1.0 equivalent) was added as a dry DMF solution (160 μL). The reaction mixture was stirred for 3 hours.

[0436] The crude material is purified by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and then freeze-dried to obtain a white solid.

[0437] The R group and m / z values ​​used in the derivatives are listed in the table below.

[0438] [Table 7]

[0439] (S)-N 1 -(4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)-N 4 Synthesis of -(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)succinimide (P15)

[0440] [ka]

[0441] (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)amino)-4-oxobutanoic acid (65 mg, 0.14 mmol, 1 equivalent), HATU (54 mg, 0.14 mmol, 1 equivalent), and 1-(2-aminoethyl)maleimide HCl (25 mg, 0.14 mmol, 1 equivalent) were dissolved in 1.5 mL of DCM and 500 μL of DMF. DIPEA (54 mg, 0.43 mmol, 3 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature for 30 minutes. Water was added, and the mixture was separated from the organic layer, then extracted three times with DCM. The crude product was dried over sodium sulfate, filtered, and evaporated. Purification of the crude product via chromatography (DCM / MeOH, 100:0 to 95:5 in 10 minutes) yielded a yellow oil (50 mg, 0.0868 mmol, 62%). MS(ES+)m / z 582.6(M+1H) 1+ Synthesis of Conjugate 66

[0442] [ka]

[0443] Dissolve SH-Cys-Asp-Lys-Asp-ESV6 (2 mg, 2.171 μmol, 1.0 equivalent) in PBS pH 7.4 (800 μL). Add maleimide-NODAGA (3.3 mg, 4.343 μmol, 2.0 equivalents) as a dry DMSO solution (200 μL). Stir the reaction mixture for 3 hours. Purify the crude material by reverse-phase HPLC (water 0.1% TFA / acetonitrile 0.1% TFA, 9.5:0.5 to 2:8 over 20 minutes) and freeze-dry to obtain a yellow solid. MS(ES+)m / z 1346.36(M+1H) 1+ General synthesis of protein-OncoFAP

[0444] [ka]

[0445] The protein is reduced overnight at 4°C using 30 equivalents of TCEP-HCl per cysteine ​​residue. The product is purified via FPLC, and the fraction containing the product is integrated. The reduced protein is then treated with (S)-N at a rate of 20 equivalents per cysteine ​​residue. 1 -(4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)quinoline-8-yl)-N 4 -(2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)ethyl)succinamide is reacted with gentle shaking at room temperature for 1 hour.

[0446] The product is then purified via FPLC and analyzed by LC / MS to confirm its identity. The described protocol allows for the functionalization of proteins, enzymes, targets, antibodies, immune cytokines, pro-inflammatory proteins, and peptides containing one or more cysteine ​​residues.

[0447] Further examples of protein conjugates according to the present invention are: proteins coupled to FAP targeting agents (e.g., conjugates 63, 63a), and proteins coupled to multiple FAP targeting agents (e.g., 69, 69a), as illustrated below. The ball represents the comprehensive protein structure.

[0448] [ka]

Claims

1. A compound represented by one of the following structures, its individual diastereoisomers, its hydrate, its solvate, its crystalline form, its individual tautomers, or any pharmaceutically acceptable salt thereof: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 。

2. The compound according to claim 1, wherein the structure is selected from those represented by numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 21, 26, 27, 30, 31, 34a, 35, 36, 37a, 38, 39, 40, 41, 42, 43, 43a, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58a, 58b, 58c, 58d, 58e, 58f, 59a, 60a, 64, 65, 66, 67, and 68.

3. The compound according to claim 1, wherein the structure is selected from those represented by numbers 2, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 21, 26, 27, 30, 31, 34a, 35, 36, 37a, 38, 39, 40, 41, 42, 43, 43a, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58a, 58b, 58c, 58d, 58e, 58f, 59a, 60a, 64, 65, 66, 67, and 68.

4. The compound according to claim 1, wherein the structure is selected from those represented by numbers 8, 9, 10, 11, 12, 13, 14, 15, 18, 21, 26, 27, 30, 35, 38, 39, 40, 41, 42, 43a, 58a, 58b, 58c, 59a, 60a, 64, 65, 66, 67, and 68.

5. The compound according to claim 1, wherein the structure is selected from those represented by numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 26.

6. The compound according to claim 1, represented by structure 8.

7. A compound represented by one of the following structures, its individual diastereoisomers, its hydrate, its solvate, its crystalline form, its individual tautomers, or any pharmaceutically acceptable salt thereof: Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 2-6 Table 2-7 Table 2-8 Table 2-9 。

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