RU2836270C2 - Compounds containing fibroblast activation protein ligand, and use thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a compound comprising a cyclic peptide and a chelator, a radionuclide chelate complex, pharmaceutical composition for inhibiting enzymatic activity of fibroblast activation protein (FAP) and use thereof for treating a disease, where the disease is cancer or tumour.

EFFECT: disclosed are compounds containing a fibroblast activation protein ligand and use thereof.

48 cl, 12 dwg, 21 tbl, 16 ex

Description

FIELD OF INVENTION

The present invention relates to a chemical compound; a fibroblast activation protein (FAP) inhibitor; a composition comprising the compound and the inhibitor, respectively; a compound, an inhibitor, and a composition, respectively, for use in a method of diagnosing a disease; a compound, an inhibitor, and a composition, respectively, for use in a method of treating a disease; a compound, an inhibitor, and a composition, respectively, for use in a method of diagnosing and treating a disease, which is also referred to as "thera(g)nosis" or "thera(g)nostics"; a compound, an inhibitor, and a composition, respectively, for use in a method of delivering an effector to a tissue expressing FAP; a method of diagnosing a disease using the compound, the inhibitor, and the composition, respectively; a method of treating a disease using the compound, the inhibitor, and the composition, respectively; a method of diagnosing and treating a disease, which is also referred to as "thera(g)nosis" or "thera(g)nostics", using the compound, the inhibitor, and the composition, respectively; a method of delivering an effector to a tissue expressing FAP, using the compound, the inhibitor, and the composition, respectively.

LEVEL OF TECHNOLOGY

Despite the increasing availability of therapeutic options, cancer remains the second leading cause of death worldwide. Therapeutic strategies have largely focused on targeting malignant cancer cells themselves, ignoring the ever-present tumor microenvironment (TME) that limits the access of therapeutic agents to cancer cells (Valkenburg, et al., Nat Rev Clin Oncol, 2018, 15: 366). The TME is part of the tumor mass and consists not only of a heterogeneous population of cancer cells but also of a variety of resident and infiltrating host cells, secreted factors, and extracellular matrix proteins (Quail, et al., Nat Med, 2013, 19: 1423). The dominant cell type found in the TME is the cancer-associated fibroblast (CAF) (Kalluri, Nat Rev Cancer, 2016, 16: 582). Many different cell types have been described as sources and origins of CAFs, such as fibroblasts, mesenchymal stem cells, smooth muscle cells, epithelial-derived cells or endothelial cells (Madar, et al., Trends Mol Med, 2013, 19: 447). CAFs exhibit mesenchymal properties and are often the dominant cell type in solid tumor mass. CAFs are gaining increasing attention as a player influencing tumor progression and homeostasis (Gascard, et al., Genes Dev, 2016, 30: 1002; LeBleu, et al., Dis ModelMech, 2018, 11).

In recent years, fibroblast activation protein (FAP) has gained prominence as a marker of CAF (Shiga, et al., Cancers (Basel), 2015, 7: 2443; Pure, et al., Oncogene, 2018, 37: 4343; Jacob, et al., Curr Mol Med, 2012, 12: 1220). Due to the ubiquitous presence of CAF and stroma in tumors, FAP has been discovered as a suitable marker for radiopharmaceutical diagnostics and as a suitable target for radiopharmaceutical therapy (Siveke, J NuclMed, 2018, 59: 1412).

Fibroblast activation protein α (FAP) is a type II transmembrane serine protease and a member of the S9 prolyl oligopeptidase family (Park, et al., J Biol Chem, 1999, 274: 36-505). The closest family member DPP4 is 53% homologous to FAP. Like other DPP enzymes (DPP4, DPP7, DPP8, DPP9), FAP has post-proline exopeptidase activity. In addition, FAP has endopeptidase activity similar to prolyl oligopeptidase/endopeptidase (POP/PREP). The FAP gene is highly conserved across species. The extracellular domain of human FAP is 90% identical to the amino acid sequence of mouse and rat FAP. Mouse FAP has 97% sequence identity to rat FAP.

Structurally, FAP is a 760 amino acid transmembrane protein consisting of a short N-terminal cytoplasmic tail (6 amino acids), one transmembrane domain (20 amino acids), and a 734 amino acid extracellular domain (Aertgeerts, et al., J Biol Chem, 2005, 280: 19441). This extracellular domain consists of an eight-lobed β-propeller and an α/β hydrolase domain. The catalytic triad consists of Ser624, Asp702, and His734 and is located at the interface of the β-propeller and hydrolase domain. The active site is accessible through a central hole in the β-propeller domain or through a narrow cavity between the β-propeller and hydrolase domain. FAP monomers are inactive but form active homodimers as well as heterodimers with DPP4 (Ghersi, et al., Cancer Res, 2006, 66: 4652). Soluble homodimeric FAP has also been described (Keane, et al., FEBS Open Bio, 2013, 4: 43; Lee, et al., Blood, 2006, 107: 1397).

FAP has dual enzymatic activity (Hamson, et al., Proteomics Clin Appl, 2014, 8: 454). Its dipeptidyl peptidase activity allows for the cleavage of two amino acids at the N-terminus after a proline residue. Substrates of FAP that are rapidly cleaved by its dipeptidyl peptidase activity include neuropeptide Y, Peptide YY, Substance P, and B-type natriuretic peptide. Collagen I and III, FGF21, and _α2- antiplasmin have been shown to be cleaved by the endopeptidase activity of FAP. Although FAP cannot cleave native collagens, predigestion with other proteases such as matrix metalloproteinases promotes further collagen degradation by FAP. Collagen processing may influence the migratory capacity of cancer cells. In addition to enhancing cancer cell invasiveness via extracellular matrix remodeling, several other FAP-mediated tumor-promoting roles have been proposed, including proliferation and enhanced angiogenesis. Furthermore, stromal FAP expression is associated with immune evasion in a variety of cancers, suggesting a role in anti-tumor immunity (Pure, et al., Oncogene, 2018, 37: 4343).

FAP is transiently expressed during normal development, but very rarely in healthy adult tissues. In transgenic mice, FAP has been shown to be expressed in adipose tissue, skeletal muscle, skin, bone, and pancreas (Pure, et al., Oncogene, 2018, 37: 4343; Roberts, et al., J Exp Med, 2013, 210: 1137). However, a FAP knockout mouse has a healthy phenotype, suggesting a redundant role under normal conditions (Niedermeyer, et al., Mol Cell Biol, 2000, 20: 1089). At sites of active tissue remodeling, including wound healing, fibrosis, arthritis, atherosclerosis, and cancer, FAP becomes highly activated in stromal cells (Pure, et al., Oncogene, 2018, 37: 4343).

FAP expression in the tumor stroma of 90% of epithelial carcinomas was first described in 1990 using the monoclonal antibody F19 (Garin-Chesa, et al., Proc Natl Acad Sci USA, 1990, 87: 7235; Rettig, et al., Cancer Res, 1993, 53: 3327). FAP-expressing stromal cells were further characterized as cancer-associated fibroblasts (CAFs) and cancer-associated pericytes (Cremasco, et al., Cancer Immunol Res, 2018, 6: 1472). FAP expression on malignant epithelial cells has also been reported, but its significance remains to be determined (Pure, et al., Oncogene, 2018, 37: 4343). The following Table 1, taken from Busek et al. (Busek, et al., Front Biosci (Landmark Ed), 2018, 23: 1933) summarized FAP expression in various malignancies, indicating tumor type and cellular expression.

FAP expression in CAFs has been shown in almost all carcinomas and sarcomas (Riege, et al., Oncogene, 2018, 37: 4343; Busek, et al., FrontBiosci (LandmarkEd), 2018, 23: 1933). In addition, CAFs are present in hematological malignancies (Raffaghello, et al., Oncotarget, 2015, 6: 2589). Thus, the use of FAP as a therapeutic target is not limited to certain tumor entities.

The abundance of FAP-expressing CAFs correlates with poor prognosis. Across a wide range of human tumor indications, FAP expression correlates with higher tumor grade and worse overall survival (Pure, et al., Oncogene, 2018, 37:4343).

As described above, FAP, as well as FAP-expressing cells present in the tumor microenvironment, are reported to significantly influence tumor progression (Hanahan, et al., Cancer Cell, 2012, 21: 309). Furthermore, due to its relatively selective expression in tumors, FAP is considered a suitable target for therapeutic and diagnostic agents, as described below (Siveke, J NuclMed, 2018, 59: 1412; Christiansen, et al, Neoplasia, 2013, 15: 348; Zi, et al., MolMed Rep, 2015, 11: 3203).

Soon after its discovery, FAP was used as a therapeutic target in cancer. To date, various strategies have been studied, including, for example, inhibition of FAP enzymatic activity, deletion of FAP-positive cells, or targeted delivery of cytotoxic compounds.

In 2007, the FAP and DPP4 inhibitor Talabastat (Val-boro-Pro, PT-100) was developed by Point Therapeutics (e.g., as described in U.S. Patent No. 6,890,904, WO9916864). Pennisi et al. (Pennisi, et al., Br J Haematol, 2009, 145:775) observed a reduction in tumor growth in an animal model of multiple myeloma as well as in syngeneic mouse models of the cancer. Additionally, several other prolyl boronic acid derivatives have been developed and reported as putative selective FAP inhibitors. These derivatives exhibit instability in aqueous media at physiological pH (Courts, et al., J Med Chem, 1996, 39:2087) and non-specific reactivity with other enzymes.

WO 2008/116054 discloses hexapeptide derivatives in which the compounds contain a C-terminal bis-amino or boronic acid functional group.

US 2017/0066800 discloses pseudopeptide inhibitors such as M83 that are effective against FAP. These inhibitors were evaluated in lung and colon cancer xenografts in immunodeficient mice. Tumor growth suppression was observed (Jackson, et al., Neoplasia, 2015, 17: 43). These pseudopeptides inhibit both prolyl oligopeptidase (POP/PREP) and FAP activity, thereby precluding their use as specific therapeutic inhibitors of FAP.

US 2008/280856 discloses a nanomolar boronic acid inhibitor. The inhibitor exhibits bispecific inhibition of FAP and PREP, which excludes their use as specific therapeutic inhibitors of FAP.

Cyclic peptide-based FAP inhibitors have been disclosed, for example, in WO 2016/146174 and WO 2006/042282. WO 2016/146174 discloses peptides for the diagnosis and treatment of FAP-expressing tumors, demonstrating specificity for FAP, wherein the closely related homologue of DPP4 is not recognized by said peptides. WO 2006/042282 discloses polypeptides for the treatment of melanoma. In nude mice, inhibition of melanoma growth and melanoma metastasis was shown.

WO 99/75151 and WO 01/68708 disclose a humanized monoclonal antibody to FAP F19 (Sibrotuzumab). In addition, an antibody to FAP F19 and humanized versions thereof are disclosed in WO 99/57151 and WO 01/68708. Development approaches have included, for example, the creation of high-affinity, cross-species cross-reactive, FAP-specific scFvs derivatized into a divalent derivative (Brocks, et al., Mol Med, 2001, 7: 461). In phase I and II clinical trials, Sibrotuzumab showed tumor specific enrichment but failed to demonstrate measurable therapeutic activity in patients with metastatic colorectal cancer, with only 2 of 17 patients having stable disease (Hofheinz, et al., Onkologie, 2003, 26: 44). This F19 antibody has not been shown to block any cellular or protease function of FAP, which would explain the lack of therapeutic effects (Hofheinz, et al., Onkologie, 2003, 26: 44; Scott, et al., Clin Cancer Res, 2003, 9: 1639).

US 2018/022822 discloses novel molecules that specifically bind to human FAP and its epitopes as human-derived antibodies and chimeric antigen receptors (CARs) useful in the treatment of FAP-induced diseases and conditions. Treatment of mice bearing orthotopic syngeneic MC38 colorectal tumors with an anti-FAP antibody reduced tumor diameter and the number of metastases. WO 2012/020006 discloses glycoengineered antibodies that carry modified oligosaccharides in the Fc region. Subsequently, bispecific antibodies specific for FAP and DR5 were developed, which are the subject of WO 2014/161845. These antibodies trigger tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma (Brunker, et al., Mol Cancer Ther, 2016, 15: 946). Antibody-drug conjugates and immunotoxins targeting FAP are described in WO 2015/118030. In vitro toxicity as well as tumor growth inhibition in vivo have been demonstrated after administration of the anti-hu/moFAP hu36:cytolysin ADC candidates. It is unclear whether these antibodies are capable of inhibiting FAP activity.

Small molecule FAP inhibitors based on (4-quinolinoyl)glycyl-2-cyanopyrrolidine, exhibiting low nanomolar inhibitory potency and high selectivity for related DPP and PREP, have been described by Jansen et al. (Jansen, et al., J Med Chem, 2014, 57: 3053; Jansen, et al., ACS Med Chem Lett, 2013, 4: 491) and disclosed in WO 2013/107820. However, the compounds are structurally unrelated to the compounds of the present invention and include an active moiety for covalently binding to FAP.

In recent years, several radiopharmaceutical approaches targeting FAP have been developed, which are described here as examples.

WO 2010/036814 discloses small molecule FAP inhibitors for use as therapeutic agents by inhibiting the enzymatic activity of FAP or as radiopharmaceuticals by binding to FAP.

WO 2019/083990 discloses small molecule FAP inhibitor imaging agents and radiotherapeutic agents described by Jansen et al. (Jansen, et al., J Med Chem, 2014, 57: 3053; Jansen, et al., ACS Med Chem Lett, 2013, 4: 491). In addition, several authors have described selective uptake of imaging and radiotherapeutic agents by tumors of cancer patients (Lindner, et al., J Nucl Med, 2018, 59: 1415; Loktev, et al., J Nucl Med, 2018, 59: 1423; Giesel, et al., J Nucl Med, 2019, 60: 386; Loktev, et al., J Nucl Med, 2019, Mar 8 (epub ahead of print); Giesel, et al., Eur J Nucl Med Mol Imaging, 2019, 46: 1754; Kratochwil, et al., J Nucl Med, 2019, 60: 801) based on the FAP inhibitors described by Jansen et al. (Jansen, et al., J Med Chem, 2014, 57: 3053; Jansen, et al., ACS Med Chem Lett, 2013, 4: 491).

Clinical evaluations of the humanized form of the F19 antibody (sibrotuzumab) labeled with ^131I revealed selective uptake by tumors but not normal tissues in patients with colorectal carcinoma or non-small cell lung cancer (Scott, et al., Clin Cancer Res, 2003, 9: 1639). This may be due to the long circulation time of the antibody, making it unsuitable for diagnostic, therapeutic, or theranostic approaches involving radionuclides.

WO 2011/040972 discloses high-affinity antibodies recognizing both human and murine FAP antigens as potent radioimmunoconjugates. ESC11 IgG1 induces down-modulation and internalization of surface FAP (Fischer, et al., Clin Cancer Res, 2012, 18: 6208). WO 2017/211809 discloses tissue-targeted thorium-227 complexes in which the targeting moiety has specificity for FAP. However, the long circulation time of the antibodies makes them unsuitable for diagnostic, therapeutic or theranostic approaches involving radionuclides.

FAP has also been described as being involved in other diseases besides oncological indications, examples of which are given below.

Fibroblast-like synoviocytes in the joints of patients with rheumatoid arthritis exhibit significantly increased FAP expression (Bauer, et al., Arthritis Res Ther, 2006, 8: R171; Milner, et al., Arthritis Res Ther, 2006, 8: R23). In rheumatoid arthritis, stromal cells play an important role in organizing the structure of the synovial tissue of the joints by producing extracellular matrix components, recruiting infiltrating immune cells, and secreting inflammatory mediators. There is considerable evidence supporting a role for these cells in the perpetuation of joint inflammation and damage (Bartok, et al., Immunol Rev, 2010, 233: 233; Turner, et al., Curr Opin Rheumatol, 2015, 27: 175). In rheumatoid arthritis, FAP plays a pathological role in cartilage turnover at least by promoting proteoglycan loss and subsequent cartilage destruction (Bauer, et al., Arthritis Res Ther, 2006, 8: R171; Waldele, et al., Arthritis Res Ther, 2015, 17: 12). Therefore, it may serve as a marker for patient stratification, for the assessment and follow-up of treatment success, or as a therapeutic target (Bauer, et al., Arthritis Res Ther, 2006, 8: R171). In mice, response to treatment was demonstrated using SPECT/CT imaging with ^99mTc -labeled anti-FAP antibody (van der Geest, et al., Rheumatology (Oxford), 2018, 57: 737; Laverman, et al., J Nucl Med, 2015, 56: 778; van der Geest, et al., J Nucl Med, 2017, 58: 151).

Furthermore, FAP has been recognized not only as a marker of activated fibroblasts in response to injury (Tillmanns, et al., Int J Cardiol, 2013, 168: 3926) but also as an important player in the wound healing process (Ramirez-Montagut, et al., Oncogene, 2004, 23: 5435). Jing et al. demonstrated a time-dependent change in FAP expression after burn wounds in rats (Jing, et al., Nan Fang Yi Ke Da Xue Xue Bao, 2013, 33: 615). Inhibition of FAP activity in reactive wound fibroblasts in keloid scars, common benign fibroproliferative reticular dermal lesions, may offer a therapeutic option to prevent disease progression (Dienus, et al., Arch Dermatol Res, 2010, 302: 725).

FAP expression has been increased in fibrotic diseases, such as idiopathic pulmonary fibrosis, Crohn's disease, and liver fibrosis. In an ex vivo model of Crohn's disease, a chronic inflammatory bowel disease characterized by excessive, unbalanced extracellular matrix (ECM) deposition, upregulated FAP expression was observed. Inhibition of FAP restored ECM homeostasis (Truffi, et al., Inflamm Bowel Dis, 2018, 24: 332). Similar observations were made by Egger et al. (Egger, et al., Eur J Pharmacol, 2017, 809: 64) using a mouse model of lung fibrosis. FAP inhibition resulted in a reduction in fibrotic pathology. FAP is also expressed in tissue remodeling regions in chronically injured liver (Wang, et al., Front Biosci, 2008, 13:3168), and FAP expression by hepatic stellate cells correlates with the histological severity of liver disease (Gorrell, et al., Adv Exp Med Biol, 2003, 524:235). Therefore, FAP is also a promising target for the treatment of liver fibrosis (Lay, et al., Front Biosci (Landmark Ed), 2019, 24:1).

FAP is expressed in arteriosclerotic lesions and is upregulated in activated vascular smooth muscle cells (Monslow, et al., Circulation, 2013, 128: A17597). Monslow et al. showed that targeted inhibition of FAP in arteriosclerotic lesions can reduce overall lesion burden, suppress inflammatory cells, and enhance lesion stability through its ability to alter lesion architecture, favoring matrix-rich lesions over inflammatory ones. More importantly, most arteriosclerotic pathologies share a common pathogenetic feature: atherosclerotic plaque rupture giving rise to arteriosclerotic lesions (Davies, et al., Br Heart J, 1985, 53: 363; Falk, Am J Cardiol, 1989, 63: 114e). Rupture of the fibrous cap in advanced atherosclerotic plaques is a critical trigger for acute coronary syndromes that can lead to myocardial infarction and sudden cardiac death. One of the key factors contributing to plaque instability is the disruption of the fibrous cap, which exposes the underlying thrombogenic plaque core to blood flow, thereby causing thrombosis and subsequent vascular occlusion (Farb, et al., Circulation, 1996, 93: 1354; Virmani, et al., J Am Coll Cardiol, 2006, 41: C13). Brokopp et al showed that FAP promotes the disruption of type I collagen in fibrous caps (Brokopp, et al., Eur Heart J, 2011, 32: 2713). A radiolabeled tracer has been developed and shown to be useful for imaging atherosclerosis (Meletta, et al., Molecules, 2015, 20: 2081).

DETAILED DESCRIPTION OF THE INVENTION

The problem underlying the present invention is to provide a compound which is suitable as a diagnostic agent and/or a pharmaceutical agent, especially if it is conjugated to a diagnostically and/or therapeutically active effector. Another problem underlying the present invention is to provide a compound which is suitable as a diagnostic agent and/or a pharmaceutical agent, especially if it is conjugated to a diagnostically and/or therapeutically active effector, whereby the compound is a potent inhibitor of FAP activity; preferably the pIC50 of the compound is 6.0 or more. Another problem underlying the present invention is to provide a compound which is suitable as a diagnostic agent and/or a pharmaceutical agent, especially if it is conjugated to a diagnostically and/or therapeutically active effector, in the diagnosis and/or therapy of a disease wherein the affected cells and/or affected tissues express FAP. Another problem underlying the present invention is to provide a compound which is suitable for delivering a diagnostically and/or therapeutically effective agent to a diseased cell and/or diseased tissue, respectively, and in particular to FAP-expressing diseased cells and/or diseased tissue, preferably the diseased tissue comprises or contains cancer-associated fibroblasts. Furthermore, the problem underlying the present invention is to provide a method for diagnosing a disease, a method for treating and/or preventing a disease, and a method for the combined diagnosis and treatment of a disease; preferably, such a disease is a disease comprising FAP-expressing cells and/or tissues, more particularly FAP-expressing diseased cells and/or diseased tissues, preferably the diseased tissue comprises or contains cancer-associated fibroblasts. Another problem underlying the present invention is to provide a method for identifying a subject in which the subject is likely to respond or likely not to respond to treatment of the disease, a method for selecting a subject from a group of subjects, where the subject is likely to respond or likely not to respond to treatment of the disease. Furthermore, the problem underlying the present invention is to provide a pharmaceutical composition containing a compound having the characteristics specified above. Furthermore, the problem underlying the present invention is to provide a kit that is suitable for use in any of the above methods.

There is a need for compounds that are suitable as a diagnostic agent and/or a pharmaceutical agent, especially if they are conjugated to a diagnostically and/or therapeutically active effector. In addition, there is a need for compounds that are suitable as a diagnostic agent and/or a pharmaceutical agent, especially if they are conjugated to a diagnostically and/or therapeutically active effector, whereby the compound is a potent inhibitor of FAP activity; preferably the pIC50 of the compound is 6.0 or greater. In addition, there is a need for compounds that are suitable as diagnostic agents and/or pharmaceutical agents, especially if they are conjugated to a diagnostically and/or therapeutically active effector, in the diagnosis and/or therapy of a disease in which the affected cells and/or affected tissues express FAP. There is also a need for a compound which is suitable for delivering a diagnostically and/or therapeutically effective agent to a diseased cell and/or diseased tissue, respectively, and more particularly to a diseased cell and/or diseased tissue expressing FAP, preferably the diseased tissue comprises or includes cancer-associated fibroblasts. There is also a need for a method for diagnosing a disease, a method for treating and/or preventing a disease and a method for combined diagnosis and treatment of a disease; preferably, such a disease is a disease comprising FAP-expressing cells and/or tissues, more particularly FAP-expressing diseased cells and/or diseased tissues, preferably the diseased tissue comprises or includes cancer-associated fibroblasts. There is also a need for a method for identifying a subject in which the subject is likely to respond or likely not to respond to treatment of a disease, a method for selecting a subject from a group of subjects, where the subject is likely to respond or likely not to respond to treatment of a disease. There is also a need for a pharmaceutical composition comprising a compound having the characteristics specified above. In addition, there is a need for a kit that is suitable for use in any of the above methods. The present invention satisfies these needs.

These and other problems are solved by the essence according to the attached claims.

These and other problems underlying the present invention are also solved by the following embodiments of the invention.

Embodiment of the invention 1. A compound selected from the group consisting of the compound Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3554) of the following formula

compounds Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3407) of the following formula

Embodiment of the invention 2. The compound according to Embodiment of the invention 1, wherein the compound is the compound Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3554) of the following formula

Embodiment of the invention 3. The compound according to Embodiment of the invention 1, wherein the compound is a Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3407) compound of the following formula

Embodiment 4. A compound according to any one of Embodiments 1-3, wherein any S atom that can be oxidized, preferably the S atoms of the thioether groups, is present as -S-, -S(O)- or -S(O ₂ )- or a mixture thereof.

Embodiment 5. A compound according to any one of Embodiments 1-4, wherein the compound is capable of binding to fibroblast activation protein (FAP).

Embodiment of the invention 6. A compound according to any one of Embodiments of the invention 1-5, wherein the compound contains a diagnostically active nuclide or a therapeutically active nuclide.

Embodiment of the invention 7. The compound according to Embodiment of the invention 6, wherein the compound is selected from the group comprising the compound Нех-[Cys(tMeBn(InDOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3590) of the following formula

compound Hex-[Cys(tMeBn(LuDOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3591) of the following formula

compound Hex-[Cys(tMeBn(GaDOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3592) of the following formula

compound Hex-[Cys(tMeBn(EuDOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3661) of the following formula

compound Hex-[Cys(tMeBn(InDOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3623) of the following formula

compound Hex-[Cys(tMeBn(LuDOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3624) of the following formula

compound Hex-[Cys(tMeBn(EuDOTA-AET))-Pro-Pro-Tlir-Gln-Phe-Cys]-OH (3BP-3662) of the following formula

compound Hex-[Cys(tMeBn(GaDOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3949) of the following formula

compound Hex-[Cys-(tMeBn(CuDOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-4293) of the following formula

compound Hex-[Cys-(tMeBn(ZnDOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-4343) of the following formula

Embodiment of the invention 8. A compound according to any one of Embodiments of the invention 6 and 7, wherein the diagnostically active nuclide is a diagnostically active radionuclide.

Embodiment 9. The compound according to Embodiment 8, wherein the diagnostically active radionuclide is selected from the group consisting of ⁴³ Sc, ⁴⁴ Sc, ⁵¹ Mn, ⁵² Mn, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^94m Tc, ^99m Tc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰¹ Tl, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, preferably ⁴³ Sc, ⁴⁴ Sc, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and most preferably ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹⁸ F, ¹²³ I and ¹²⁴ I.

Embodiment of the invention 10. A compound according to any one of Embodiments of the invention 6 and 7, wherein the therapeutically active nuclide is a therapeutically active radionuclide.

Embodiment 11. The compound of Embodiment 10, wherein the therapeutically active radionuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, 226 Th, ²²⁷ Th ^{, 131 I} , ²¹¹ At, preferably ⁴⁷ Sc, ⁶⁷ Cu, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I, ²¹¹ At and most preferably ⁹⁰ Y, ¹⁷⁷ Lu, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I and ²¹¹ At.

Embodiment 12. A compound according to any one of Embodiments 1-11, wherein the compound interacts with a fibroblast activation protein (FAP), preferably with a human FAP having the amino acid sequence of SEQ ID NO: 1 or a homologue thereof, wherein the amino acid sequence of the homologue is at least 85% identical to the amino acid sequence of SEQ ID NO: 1.

Embodiment 13. The compound according to Embodiment 12, wherein the compound is an inhibitor of fibroblast activation protein (FAP).

Embodiment of the invention 14. A compound according to any one of Embodiments of the invention 1-13 for use in a method for diagnosing a disease.

Embodiment 15. A compound for use according to Embodiment 14, wherein the disease is a disease involving fibroblast activation protein (FAP), preferably increased expression of fibroblast activation protein (FAP).

Embodiment 16. A compound for use according to any one of Embodiments 14-15, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP), preferably diseased tissue containing cells exhibiting increased expression of fibroblast activation protein (FAP), more preferably the disease involves tumor-associated fibroblasts.

Embodiment of the invention 17. A compound for use according to any one of Embodiments of the invention 14-16, wherein the disease is a neoplasm, preferably cancer or a tumor.

Embodiment of the invention 18. A compound for use according to Embodiment of the invention 17, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma and thyroid cancer.

Embodiment of the invention 19. A compound for use according to Embodiment of the invention 18, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, head and neck cancer, lung cancer, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and squamous cell carcinoma.

Embodiment 20. A compound for use according to any one of Embodiments 14-16, wherein the disease is selected from the groups comprising an inflammatory disease, a cardiovascular disease, an autoimmune disease, and a fibrotic disease.

Embodiment of the invention 21. A compound for use according to Embodiment of the invention 20, wherein the disease is an inflammatory disease.

Embodiment of the invention 22. A compound for use according to Embodiment of the invention 21, wherein the disease is atherosclerosis, arthritis or rheumatoid arthritis.

Embodiment 23. A compound for use according to Embodiment 20, wherein the disease is a cardiovascular disease.

Embodiment 24. A compound for use according to Embodiment 23, wherein the disease is a cardiovascular disease involving atherosclerotic plaques.

Embodiment of the invention 25. A compound for use according to Embodiment of the invention 24, wherein the disease is an atherosclerotic pathology caused by plaque rupture, acute coronary syndrome, myocardial infarction, thrombosis or vascular occlusion.

Embodiment 26. A compound for use according to Embodiment 20, wherein the disease is a fibrotic disease.

Embodiment of the invention 27. A compound for use according to Embodiment of the invention 26, wherein the disease is selected from the group comprising idiopathic pulmonary fibrosis, Crohn's disease and liver fibrosis.

Embodiment of the invention 28. A compound for use according to any one of Embodiments of the invention 14-27, wherein the compound contains a diagnostically active nuclide, preferably a diagnostically active radionuclide.

Embodiment 29. A compound for use according to Embodiment 28, wherein the diagnostically active nuclide is selected from the group consisting of ⁴³ Sc, ⁴⁴ Sc, ⁵¹ Mn, ⁵² Mn, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^94m Tc, ^99m Tc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰¹ Tl, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, preferably ⁴³ Sc, ⁴⁴ Sc, ⁶⁴ Cu, 67 Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^99m Tc, ^{111 In} , ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and most preferably ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹⁸ F, ¹²³ I and ¹²⁴ I.

Embodiment 30. A compound for use according to any one of Embodiments 14-29, wherein the diagnostic method is a visualization method.

Embodiment 31. A compound for use according to Embodiment 30, wherein the imaging method is selected from the group consisting of scintigraphy, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET).

Embodiment 32. A compound for use according to any one of Embodiments 14-31, wherein the method comprises administering a diagnostically effective amount of the compound to a subject, preferably a mammal, wherein the mammal is selected from the group consisting of a human, companion animals, domestic animals and livestock, more preferably the subject is selected from the group consisting of a human, a dog, a cat, a horse and a cow, and most preferably the subject is a human.

Embodiment of the invention 33. A compound according to any one of Embodiments of the invention 1-13 for use in a method for treating a disease.

Embodiment 34. A compound for use according to Embodiment 34, wherein the disease is a disease involving fibroblast activation protein (FAP), preferably increased expression of fibroblast activation protein (FAP).

Embodiment 35. A compound for use according to any one of Embodiments 33-34, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP), preferably diseased tissue containing cells exhibiting increased expression of fibroblast activation protein (FAP), more preferably the disease involves tumor-associated fibroblasts.

Embodiment 36. A compound for use according to any one of Embodiments 33-35, wherein the disease is a neoplasm, preferably cancer or a tumor.

Embodiment 37. A compound for use according to Embodiment 36, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma and thyroid cancer.

Embodiment 38. A compound for use according to Embodiment 37, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, head and neck cancer, lung cancer, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and squamous cell carcinoma.

Embodiment 39. A compound for use according to any one of Embodiments 33-35, wherein the disease is selected from the groups comprising an inflammatory disease, a cardiovascular disease, an autoimmune disease, and a fibrotic disease.

Embodiment 40. A compound for use according to Embodiment 39, wherein the disease is an inflammatory disease.

Embodiment 41 of the invention. A compound for use according to Embodiment 40 of the invention, wherein the disease is atherosclerosis, arthritis or rheumatoid arthritis.

Embodiment 42. A compound for use according to Embodiment 39, wherein the disease is a cardiovascular disease.

Embodiment 43. A compound for use according to Embodiment 42, wherein the disease is a cardiovascular disease involving atherosclerotic plaques.

Embodiment of the invention 44. A compound for use according to Embodiment of the invention 43, wherein the disease is an atherosclerotic pathology caused by plaque rupture, acute coronary syndrome, myocardial infarction, thrombosis or vascular occlusion.

Embodiment 45. A compound for use according to Embodiment 39, wherein the disease is a fibrotic disease.

Embodiment of the invention 46. A compound for use according to Embodiment of the invention 45, wherein the disease is selected from the group comprising idiopathic pulmonary fibrosis, Crohn's disease and liver fibrosis.

Embodiment 47. A compound for use according to any one of Embodiments 33-38, wherein the compound comprises a therapeutically active nuclide, preferably a therapeutically active radionuclide.

Embodiment 48. A compound for use according to Embodiment 47, wherein the therapeutically active nuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, 226 Th, ²²⁷ Th, ¹³¹ I, ²¹¹ At, preferably ⁴⁷ Sc, ⁶⁷ Cu, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, 225 Ac, ²²⁷ Th, ^{131 I} , ²¹¹ At and most preferably ^{90 Y} , ¹⁷⁷ Lu, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I and ²¹¹ At.

Embodiment 49. A compound for use according to any one of Embodiments 33-48, wherein the method comprises administering a therapeutically effective amount of the compound to a subject, preferably a mammal, wherein the mammal is selected from the group consisting of a human, companion animals, domestic animals and livestock, more preferably the subject is selected from the group consisting of a human, a dog, a cat, a horse and a cow, and most preferably the subject is a human.

Embodiment 50. A compound according to any one of Embodiments 1-13 for use in a method of identifying a subject where the subject is likely to respond or likely not to respond to treatment for a disease, where the method of identifying a subject comprises performing a diagnostic method using the compound according to any one of Embodiments 1-13, preferably a method of diagnosing a disease as described in any one of Embodiments 14-33.

Embodiment 51. A compound according to any one of Embodiments 1-13 for use in a method of selecting a subject from a group of subjects wherein the subject is likely to respond or is likely not to respond to treatment for a disease, wherein the method of selecting a subject from a group of subjects comprises performing a diagnostic method using the compound according to any one of Embodiments 1-13, preferably a method of diagnosing a disease as described in any one of Embodiments 14-32.

Embodiment 52. A compound according to any one of Embodiments 1-13 for use in a method of stratifying a group of subjects into subjects likely to respond to treatment for a disease and subjects likely not to respond to treatment for a disease, wherein the method of stratifying a group of subjects comprises performing a diagnostic method using the compound according to any one of Embodiments 1-13, preferably a method of diagnosing a disease as described in any one of Embodiments 14-32.

Embodiment 53. A compound for use according to any one of Embodiments 50-52, wherein the disease is a disease involving fibroblast activation protein (FAP), preferably increased expression of fibroblast activation protein (FAP).

Embodiment 54. A compound for use according to any one of Embodiments 50-53, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP), preferably diseased tissue containing cells exhibiting increased expression of fibroblast activation protein (FAP), more preferably a disease involving tumor-associated fibroblasts.

Embodiment 55. A compound for use according to any one of Embodiments 50-54, wherein the disease is a neoplasm, preferably cancer or a tumor.

Embodiment 56. A compound for use according to Embodiment 55, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma and thyroid cancer.

Embodiment 57. A compound for use according to Embodiment 56, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, head and neck cancer, lung cancer, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and squamous cell carcinoma.

Embodiment 58. A compound for use according to any one of Embodiments 50-54, wherein the disease is selected from the groups comprising an inflammatory disease, a cardiovascular disease, an autoimmune disease, and a fibrotic disease.

Embodiment 59. A compound for use according to Embodiment 58, wherein the disease is an inflammatory disease.

Embodiment 60. A compound for use according to Embodiment 59, wherein the disease is atherosclerosis, arthritis or rheumatoid arthritis.

Embodiment 61. A compound for use according to Embodiment 58, wherein the disease is a cardiovascular disease.

Embodiment 62. A compound for use according to Embodiment 61, wherein the disease is a cardiovascular disease involving atherosclerotic plaques.

Embodiment of the invention 63. A compound for use according to Embodiment of the invention 62, wherein the disease is an atherosclerotic pathology caused by plaque rupture, acute coronary syndrome, myocardial infarction, thrombosis or vascular occlusion.

Embodiment 64. A compound for use according to Embodiment 58, wherein the disease is a fibrotic disease.

Embodiment 65. A compound for use according to Embodiment 64, wherein the disease is selected from the group consisting of idiopathic pulmonary fibrosis, Crohn's disease, and liver fibrosis.

Embodiment 66. A compound for use according to any one of Embodiments 50-65, wherein the diagnostic method is a visualization method.

Embodiment 67. A compound for use according to Embodiment 66, wherein the imaging method is selected from the group consisting of scintigraphy, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET).

Embodiment 68. A compound for use according to any one of Embodiments 50-67, wherein the compound comprises a diagnostically active nuclide, preferably a diagnostically active radionuclide.

Embodiment 69. A compound for use according to Embodiment 68, wherein the diagnostically active nuclide is selected from the group consisting of ⁴³ Sc, ⁴⁴ Sc, ⁵¹ Mn, ⁵² Mn, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^94m Tc, ^99m Tc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰¹ Tl, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, preferably ⁴³ Sc, ⁴⁴ Sc, ⁶⁴ Cu, 67 Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^99m Tc, ^{111 In} , ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and most preferably ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹⁸ F, ¹²³ I and ¹²⁴ I.

Embodiment of the invention 70. A compound according to any one of Embodiments of the invention 1-13 for use in a method of delivering an effector to a fibroblast activation protein (FAP), preferably a human fibroblast activation protein (FAP), wherein the effector is selected from the group consisting of a diagnostically active agent and a therapeutically active agent.

Embodiment of the invention 71. A compound for use according to Embodiment of the invention 70, wherein the effector is selected from the group comprising a diagnostically active nuclide and a therapeutically active nuclide.

Embodiment of the invention 72. A compound for use according to Embodiment of the invention 71, wherein the diagnostically active nuclide is a diagnostically active radionuclide.

Embodiment 73. A compound for use according to Embodiment 72, wherein the diagnostically active radionuclide is selected from the group consisting of ⁴³ Sc, ⁴⁴ Sc, ⁵¹ Mn, ⁵² Mn, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^94m Tc, ^99m Tc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰¹ Tl, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, preferably ⁴³ Sc, ⁴⁴ Sc, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and most preferably ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ^99m Tc, ¹¹¹ In, ¹⁸ F, ¹²³ I and ¹²⁴ I.

Embodiment 74. A compound for use according to any one of Embodiments 70-73, wherein the fibroblast activation protein (FAP) is expressed by a cell, preferably a fibroblast, a mesenchymal stem cell, a smooth muscle cell, a cell of epithelial origin or an endothelial cell, more preferably a human fibroblast, a mesenchymal stem cell, a smooth muscle cell, a cell of epithelial origin or an endothelial cell, most preferably the human fibroblast, mesenchymal stem cell, smooth muscle cell, cell of epithelial origin or endothelial cell each exhibits increased expression of the fibroblast activation protein (FAP).

Embodiment 75. A compound for use according to Embodiment 74, wherein the cell is contained in tissue or a portion thereof, preferably in diseased tissue of a subject suffering from a disease.

Embodiment 76. A compound for use according to Embodiment 75, wherein the disease comprises cells exhibiting increased expression of fibroblast activation protein (FAP), preferably diseased tissue containing cells exhibiting increased expression of fibroblast activation protein (FAP), more preferably a disease involving tumor-associated fibroblasts.

Embodiment 77. A compound for use according to any one of Embodiments 75-76, wherein the disease is a neoplasm, preferably cancer or a tumor.

Embodiment 78. A compound for use according to Embodiment 77, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma and thyroid cancer.

Embodiment 79. A compound for use according to Embodiment 78, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, head and neck cancer, lung cancer, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and squamous cell carcinoma.

Embodiment 80. A compound for use according to any one of Embodiments 75-76, wherein the disease is selected from the groups comprising an inflammatory disease, a cardiovascular disease, an autoimmune disease, and a fibrotic disease.

Embodiment 81. A compound for use according to Embodiment 80, wherein the disease is an inflammatory disease.

Embodiment 82. A compound for use according to Embodiment 81, wherein the disease is atherosclerosis, arthritis or rheumatoid arthritis.

Embodiment 83. A compound for use according to Embodiment 80, wherein the disease is a cardiovascular disease.

Embodiment 84. A compound for use according to Embodiment 83, wherein the disease is a cardiovascular disease comprising atherosclerotic plaques.

Embodiment 85. A compound for use according to Embodiment 84, wherein the disease is an atherosclerotic pathology caused by plaque rupture, acute coronary syndrome, myocardial infarction, thrombosis or vascular occlusion.

Embodiment 86. A compound for use according to Embodiment 80, wherein the disease is a fibrotic disease.

Embodiment of the invention 87. A compound for use according to Embodiment of the invention 86, wherein the disease is selected from the group comprising idiopathic pulmonary fibrosis, Crohn's disease and liver fibrosis.

Embodiment of the invention 88. A compound for use according to Embodiment of the invention 71, wherein the therapeutically active nuclide is a therapeutically active radionuclide.

Embodiment 89. A compound for use according to Embodiment 88, wherein the therapeutically active radionuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁶ Th, ²²⁷ Th, ¹³¹ I, ²¹¹ At, preferably ⁴⁷ Sc, ⁶⁷ Cu, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I, ²¹¹ At and most preferably ⁹⁰ Y, ¹⁷⁷ Lu, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I and ²¹¹ At.

Embodiment 90. A compound for use according to any one of Embodiments 88-89, wherein the fibroblast activation protein (FAP) is expressed by a cell, preferably a fibroblast, a mesenchymal stem cell, a smooth muscle cell, a cell of epithelial origin or an endothelial cell, more preferably a human fibroblast, a mesenchymal stem cell, a smooth muscle cell, a cell of epithelial origin or an endothelial cell, most preferably a human fibroblast, a mesenchymal stem cell, a smooth muscle cell, a cell of epithelial origin or an endothelial cell exhibits increased expression of the fibroblast activation protein (FAP).

Embodiment 91. A compound for use according to Embodiment 90, wherein the cell is contained in tissue or a portion thereof, preferably in diseased tissue of a subject suffering from a disease.

Embodiment 92. A compound for use according to Embodiment 91, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP), preferably diseased tissue containing cells exhibiting increased expression of fibroblast activation protein (FAP), more preferably the disease involves tumor-associated fibroblasts.

Embodiment 93. A compound for use according to any of Embodiments 90-92, wherein the disease is a neoplasm, preferably cancer or a tumor.

Embodiment 94. A compound for use according to Embodiment 93, wherein the neoplasm, cancer and tumor are each and individually selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma and thyroid cancer.

Embodiment of the invention 95. A composition, preferably a pharmaceutical composition, wherein the composition comprises a compound according to any one of Embodiments of the invention 1-13 and a pharmaceutically acceptable excipient.

Embodiment 96. A composition according to Embodiment 95 for use in any method as defined in any of the preceding claims.

Embodiment of the invention 97. A method for diagnosing a disease in a subject, comprising administering to the subject a diagnostically effective amount of a compound according to any one of Embodiments of the invention 1-13.

Embodiment 98. The method according to Embodiment 97, wherein the compound comprises a diagnostically active agent, wherein the agent is preferably a radionuclide.

Embodiment 99. A method for treating a disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a compound according to any one of Embodiments 1-13.

Embodiment 100. The method according to Embodiment 99, wherein the compound comprises a therapeutically active agent, wherein the agent is preferably a radionuclide.

Embodiment 101. The method according to any one of Embodiments 97-100, wherein the disease is a disease involving fibroblast activation protein (FAP), preferably increased expression of fibroblast activation protein (FAP).

Embodiment 102. The method according to any one of Embodiments 97-101, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP), preferably diseased tissue containing cells exhibiting increased expression of fibroblast activation protein (FAP), more preferably the disease involves tumor-associated fibroblasts.

Embodiment of the invention 103. The method according to any one of Embodiments of the invention 97-102, wherein the disease is selected from the groups including neoplasms, preferably cancer or tumors, and an inflammatory disease, a cardiovascular disease, an autoimmune disease, and a fibrotic disease.

Embodiment 104. A kit comprising a compound according to any one of Embodiments 1-13, one or more optional excipients, and optionally one or more devices, wherein the device is selected from the group consisting of a labeling device, a purification device, a processing device, a radioprotection device, an assay device, or an administration device.

Embodiment of the invention 105. A kit according to Embodiment of the invention 104 for use in any method as defined in any of the preceding paragraphs.

More specifically, the problem underlying the present invention is solved in a first aspect by a compound selected from the group consisting of

compounds Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3554) of the following formula

compounds Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH2 (3BP-3407) of the following formula

More specifically, the problem underlying the present invention is solved in a second aspect by a compound according to the first aspect, including any embodiment thereof, for use in a method for diagnosing a disease.

More specifically, the problem underlying the present invention is solved in a third aspect by a compound according to the first aspect, including any embodiment thereof, for use in a method for treating a disease.

More specifically, the problem underlying the present invention is solved in a fourth aspect by a compound according to the first aspect, including any embodiment thereof, for use in a method for identifying a subject, where the subject is likely to respond or is likely not to respond to treatment for a disease, where the method for identifying a subject comprises performing a diagnostic method using the compound according to the first aspect, including any embodiment thereof.

More specifically, the problem underlying the present invention is solved in a fifth aspect by a compound according to the first aspect, including any embodiment thereof, for use in a method for selecting a subject from a group of subjects, wherein the subject is likely to respond or is likely not to respond to treatment for a disease, wherein the method for selecting a subject from a group of subjects comprises performing a diagnostic method using the compound according to the first aspect, including any embodiment thereof.

More specifically, the problem underlying the present invention is solved in a sixth aspect by a compound according to the first aspect, including any embodiment thereof, for use in a method for stratifying a group of subjects into subjects who are likely to respond to treatment for a disease and subjects who are likely not to respond to treatment for a disease, wherein the method for stratifying a group of subjects comprises performing a diagnostic method using the compound according to the first aspect, including any embodiment thereof.

More specifically, the problem underlying the present invention is solved in a seventh aspect by a composition, preferably a pharmaceutical composition, wherein the composition comprises a compound according to the first aspect, including any embodiment thereof, and a pharmaceutically acceptable excipient.

More specifically, the problem underlying the present invention is solved in an eighth aspect by a method for diagnosing a disease in a subject, wherein the method comprises administering to the subject a diagnostically effective amount of a compound according to the first aspect, including any embodiment thereof.

More specifically, the problem underlying the present invention is solved in a ninth aspect by a method for treating a disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a compound according to the first aspect, including any embodiment thereof.

More specifically, the problem underlying the present invention is solved in a tenth aspect by a kit comprising a compound according to the first aspect, including any embodiment thereof, one or more optional excipients and optionally one or more devices, wherein the device(s) is(are) selected from the group comprising a labeling device, a purification device, a processing device, a radioprotection device, an assay device or an administration device.

One skilled in the art will appreciate that any compound or specific compound of the invention is any compound disclosed herein, including, but not limited to, any compound described in any of the above embodiments and any of the following embodiments.

One skilled in the art will appreciate that any method or particular method of the invention is any method disclosed herein, including, but not limited to, any method described in any of the above embodiments and any of the following embodiments.

One skilled in the art will appreciate that any composition or particular composition of the invention is any composition described herein, including, but not limited to, any composition described in any of the above embodiments and any of the following embodiments.

One skilled in the art will appreciate that any kit or particular kit of the invention is any kit disclosed herein, including, but not limited to, any kit described in any of the above embodiments and any of the following embodiments.

The present invention is based on the unexpected discovery of the present inventors that the compound of the invention and, more particularly, its cyclic peptide, provides highly specific binding of the compound containing such cyclic peptide to fibroblast activation protein (FAP), and FAP-specific inhibitors based on cyclic peptides with nanomolar affinity have not been described so far.

Finally, the present inventors have found that the compounds of the invention are unexpectedly stable in blood plasma and unexpectedly useful as imaging agents and effective in reducing tumor size.

In an embodiment of the invention and as preferably used herein, the chelator is a compound that is capable of forming a chelate, wherein the chelate is a compound, preferably a cyclic compound, in which a metal or a moiety having an electron gap or an unshared pair of electrons participates in the formation of a ring. More preferably, the chelator is a type of compound in which one ligand occupies more than one coordination site at the central atom.

In an embodiment of the invention and as preferably used herein, the diagnostically active compound is a compound that is suitable or usable for diagnosing a disease.

In an embodiment of the invention and as preferably used herein, the diagnostic agent or diagnostically active agent is a compound that is suitable or usable for diagnosing a disease.

In an embodiment of the invention and as preferably used herein, the therapeutically active compound is a compound that is suitable or applicable for treating a disease.

In an embodiment of the invention and as preferably used herein, the therapeutic agent or therapeutically active agent is a compound that is suitable or applicable for treating a disease.

In an embodiment of the invention and as preferably used herein, the theranostically active compound is a compound that is suitable or usable for both diagnosis and therapy of a disease.

In an embodiment of the invention and as preferably used herein, a theranostic agent or theranostically active agent is a compound that is suitable or usable for both diagnosis and therapy of a disease.

In an embodiment of the invention and as preferably used herein, theranostics is a method for combined diagnosis and therapy of a disease; preferably, the combined diagnostic and therapeutically active compounds used in theranostics are radiolabeled.

In an embodiment of the invention and as preferably used herein, treatment of a disease is the treatment and/or prevention of the disease.

In an embodiment of the invention and as preferably used herein, a disease involving FAP is a disease in which cells, including but not limited to fibroblasts, preferably expressing upregulated FAP, and tissue either expressing FAP or including or containing cells, such as fibroblasts, preferably expressing upregulated FAP, respectively, are either the cause of the disease and/or symptoms of the disease or part of the pathology underlying the disease. A preferred cell expressing FAP is a cancer-associated fibroblast (CAF). In an embodiment of the disease, preferably when used in connection with the treatment, cure and/or therapy of the disease, the effect on the cells, tissue and pathology, respectively, results in a cure, treatment or amelioration of the disease and/or symptoms of the disease. In a variant of the disease, preferably when used in connection with the diagnosis and/or diagnostics of the disease, the labeling of FAP-expressing cells and/or FAP-expressing tissue allows to distinguish or differentiate said cells and/or said tissue from healthy or non-FAP-expressing cells and/or healthy or non-FAP-expressing tissue. More preferably, such distinction or difference forms the basis for said diagnosis and diagnosis, respectively. In an embodiment, the labeling means the interaction of a detectable label directly or indirectly with FAP-expressing cells and/or with FAP-expressing tissue or tissue containing such FAP-expressing cells; more preferably, such interaction includes or is based on the interaction of the label or a compound bearing such a label with FAP.

In an embodiment of the invention and as preferably used herein, the target cell is a cell that expresses FAP and is either the cause of the disease and/or symptoms of the disease or part of the pathology underlying the disease.

In an embodiment of the invention and as preferably used herein, a non-target cell is a cell that either does not express FAP and/or is not the cause of the disease and/or disease symptoms or is part of the pathology underlying the disease.

In an embodiment of the invention and as preferably used herein, the neoplasm is an abnormal new growth of cells. The cells in the neoplasm grow faster than normal cells and will continue to grow if left untreated. The neoplasm may be benign or malignant.

In an embodiment of the invention and as preferably used herein, the tumor is a mass lesion, which may be benign or malignant.

In an embodiment of the invention and as preferably used herein, cancer is a malignant neoplasm.

The amino acid sequences of the peptides provided herein are depicted in a typical peptide sequence format, as will be understood by one of ordinary skill in the art. For example, a three-letter code for a conventional amino acid or a code for an unconventional amino acid or abbreviations for additional building blocks indicates the presence of an amino acid or building block at a given position in the peptide sequence. Each amino acid or building block code is linked to the code for the next and/or previous amino acid or building block in the sequence by a hyphen, which (usually represents an amide bond).

If an amino acid contains more than one amino and/or carboxyl group, all orientations of this amino acid are in principle possible, but in an α-amino acid the use of the α-amino and α-carboxyl group is preferred and otherwise the preferred orientations are explicitly indicated.

For amino acids, in their abbreviations, the first letter indicates the stereochemistry of the C-α atom, if applicable. For example, the first capital letter indicates that the L-form of the amino acid is present in the peptide sequence, and the first lowercase letter indicates that the D-form of the corresponding amino acid is present in the peptide sequence.

In an embodiment of the invention and as preferably used herein, the aromatic L-α-amino acid is any kind of L-α-amino acid that contains an aryl group.

In an embodiment of the invention and as preferably used herein, the heteroaromatic L-α-amino acid is any type of L-α-amino acid that contains a heteroaryl group.

Unless otherwise stated, amino acid sequences are presented herein in the N- to C-terminal direction.

The compounds of the invention generally comprise the amino acid sequences presented herein. Common amino acids, also referred to as natural amino acids, are identified according to their standard three-letter codes and single-letter abbreviations, as set forth in Table 2.

Unconventional amino acids, also called non-naturally occurring amino acids, are any kind of non-oligomeric compound that includes an amino group and a carboxyl group and is not a normal amino acid.

Examples of non-traditional amino acids and other building blocks used to construct the compounds of the invention are identified by their abbreviation or name, as listed in Table 3. The structures of some building blocks are shown with a typical reagent for introducing the building block into a peptide (e.g., as a carboxylic acid), or these building blocks are shown as a residue that is fully attached to another structure, such as a peptide or amino acid. The amino acid structures are shown as complete amino acids, rather than as amino acid residues as they appear after implementation into the peptide sequence. Some larger chemical fragments consisting of more than one fragment are also shown for clarity.

In accordance with this application, DOTA means 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid.

It will be appreciated by those skilled in the art that the presence of a chelator in a compound of the invention includes, unless otherwise stated, the possibility that the chelator forms a complex with any partner of the metal complex, i.e. with any metal that can in principle be complexed with the chelator. An explicitly mentioned chelator of a compound of the invention or a chelator in general in connection with a compound of the invention refers either to a non-complexed chelator as such or to a chelator to which any partner of the metal complex is bound, where the partner of the metal complex is any radioactive or non-radioactive partner of the metal complex. Preferably, the chelator metal complex, i.e. the chelator to which the partner of the metal complex is bound, is a stable chelator metal complex.

Non-radioactive metal chelator complexes have several applications, e.g., to assess properties such as stability or activity that are otherwise difficult to determine. One aspect is that cold versions of radioactive versions of the metal complex partner (e.g., non-radioactive Gallium, Lutetium or Indium complexes, as described in the examples) can act as surrogates for radioactive compounds. In addition, they are valuable tools for identifying metabolites in vitro or in vivo, as well as for assessing the toxicity properties of the compounds of the invention. In addition, metal chelator complexes can be used in binding assays using the fluorescent properties of certain metal complexes with various ligands (e.g., Europium salts).

It will be understood by a person skilled in the art that the radioactive nuclide which is or which is to be attached to the compound of the invention is selected taking into account the disease to be treated and/or the disease to be diagnosed, respectively, and/or the characteristics of the patient and the group of patients, respectively, to be treated and to be diagnosed, respectively.

In an embodiment of the present invention, a radioactive nuclide is also called a radionuclide. Radioactive decay is a process in which the atomic nucleus of an unstable atom loses energy by emitting ionizing particles (ionizing radiation). There are different types of radioactive decay. Decay or loss of energy occurs when an atom with one type of nucleus, called the parent radionuclide, is transformed into an atom with a nucleus in a different state or into another nucleus containing a different number of protons and neutrons. Either product is called a daughter nuclide. In some decays, the parent and daughter are different chemical elements, and thus the decay process results in nuclear transmutation (creation of an atom of a new element). For example, radioactive decay can be alpha decay, beta decay, and gamma decay. Alpha decay occurs when the nucleus emits an alpha particle (helium nucleus). This is the most common nucleon emission process, but in rarer types of decay, nuclei may emit protons or certain nuclei of other elements (in a process called cluster decay). Beta decay occurs when a nucleus emits an electron (β decay) or a positron (β ⁺ decay) and a type of neutrino in a process that converts a proton to a neutron or vice versa. In contrast, there are radioactive decay processes that do not result in transmutation. The energy of an excited nucleus may be emitted as a gamma ray in gamma decay, or used to eject an orbital electron by interacting with the excited nucleus in a process called internal conversion, or used to absorb an inner atomic electron from an electron shell, whereby the change of a nuclear proton to a neutron causes the emission of an electron neutrino in a process called electron capture (EC), or may be emitted without changing the number of protons and neutrons in a process called an isomeric transition (IT). Another form of radioactive decay, spontaneous fission (SF), is found only in very heavy chemical elements, resulting in spontaneous disintegration into smaller nuclei and a few isolated nuclear particles.

In a preferred embodiment of the present invention, a radionuclide can be used to label a compound of the invention.

In an embodiment of the present invention, the radionuclide is suitable for forming a complex with a chelating agent, resulting in the formation of a radionuclide chelate complex.

In a further embodiment of the invention, one or more atoms of the compound of the invention have a non-natural isotopic composition, preferably these atoms are radionuclides; more preferably radionuclides of carbon, oxygen, nitrogen, sulfur, phosphorus and halogens: these radioactive atoms are typically part of amino acids, in some cases halogenated amino acids, and/or building blocks and in some cases halogenated building blocks of each compound of the invention.

In a preferred embodiment of the present invention, the radionuclide has a half-life that allows it to be used for diagnostic and/or therapeutic medical applications. In particular, the half-life is from 1 min to 100 days.

In a preferred embodiment of the present invention, the radionuclide has a decay energy that allows it to be used for diagnostic and/or therapeutic medical applications. In particular, for γ-emitting isotopes, the decay energy is from 0.004 to 10 MeV, preferably from 0.05 to 4 MeV, for diagnostic applications. For positron-emitting isotopes, the decay energy is from 0.6 to 13.2 MeV, preferably from 1 to 6 MeV, for diagnostic applications. For particle-emitting isotopes, the decay energy is from 0.039 to 10 MeV, preferably from 0.4 to 6.5 MeV, for therapeutic applications.

In a preferred embodiment of the invention, the radionuclide is produced on an industrial scale for medical use. In particular, the radionuclide is available in accordance with GMP quality requirements.

In a preferred embodiment of the invention, the daughter nuclide(s) after radioactive decay of the radionuclide are compatible with diagnostic and/or therapeutic medical use. In addition, the daughter nuclides are either stable or continue to decay in a manner that does not interfere with or even support diagnostic and/or therapeutic medical use. Typical radionuclides that can be used in connection with the present invention are summarized in Table 4.

In an embodiment of the present invention, the radionuclide is used for diagnostics. Preferably, the radioactive isotope is selected from the group consisting of, but not limited to, ⁴³ Sc, ⁴⁴ Sc, ⁵¹ Mn, ⁵² Mn, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁴ mTc, ⁹⁹ mTc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ¹⁷⁷ Lu, ²⁰¹ Tl, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I. More preferably, the radionuclide is selected from the group consisting of ⁴³ Sc, ⁴⁴ Sc, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁹ mTc, ¹¹¹ In, ¹⁵² Tb, ¹⁵⁵ Tb, ²⁰³ Pb, ¹⁸ F, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I. Even more preferably, the radionuclide is selected from the group consisting of ⁶⁴ Cu, ⁶⁸ Ga, ⁸⁹ Zr, ⁹⁹ mTc, ¹¹¹ In, ¹⁸ F, ¹²³ I and ¹²⁴ I. However, it will also be clear to one skilled in the art that the use of said radionuclide is not limited to diagnostic purposes, but also covers their use in therapy and theranostics when conjugated to a compound of the invention.

In an embodiment of the present invention, the radionuclide is used for therapy. Preferably, the radioactive isotope is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹¹¹ In, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁶ Th, ²²⁷ Th, ¹³¹ I, ²¹¹ At. More preferably, the radioactive isotope is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I, ²¹¹ At. Even more preferably, the radionuclide is selected from the group consisting of ⁹⁰ Y, ¹⁷⁷ Lu, ²²⁵ Ac, ²²⁷ Th, ¹³¹ I and ²¹¹ At. However, it will also be clear to a person skilled in the art that the use of said radionuclide is not limited to therapeutic purposes, but also covers their use in diagnostics and theranostics when conjugated with a compound of the invention.

In an embodiment of the invention, the compound of the invention is present in the form of a pharmaceutically acceptable salt.

A "pharmaceutically acceptable salt" of a compound of the present invention is preferably an acid salt or a basic salt that is generally recognized in the art as suitable for use in contact with human or animal tissues without excessive toxicity or carcinogenicity, and preferably without irritation, allergic reaction or other problem or complication. Such salts include salts of mineral and organic acids and basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. The compounds of the invention are capable of forming internal salts that are also pharmaceutically acceptable salts.

Suitable pharmaceutically acceptable salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethanedisulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-( _CH2 ) _n -COOH, where n is any integer from 0 to 4, i.e. 0, 1, 2, 3 or 4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium. Other pharmaceutically acceptable salts of the compounds provided herein will be apparent to those skilled in the art. Generally, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of a suitable base or acid in water or in an organic solvent, or a mixture of both. Generally, the use of non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred.

A "pharmaceutically acceptable solvate" of a compound of the invention is preferably a solvate of the compound of the invention formed by association of one or more molecules of a solvent with one or more molecules of the compound of the invention. Preferably, the solvent is a solvent that is generally considered suitable for use in contact with the tissues of humans or animals without excessive toxicity or carcinogenicity and preferably without irritation, allergic reaction or other problem or complication. Such a solvent includes an organic solvent such as alcohols, ethers, esters and amines.

A "hydrate" of a compound of the invention is formed by association of one or more molecules of water with one or more molecules of the compound of the invention. Such a hydrate includes, but is not limited to, a hemihydrate, a monohydrate, a dihydrate, a trihydrate, and a tetrahydrate. Regardless of the composition of the hydrate, all hydrates are generally considered pharmaceutically acceptable.

The compound of the invention has a high binding affinity to FAP and a high inhibitory activity against FAP. Due to this high binding affinity, the compound of the invention is effective, useful and/or suitable as a targeting agent and, if conjugated to another moiety, as a targeting moiety. As preferably used herein, a targeting agent is an agent that interacts with a target molecule, which in this case is said FAP. With regard to the cells and tissues to which the compound of the invention is thus targeted, any cell and tissue, respectively, expressing said FAP is targeted or can be targeted.

In an embodiment of the invention, the compound interacts with a fibroblast activation protein (FAP), preferably with human FAP having the amino acid sequence of SEQ ID NO: 1, or a homologue thereof, wherein the amino acid sequence of the FAP homologue is at least 85% identical to the amino acid sequence of SEQ ID NO: 1. In preferred embodiments of the invention, the identity is 90%, preferably 95%, 96%, 97%, 98% or 99%.

The identity between two nucleic acid molecules can be determined as known to a person skilled in the art. More specifically, a sequence comparison algorithm can be used to calculate the percentage of sequence homology for a test sequence(s) relative to a reference sequence based on established program parameters. The test sequence is preferably a sequence or a protein or polypeptide that is considered identical or to be tested, whether and to what extent it is identical to another protein or polypeptide, whereby such other protein or polypeptide is also referred to as a reference sequence and is preferably a wild-type protein or polypeptide, more preferably human FAP SEQ ID NO: 1.

Optimal alignment of sequences for comparison can be accomplished, for example, by the local homology algorithm of Smith & Waterman (Smith, et al., Advances in Applied Mathematics, 1981, 2: 482), by the homology alignment algorithm of Needleman & Wunsch (Needleman, et al., J Mol Biol, 1970, 48: 443), by the similarity search of Pearson & Lipman (Pearson, et al., Proc Natl Acad Sci USA, 1988, 85: 2444), by computerized applications of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.

One example of an algorithm that is suitable for determining percent sequence identity is that used in the Basic Local Alignment Search Tool (hereafter "BLAST"), see, e.g., Altschul et al., 1990 (Altschul, et al., J Mol Biol, 1990, 215: 403) and Altschul et al., 1997 (Altschul, et al., Nucleic Acids Res, 1997, 25: 3389). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (hereafter "NCBI"). Default parameters used to determine sequence identity using software available from NCBI, e.g., BLASTN (for nucleotide sequences) and BLASTP (for amino acid sequences), are described in McGinnis et al. (McGinnis, et al., Nucleic Acids Res, 2004, 32: W20).

Within the scope of the present invention, the compound of the invention is used or intended for use in a method for treating a disease as disclosed herein. Such a method preferably comprises the step of administering to a subject in need thereof a therapeutically effective amount of the compound of the invention. Such a method includes, but is not limited to, curative or adjuvant treatment of cancer. It is used as a palliative treatment when a cure is not possible and the goal is local control of the disease or relief of symptoms, or as a therapeutic treatment when the therapy has a survival benefit and can be curative.

The method of treating a disease disclosed herein includes treating the disease disclosed herein, including tumors and cancer, and may be used either as primary therapy or as second, third, fourth or last line therapy. The present invention also includes combining a compound of the invention with other therapeutic approaches. It is well known to those skilled in the art that the precise purpose of treatment, including curative, adjuvant, neoadjuvant, therapeutic or palliative treatment, will depend on the type, location and stage of the tumor, as well as the general health of the patient.

In an embodiment of the present invention, the disease is selected from the group consisting of: a neoplasm NEC, a benign neoplasm, a benign or malignant neoplasm of unspecified nature, a malignant neoplasm, a metastatic neoplasm, a primary or metastatic malignant neoplasm of unspecified nature, benign tumor cells, benign or malignant tumor cells of unspecified nature, malignant tumor cells, a malignant tumor of the small cell type, a malignant tumor of the giant cell type, a malignant tumor of the spindle cell type, epithelial neoplasms NEC, benign epithelial tumor, carcinoma in situ NEC, carcinoma NEC, metastatic carcinoma NEC, carcinomatosis, benign epithelioma, malignant epithelioma, large cell carcinoma NEC, carcinoma of the undifferentiated type NEC, carcinoma of the anaplastic type, pleomorphic carcinoma, giant cell and spindle cell carcinoma, giant cell carcinoma, spindle cell carcinoma, pseudosarcomatous carcinoma, polygonal cell carcinoma, spheroid cell carcinoma, microtumor, small cell carcinoma NEC, oat cell carcinoma, small cell carcinoma of spindle cell type, papillary and squamous cell neoplasms, papilloma NEC, papillary carcinoma in situ, papillary carcinoma NEC, warty papilloma, warty carcinoma NEC, squamous cell papilloma, papillary squamous cell carcinoma, inverted papilloma, papillomatosis NEC, squamous cell carcinoma in situ NEC, squamous cell carcinoma NEC, metastatic squamous cell carcinoma NEC, squamous cell carcinoma, keratinizing type NEC, squamous cell carcinoma, large cell nonkeratinizing type, squamous cell carcinoma, small cell nonkeratinizing type, squamous cell carcinoma, spindle cell type, adenoid squamous cell carcinoma, squamous cell carcinoma in situ with questionable stromal invasion, squamous cell carcinoma, microinvasive, erythroplasia of Queyrat, Bowen's disease, lymphoepithelial carcinoma, basal cell neoplasms, basal cell tumor, basal cell carcinoma NEC, multicentric basal cell carcinoma, basal cell carcinoma of focal type, basal cell carcinoma of fibroepithelial type, basal squamous cell carcinoma, metatypic carcinoma, intraepidermal epithelioma Jadassohn, trichoepithelioma, trichofolliculoma, trichilemmoma, pilomatrixoma, transitional cell papillomas and carcinomas, transitional cell papilloma NEC, urothelial papilloma, transitional cell carcinoma in situ, transitional cell carcinoma NEC, Schneiderian papilloma, inverted transitional cell papilloma, Schneiderian carcinoma, spindle cell transitional cell carcinoma, basaloid carcinoma, cloacogenic carcinoma, papillary transitional cell carcinoma, adenomas and adenocarcinomas, adenoma NEC, bronchial adenoma NEC, adenocarcinoma in situ, adenocarcinoma NEC, metastatic adenocarcinoma NEC, plastic linitis, superficial spreading adenocarcinoma, intestinal-type adenocarcinoma, diffuse-type carcinoma, monomorphic adenoma, basal cell adenoma, islet cell adenoma, islet cell carcinoma, insulinoma NEC, insulinoma, malignant, glucagonoma NEC, malignant glucagonoma, gastrinoma NEC, malignant gastrinoma, mixed islet cell and exocrine adenocarcinoma, bile duct adenoma, cholangiocarcinoma, bile duct cystadenoma, bile duct cystadenocarcinoma, hepatocellular carcinoma NEC, benign hepatocholangioma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenoma, trabecular adenocarcinoma, embryonal adenoma, eccrine dermal cylindroma, adenoid cystic carcinoma, cribriform carcinoma, adenomatous polyp NEC, adenocarcinoma in adenomatous polyp, tubular adenoma NEC, tubular adenocarcinoma, adenomatous polyposis coli, adenocarcinoma in adenomatous polyposis coli, multiple adenomatous polyps, solid carcinoma NEC, simple carcinoma, carcinoid tumor NEC, carcinoid tumor malignant, carcinoid tumor argentaffin NEC, carcinoid tumor argentaffin malignant, carcinoid tumor non-argentaffin NEC, carcinoid tumor non-argentaffin malignant, mucocarcinoid tumor malignant, complex carcinoid, pulmonary adenomatosis, bronchioloalveolar adenocarcinoma, alveolar adenoma, alveolar adenocarcinoma, papillary adenoma NEC, papillary adenocarcinoma NEC, villous adenoma NEC, adenocarcinoma in villous adenoma, villous adenocarcinoma, tubulovillous adenoma, chromophobe adenoma, chromophobe carcinoma, acidophilic adenoma, acidophilic carcinoma, mixed acidophilic-basophilic adenoma, mixed acidophilic-basophilic carcinoma, oxyphilic adenoma, oxyphilic adenocarcinoma, basophilic adenoma, basophilic carcinoma, clear cell adenoma, clear cell adenocarcinoma NEC, hypernephroid tumor, renal cell carcinoma, clear cell adenofibroma, granular cell carcinoma, chief cell adenoma, water-clear cell adenoma, water-clear cell adenocarcinoma, mixed cell adenoma, mixed cell adenocarcinoma, lipoadenoma, follicular adenoma, follicular adenocarcinoma NEC, follicular adenocarcinoma, well-differentiated type, follicular adenocarcinoma, trabecular type, microfollicular adenoma, macrofollicular adenoma, papillary and follicular adenocarcinoma, unencapsulated sclerosing carcinoma, multiple endocrine adenomas, juxtaglomerular tumor, adenoma adrenal cortex NEC, adrenal cortex carcinoma, adrenal cortex adenoma of compact cell type, adrenal cortex adenoma of heavily pigmented variant, adrenal cortex adenoma of clear cell type, adrenal cortex adenoma of glomerular cell type, adrenal cortex adenoma of mixed cell type, endometrioid adenoma NEC, endometrioid adenoma, borderline malignancy, endometrioid carcinoma, endometrioid adenofibroma NEC, endometrioid adenofibroma of borderline malignancy, endometrioid adenofibroma, malignant, neoplasms of adnexa and appendages of skin, adenoma of skin appendages, carcinoma of skin appendages, adenoma of sweat glands, sweat gland tumor NEC, sweat gland adenocarcinoma, apocrine adenoma, apocrine adenocarcinoma, eccrine acrospiroma, eccrine spiradenoma, hidrocystoma, papillary hidradenoma, papillary syringadenoma, syringoma NEC, sebaceous adenoma, sebaceous adenocarcinoma, ceruminous adenoma, ceruminous adenocarcinoma, mucoepidermoid neoplasms, mucoepidermoid tumor, mucoepidermoid carcinoma cystic, mucinous and serous neoplasms, cystadenoma NEC, cystadenocarcinoma NEC, serous cystadenoma NEC, serous cystadenoma of borderline malignancy, serous cystadenocarcinoma NEC, papillary cystadenoma NEC, papillary cystadenoma of borderline malignancy, papillary cystadenocarcinoma NEC, papillary serous cystadenoma NEC, papillary serous cystadenoma of borderline malignancy, papillary serous cystadenoma, serous superficial papilloma NEC, serous superficial papilloma of borderline malignancy, serous superficial papillary carcinoma, mucinous cystadenoma NEC, mucinous cystadenoma of borderline malignancy, mucinous cystadenocarcinoma NEC, papillary mucinous cystadenoma, papillary mucinous cystadenoma of borderline malignancy, papillary mucinous cystadenocarcinoma, mucinous adenoma, mucinous adenocarcinoma, pseudomyxoma peritonei, mucin-producing adenocarcinoma, signet ring cell carcinoma, metastatic signet ring cell carcinoma, ductal, lobular, and medullary neoplasms, intraductal carcinoma, noninfiltrating, infiltrating ductal carcinoma, comedocarcinoma, noninfiltrating, comedocarcinoma NEC, juvenile breast carcinoma, intraductal papilloma, noninfiltrating intraductal papillary adenocarcinoma, intracystic papillary adenoma, noninfiltrating intracystic carcinoma, intraductal papillomatosis NEC, subareolar ductal papillomatosis, medullary carcinoma NEC, medullary carcinoma with amyloid stroma, medullary carcinoma with lymphoid stroma, lobular carcinoma in situ, lobular carcinoma NEC, infiltrating ductal carcinoma, inflammatory carcinoma, Paget's disease of the breast, Paget's disease and infiltrating ductal carcinoma of the breast, extramammary Paget's disease, acinar cell neoplasms, acinar cell adenoma, acinar cell tumor, acinar cell carcinoma, complex epithelial neoplasms, adenosquamous carcinoma, adenolymphoma, adenocarcinoma with squamous cell metaplasia, adenocarcinoma with cartilaginous and bone metaplasia, adenocarcinoma with spindle cell metaplasia, adenocarcinoma with apocrine metaplasia, thymoma benign, thymoma, malignant, specialized neoplasms of gonadal tissue, stromal germ cell tumor, thecoma NEC, thecal cell carcinoma, luteoma NEC, granulosa cell tumor NEC, granulosa cell tumor, malignant, granulosa cell thecal cell tumor, androblastoma, benign, androblastoma NEC, malignant androblastoma, Sertoli-Leydig cell tumor, gynandroblastoma, tubular androblastoma NEC, Sertoli cell carcinoma, tubular androblastoma with lipid accumulation, benign Leydig cell tumor, Leydig cell tumor NEC, malignant Leydig cell tumor, hilar cell tumor, lipid cell tumor of ovary, adrenal quiescent tumor, paraganglioma and glomus tumors, paraganglioma NEC, paraganglioma, malignant, sympathetic paraganglioma, parasympathetic paraganglioma, jugular glomus tumor, aortic body tumor, carotid body tumor, extra-adrenal paraganglioma NEC, extra-adrenal paraganglioma, malignant, pheochromocytoma NEC, pheochromocytoma, malignant, glomangiosarcoma, glomus tumor, glomangioma, nevi and melanomas, pigmented nevi NEC, malignant melanoma NEC, nodular melanoma, balloon cell nevus, balloon cell melanoma, halo nevus, fibrous papule NEC, neuronevus, large cell nevus, amelanotic nevus, junctional nevus, malignant melanoma in junctional nevus, premalignant melanosis NEC, malignant melanoma in premalignant melanosis, Hutchinson's melanotic freckle, malignant melanoma in Hutchinson's melanotic freckle, superficial spreading melanoma, intradermal nevus, compound nevus, giant pigmented nevus, malignant melanoma in giant pigmented nevus, epithelioid and spindle cell nevus, epithelioid cell melanoma, spindle cell melanoma NEC, spindle cell melanoma type a, spindle cell melanoma type b, mixed epithelioid and spindle cell melanoma, blue nevus NEC, blue nevus, malignant, cellular blue nevus, tumors and sarcomas of soft tissue NEC, soft tissue tumor, benign, sarcoma NEC, sarcomatosis NEC, spindle cell sarcoma, giant cell sarcoma, small cell sarcoma, epithelioid cell sarcoma, fibromatous neoplasms, fibroma NEC, fibrosarcoma NEC, fibromyxoma, fibromyxosarcoma, periosteal fibroma, periosteal fibrosarcoma, fascial fibroma, fascial fibrosarcoma, infantile fibrosarcoma, elasto fibroma, aggressive fibromatosis, abdominal fibromatosis, desmoplastic fibroma, fibrous histiocytoma NEC, atypical fibrous histiocytoma, fibrous histiocytoma, malignant, fibroxanthoma NEC, atypical fibroxanthoma, malignant fibroxanthoma, dermatofibroma NEC, dermatofibroma protuberans, dermatofibrosarcoma NEC, myxomatous neoplasms, myxoma NEC, myxosarcoma, lipomatous neoplasms, lipoma NEC, liposarcoma NEC, fibrolipoma, well-differentiated liposarcoma, fibromyxolipoma, myxoid liposarcoma, round cell liposarcoma, pleomorphic liposarcoma, mixed-type liposarcoma, intramuscular lipoma, spindle cell lipoma, angiomyolipoma, angiomyoliposarcoma, angiolipoma NEC, angiolipoma infiltrating, myelolipoma, hibernoma, lipoblastomatosis, myomatous neoplasms, leiomyoma NEC, intravascular leiomyomatosis, leiomyosarcoma NEC, epithelioid leiomyoma, epithelioid leiomyosarcoma, cellular leiomyoma, abnormal leiomyoma, angiomyoma, angiomyosarcoma, myoma, myosarcoma, rhabdomyoma NEC, rhabdomyosarcoma NEC, pleomorphic rhabdomyosarcoma, mixed rhabdomyosarcoma, fetal rhabdomyoma, adult rhabdomyoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, complex mixed and stromal neoplasms, endometrial stromal sarcoma, endolymphatic stromal myosis, adenomyoma, pleomorphic adenoma, mixed tumor malignant NEC, Müllerian mixed tumor, mesodermal mixed tumor, mesoblastic nephroma, nephroblastoma NEC, epithelial nephroblastoma, mesenchymal nephroblastoma, hepatoblastoma, carcinosarcoma NEC, embryonal carcinosarcoma, myoepithelioma, mesenchymoma, benign, mesenchymoma NEC, malignant mesenchymoma, embryonal sarcoma, fibroepithelial neoplasms, Brenner tumor NEC, Brenner tumor of borderline malignancy, malignant Brenner tumor, fibroadenoma NEC, intratubular fibroadenoma NEC, peritubular fibroadenoma, adenofibroma NEC, serous adenofibroma, mucinous adenofibroma, cellular intratubular fibroadenoma, cystosarcoma phyllodes NEC, cystosarcoma phyllodes malignant, juvenile fibroadenoma, synovial neoplasms, benign synovioma, synovial sarcoma NEC, spindle cell synovial sarcoma, epithelioid cell synovial sarcoma, biphasic synovial sarcoma, clear cell sarcoma of tendons and aponeuroses, mesothelial neoplasms, mesothelioma, benign, mesothelioma, malignant, fibrous mesothelioma, benign, fibrous mesothelioma, malignant, epithelioid mesothelioma, benign, epithelioid mesothelioma, malignant, biphasic mesothelioma, benign, malignant, adenomatoid tumor NEC, germ cell neoplasms, dysgerminoma, seminoma NEC, anaplastic seminoma, spermatocytic seminoma, germinoma, embryonal carcinoma NEC, endodermal tumor sinus, polyembryoma, gonadoblastoma, benign teratoma, teratoma NEC, teratoma malignant NEC, teratocarcinoma, malignant teratoma of undifferentiated type, malignant teratoma of intermediate type, dermoid cyst, dermoid cyst with malignant transformation, goiter of ovary NEC, malignant goiter of ovary, strumal carcinoid, trophoblastic neoplasms, hydatidiform mole NEC, invasive hydatidiform mole, choriocarcinoma, choriocarcinoma combined with teratoma, malignant teratoma trophoblastic, mesonephromas, mesonephroma benign, mesonephric tumor, mesonephroma malignant, endosalpingioma, tumors of blood vessels, hemangioma NEC, hemangiosarcoma, cavernous hemangioma, venous hemangioma, hemangioma cystic, Kupffer cell sarcoma, hemangioendothelioma, benign, hemangioendothelioma NEC, hemangioendothelioma, malignant, capillary hemangioma, intramuscular hemangioma, Kaposi's sarcoma, angiokeratoma, verrucous keratotic hemangioma, hemangiopericytoma, benign, hemangiopericytoma NEC, hemangiopericytoma, malignant, angiofibroma NEC, hemangioblastoma, tumors of the lymphatic vessels, lymphangioma NEC, lymphangiosarcoma, capillary lymphangioma, cavernous lymphangioma, cystic lymphangioma, lymphangiomyoma, lymphangiomyomatosis, hemolymphangioma, osteomas and osteosarcomas, osteoma NEC, osteosarcoma NEC, chondroblastic osteosarcoma, fibroblastic osteosarcoma, telangiectatic osteosarcoma, osteosarcoma in Paget's disease of bone, juxtacortical osteosarcoma, osteoid osteoma NEC, osteoblastoma, chondromatous neoplasms, osteochondroma, osteochondromatosis NEC, chondroma NEC, chondromatosis NEC, chondrosarcoma NEC, juxtacortical chondroma, juxtacortical chondrosarcoma, chondroblastoma NEC, malignant chondroblastoma, mesenchymal chondrosarcoma, chondromyxoid fibroma, giant cell tumors, giant cell tumor of bone NEC, giant cell tumor of bone malignant, giant cell tumor of the soft tissues tissues NEC, malignant giant cell tumor of soft tissue, miscellaneous bone tumors, Ewing's sarcoma, adamantinoma of long bones, ossifying fibroma, odontogenic tumors, odontogenic tumor benign, odontogenic tumor NEC, odontogenic tumor malignant, dentinoma, cementoma NEC, cementoblastoma benign, cementing fibroma, gigantoform cementoma, odontoma NEC, compound odontoma, complex odontoma, ameloblastic fibroodontoma, ameloblastic odontosarcoma, adenomatoid odontogenic tumor, calcifying odontogenic cyst, ameloblastoma NEC, malignant ameloblastoma, odontoma-ameloblastoma, squamous cell odontogenic tumor, odontogenic myxoma, odontogenic fibroma NEC, ameloblastic fibroma, ameloblastic fibrosarcoma, calcifying epithelial odontogenic tumor, miscellaneous tumors, craniopharyngioma, pinealoma, pineocytoma, pineoblastoma, melanotic neuroectodermal tumor, chordoma, gliomas, malignant glioma, gliomatosis cerebri, mixed glioma, subependymal glioma, subependymal giant cell astrocytoma, choroid plexus papilloma NEC, malignant choroid plexus papilloma, ependymoma NEC, anaplastic ependymoma, papillary ependymoma, myxopapillary ependymoma, astrocytoma NEC, anaplastic astrocytoma, protoplasmic astrocytoma, gemistocytic astrocytoma, fibrillary astrocytoma, pilocytic astrocytoma, spongioblastoma NEC, polar spongioblastoma, astroblastoma, glioblastoma NEC, giant cell glioblastoma, glioblastoma with sarcomatous component, primitive polar spongioblastoma, oligodendroglioma NEC, oligodendroglioma of the anaplastic type, oligodendroblastoma, medulloblastoma NEC, desmoplastic medulloblastoma, medullomyoblastoma, cerebellar sarcoma NEC, monster ocellular sarcoma, neuroepitheliomatous neoplasms, ganglioneuroma, ganglioneuroblastoma, ganglioneuromatosis, neuroblastoma NEC, medulloepithelioma NEC, teratoid medulloepithelioma, neuroepithelioma NEC, spongioneuroblastoma, ganglioglioma, neurocytoma, pacinian tumor, retinoblastoma NEC, retinoblastoma, differentiated type, retinoblastoma, undifferentiated type, olfactory neurogenic tumor, esthesioneurocytoma, esthesioneuroblastoma, esthesioneuroepithelioma, meningiomas, meningioma NEC, meningiomatosis NEC, malignant meningioma, meningotheliomatous meningioma, fibrous meningioma, psammomatous meningioma, angiomatous meningioma, hemangioblastic meningioma, hemangiopericytic meningioma, transitional meningioma, papillary meningioma, meningeal sarcomatosis, nerve sheath tumor, neurofibroma NEC, neurofibromatosis NEC, neurofibrosarcoma, melanotic neurofibroma, plexiform neurofibroma, neurilemmoma NEC, neuromatosis, neurilemmoma malignant, neuroma NEC, granular cell tumors and alveolar sarcoma of soft tissue, granular cell tumor NEC, granular cell tumor malignant, alveolar sarcoma of soft tissue, lymphomas NEC or diffuse, benign lymphomatous tumor, malignant lymphoma NEC, malignant lymphoma of non-Hodgkin's type, malignant lymphoma of undifferentiated cell type NEC, malignant lymphoma of stem cell type, malignant lymphoma of convoluted cell type NEC, lymphosarcoma NEC, malignant lymphoma of lymphoplasmacytoid type, malignant lymphoma of immunoblastic type, malignant lymphoma mixed lymphocytic-histiocytic lymphoma NEC, malignant lymphoma centroblastic-centrocytic diffuse, malignant lymphoma with follicular central cells NEC, malignant lymphoma lymphocytic well-differentiated NEC, malignant lymphoma lymphocytic intermediate differentiated NEC, malignant lymphoma centrocytic malignant lymphoma with follicular central cells cleaved NEC, malignant lymphoma lymphocytic poorly differentiated NEC, prolymphocytic lymphosarcoma, malignant lymphoma of centroblastic type NEC, malignant lymphoma with follicular central cells uncleaved NEC, reticulosarcomas, reticulosarcoma NEC, reticulosarcoma pleomorphic cell type, reticulosarcoma nodular, Hodgkin's disease, Hodgkin's disease NEC, Hodgkin's disease with lymphocyte predominance, Hodgkin's disease with mixed cellularity, Hodgkin's disease with lymphocyte depletion NEC, Hodgkin's disease with lymphocytic depletion of diffuse fibrosis, Hodgkin's disease with lymphocytic depletion of the reticular type, Hodgkin's disease with nodular sclerosis NEC, Hodgkin's disease with nodular sclerosis of the cellular phase, Hodgkin's paragranuloma, Hodgkin's granuloma, Hodgkin's sarcoma, nodular or follicular lymphomas, malignant nodular lymphoma NEC, malignant lymphoma mixed lymphocytic-histiocytic nodular, malignant lymphoma centroblastic-centrocytic follicular, malignant lymphoma lymphocytic well-differentiated nodular, malignant lymphoma lymphocytic of intermediate differentiation nodular, malignant lymphoma with follicular central cells cleaved follicular, malignant lymphoma lymphocytic poorly differentiated nodular, malignant lymphoma of centroblastic type follicular Malignant lymphoma, follicular-centro-cell, noncleaved follicular, mycosis fungoides, mycosis fungoides, Sezary disease, miscellaneous reticuloendothelial neoplasms, microglioma, malignant histiocytosis, histiocytic medullary reticulosis, Letter-Siwe disease, plasma cell tumors, plasma cell myeloma, plasma cell tumor, benign, plasmacytoma NEC, malignant tumor of plasma cells, mast cell tumors, mastocytoma NEC, mast cell sarcoma, malignant mastocytosis, Burkitt's tumor, Burkitt's tumor, leukemias, leukemias NEC, leukemia NEC, acute leukemia NEC, subacute leukemia NEC, chronic leukemia NEC, aleukemic leukemia NEC, complex leukemias, complex leukemia, lymphoid leukemia, lymphoid leukemia NEC, acute lymphoid leukemia, subacute lymphoid leukemia, chronic lymphoid leukemia, aleukemic lymphoid leukemia, prolymphocytic leukemia, plasma cell leukemia, plasma cell leukemia, erythroleukemia, erythroleukemia, acute erythremia, chronic erythremia, lymphosarcoma cell leukemia, lymphosarcoma cell leukemia, myeloid leukemia, myeloid leukemia NEC, acute myeloid leukemia, subacute myeloid leukemia, chronic myeloid leukemia, aleukemic myeloid leukemia, neutrophilic leukemia, acute promyelocytic leukemia, basophilic leukemia, basophilic leukemia, eosinophilic leukemias, eosinophilic leukemia, monocytic leukemias, monocytic leukemia NEC, acute monocytic leukemia, subacute monocytic leukemia, chronic monocytic leukemia, aleukemic monocytic leukemia, various leukemias, mast cell leukemia, megakaryocytic leukemia, megakaryocytic myelosis, myeloid sarcoma, hairy cell leukemia, various myeloproliferative and lymphoproliferative disorders, polycythemia vera, acute panmyelosis, chronic myeloproliferative disorder, myelosclerosis with myeloid metaplasia, idiopathic thrombocythemia, chronic lymphoproliferative disorder.

In an embodiment of the present invention, the disease is selected from the group consisting of: pancreatic tumors, pancreatic adenocarcinoma, tumors of the head of the pancreas, body of the pancreas, tail of the pancreas, pancreatic duct, islets of Langerhans, neck of the pancreas, prostate tumor, prostate adenocarcinoma, prostate, neuroendocrine tumors, breast cancer, tumor of the central part of the breast, upper inner quadrant of the breast, lower inner quadrant of the breast, upper outer quadrant of the breast, lower outer quadrant of the breast, axillary tail of the breast, overlapping lesion of the breast, juvenile breast carcinoma, parathyroid tumors, myeloma, lung cancer, small cell lung cancer, non-small cell lung cancer, tumor of the main bronchus, upper lobe of the lung, middle lobe of the lung, lower lobe of the lung, colorectal carcinoma, ascending colon tumor, hepatic flexure of the colon, transverse colon, splenic flexure of the colon, descending colon, sigmoid colon, crossed colon, small intestine, liver tumors, hepatocellular adenoma, hepatocellular carcinoma, hepatocholangioma, combined hepatocellular carcinoma and cholangiocarcinoma, hepatoblastoma, ovarian carcinoma, sarcoma, osteosarcoma, fibrosarcoma, gastrointestinal stromal tumors, gastrointestinal tract, gastric carcinoma, thyroid carcinoma, medullary thyroid carcinoma, thyroid gland, renal cell carcinoma, renal pelvis, bladder tumors, urinary bladder carcinoma bladder, tumors of the trigone of the bladder, dome of the bladder, lateral wall of the bladder, posterior wall of the bladder, ureteral orifice, urachus, overlapping lesion of the bladder, basal cell carcinoma, basal cell neoplasms, basal cell tumor, basal cell carcinoma, multicentric basal cell carcinoma, basaloid carcinoma, basal cell adenoma, squamous cell carcinoma, squamous cell carcinoma of the oral cavity, squamous cell carcinoma of the larynx, carcinoma of the cervix, tumors of the exocervix, overlapping lesion of the cervix, cervix, isthmus of the uterus, tumors of the ovary, tumors of the cervical esophagus, thoracic esophagus, abdominal esophagus, upper third of the esophagus, middle third of the esophagus, lower third of the esophagus, overlapping lesion of the esophagus, carcinoma endometrial, head and neck cancer, lymphoma, malignant mesothelioma, mesothelial neoplasms, mesothelioma, fibrous mesothelioma, fibrous mesothelioma, epithelioid mesothelioma, epithelioid mesothelioma, duodenal carcinoma, neuroendocrine tumors, neuroendocrine tumors of the lung, neuroendocrine tumors of the pancreas, neuroendocrine tumors of the foregut, neuroendocrine tumors of the midgut, neuroendocrine tumors of the hindgut, gastroenteropancreatic neuroendocrine tumors, neuroendocrine carcinomas, neuroendocrine tumors of the breast, neuroendocrine tumors of the ovary, testicular cancer, thymus carcinoma, tumors of the stomach, gastric fundus, gastric body, gastric antrum, pylorus, lesser curvature of the stomach, greater curvature of the stomach, overlapping lesion of the stomach, paragangliomas, ganglioma, melanomas, malignant melanoma, nodular melanoma, amelanotic melanoma, superficial spreading melanoma, epithelioid cell melanoma, spindle cell melanoma, mixed epithelioid and spindle cell melanoma.

In another embodiment, the above-mentioned indications can occur in organs and tissues selected from the group consisting of: the outer upper lip, the outer lower lip, the outer lip NDU, the mucous membrane of the upper lip, the mucous membrane of the lower lip, the mucous membrane of the lip NDU, the commissure of the lip, an overlapping lesion of the lip, the base of the tongue NDU, the dorsal surface of the tongue NDU, the edge of the tongue, the ventral surface of the tongue NDU, the anterior 2/3 of the tongue NDU, the lingual tonsil, an overlapping lesion of the tongue, the tongue NDU, the upper gingiva, the lower gingiva, the gingiva NDU, the anterior part of the floor of the mouth, the lateral part of the floor of the mouth, an overlapping lesion of the floor of the mouth, the floor of the mouth NDU, the hard palate, the soft palate NDU, the uvula, an overlapping lesion of the palate, the palate NDU, the buccal mucosa, the vestibule of the mouth, the retromolar region, an overlapping lesion of others, and unspecified parts of mouth, mouth NEC, parotid gland, submandibular gland, sublingual gland, overlapping lesion of major salivary glands, major salivary gland NEC, tonsillar fossa, tonsil arch, overlapping lesion of tonsil, tonsil NEC, vallecula, anterior surface of epiglottis, lateral wall of oropharynx, posterior wall of oropharynx, branchial cleft, overlapping lesion of oropharynx, oropharynx NEC, superior wall of nasopharynx, posterior wall of nasopharynx, lateral wall of nasopharynx, anterior wall of nasopharynx, overlapping lesion of nasopharynx, nasopharynx NEC, pyriform sinus, retrocrinoid region, hypopharyngeal portion of laryngeal fold, posterior wall of laryngopharynx, overlapping lesion of laryngopharynx, laryngopharynx NEC, pharynx NEC, laryngopharynx, Waldeyer's ring, overlapping lesion of labium oropharynx, cervical esophagus, thoracic esophagus, peritoneal esophagus, upper esophagus, middle esophagus, lower esophagus, overlapping lesion of esophagus, esophagus NEC, cardiac region NEC, fundus of stomach, body of stomach, antrum of stomach, pylorus, lesser curvature of stomach NEC, greater curvature of stomach NEC, overlapping lesion of stomach, stomach NEC, duodenum, jejunum, ileum, Meckel's diverticulum, overlapping lesion of small intestine, small intestine NEC, cecum, appendix, ascending colon, hepatic flexure of colon, transverse colon, splenic flexure of colon, descending colon colon, sigmoid colon, overlapping lesion of colon, colon NEC, rectosigmoid junction, rectum NEC, anus NEC, anal canal, cloacogenic zone, overlapping lesion of rectum and anal canal, liver, intrahepatic bile ducts, gallbladder, extrahepatic bile ducts, ampulla of Vater, overlapping lesion of biliary tract, biliary tract NEC, head of pancreas, body of pancreas, tail of pancreas, pancreatic duct, islets of Langerhans, neck of pancreas, overlapping lesion of pancreas, pancreas NEC, intestinal tract NEC, overlapping lesion of digestive system, gastrointestinal tract NEC, nasal cavity, middle ear, maxillary sinus, ethmoid sinus, frontal sinus, sphenoid sinus, overlapping lesion of paranasal sinuses, paranasal sinus NEC, glottis, epiglottis, subglottic region, laryngeal cartilage, overlapping lesion of larynx, larynx NEC, trachea, main bronchus, upper lobe of lung, middle lobe of lung, lower lobe of lung, overlapping lesion of lung, lung NEC, thymus, heart, anterior mediastinum, posterior mediastinum, mediastinum NEC, pleura NEC, overlapping lesion of mediastinum of heart and pleura, upper respiratory tract NEC, overlapping lesion of respiratory system and intrathoracic organs, respiratory tract NEC, joints of long bones of upper limb, joints of short bones of upper limb, joints of long bones of lower limb, joints of short bones of lower limb, overlapping lesion of bones, joints and articular cartilages of extremities, bone of extremity NEC, skull and facial bone, mandible, spine, ribs sternum clavicles, pelvic bones, overlapping lesion of bones, joints and articular cartilages, bone NEC, blood, bone marrow, spleen, reticuloendothelial system NEC, hematopoietic system NEC, skin of lips NEC, eyelids NEC, external ear, skin of face, skin of scalp of neck, skin of trunk, skin of upper limb, skin of lower limb, peripheral nerve of neck of head, peripheral nerve of shoulder, peripheral nerve of leg, peripheral nerve of thorax, peripheral nerve of abdomen, peripheral nerve of pelvis, peripheral nerve of spine, overlapping lesion of peripheral nerves and autonomic nervous system, autonomic nervous system NEC, retroperitoneum, peritoneum NEC, overlapping lesion retroperitoneum and peritoneum, connective tissue of head, connective tissue of shoulder, connective tissue of leg, connective tissue of chest, connective tissue of abdomen, connective tissue of pelvis, connective tissue of trunk NEC, overlapping lesion of connective subcutaneous and other soft tissues, connective tissue NEC, nipple, central part of breast, upper inner quadrant of breast, lower inner quadrant of breast, upper outer quadrant of breast, lower outer quadrant of breast, axillary tail of breast, overlapping lesion of breast, breast NEC, labium majora, labium minora, clitoris, overlapping lesion of vulva, vulva NEC, vagina NEC, endocervix, exocervix, overlapping lesion of cervix, cervix, isthmus of uterus, endometrium, myometrium, fundus of uterus, overlapping lesion of corpus uteri, corpus uteri, uterine cavity NEC, ovary, fallopian tube, broad ligament, round ligament, parametrium, uterine appendages, wolffian body, overlapping lesion of female genital organs, female genital organs NEC, foreskin, glans penis, shaft of penis, overlapping lesion of penis, penis NEC, prostate gland, undescended testicle, descended testicle, testicle NEC, epididymis, spermatic cord, scrotum NEC, tunica vaginalis, overlapping lesion of male genital organs, male genital organs NEC, kidneys NEC, renal pelvis, ureter, trigone of bladder, dome of bladder, lateral wall of bladder, posterior wall of bladder, orifice of ureter, urinary bladder, overlapping lesion of bladder, urinary bladder NEC, urethra, paraurethral gland, overlapping lesion of urinary organs, urinary system NEC, conjunctiva, cornea NEC, retina, choroid, ciliary body, lacrimal gland, orbit NEC, overlapping lesion of eye and adnexa, eye NEC, meninges, meninges of spinal cord, meninges NEC, cerebrum, frontal lobe, temporal lobe, parietal lobe, occipital lobe, ventricle NEC, cerebellum NEC, brain stem, overlapping lesion of brain, brain NEC, spinal cord, cauda equina, olfactory nerve, optic nerve, auditory nerve, cranial nerve NEC, overlapping lesion of brain and central nervous system, nervous system NEC, thyroid gland, adrenal cortex, adrenal medulla, adrenal glands NEC, parathyroid gland, pituitary gland, craniopharyngeal duct, pineal gland, carotid body, body of aorta, overlapping lesion of endocrine glands and related structures, endocrine glands NEC, face head or neck NEC, thorax NEC, abdomen NEC, pelvis NEC, upper limbs NEC, lower limbs NEC, other ill-defined sites, overlapping ill-defined sites, lymph node of face, head, neck, intrathoracic lymph node, intra-abdominal lymph nodes, lymph node of axilla, lymph node of inguinal region of leg, pelvic lymph node, lymph nodes of multiple regions, lymph nodes NEC, primary site unknown.

Subjects treated with the compounds disclosed and claimed herein may be treated in combination with other non-surgical antiproliferative (e.g., anticancer) drug therapy. In one embodiment, the compounds may be administered in combination with an anticancer compound, such as a cytostatic compound. A cytostatic compound is a compound (e.g., a small molecule, nucleic acid, or protein) that inhibits the growth and/or proliferation of cells. In some embodiments, the cytostatic compound is directed to malignant tumor cells. In yet other embodiments, the cytostatic compound is a compound that inhibits the growth and/or proliferation of vascular smooth muscle cells or fibroblasts.

Suitable antiproliferative drugs or cytostatic compounds for use in combination with the compounds disclosed and claimed herein include anticancer drugs. Numerous anticancer drugs that can be used are well known and include, but are not limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacytidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Biselesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Karacemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cyrolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Desaguanine; Desaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidin; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Sodium Phosphate; Ethanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Fluorcitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alpha-2a; Interferon Alpha-2b; Interferon Alpha-n1; Interferon Alpha-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Methoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromine; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Niraparib; Nocodazole; Nogalamycin; Olaparib; Ormaplatin; Oxysuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Pyroxantrone Hydrochloride; Plicamycin; Plomestan; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Roglethimide; Rucaparib; Safingol; Safingol Hydrochloride; Semustine; Simtrazen; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talazoparib; Talisomycin; Taxol; Taxotere; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Velaparib; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; and Zorubicin Hydrochloride.

Other anticancer agents include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; acylfulvene; adecipenol; adozelesin; ALL-TK antagonists; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; anagrelide; andrographolide; angiogenesis inhibitors; D antagonist; G antagonist; antarelix; antidorsalizing morphogenetic protein-1; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; regulators of apoptosis; apurinic acid; apa-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorines; benzoylstaurosporine; beta-lactam derivatives; beta-aletin; betaclamycin B; betulinic acid; bFGF inhibitor; bisaziridinylspermine; bisnafide; bistraten A; breflate; budotitan; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarinox IL-2; capecitabine; carboxamide - aminotriazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage origin inhibitor; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chlorquinoxaline sulfonamide; cicaprost; cis-porphyrin; clomiphene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambecidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanethraquinones; cycloplatam; cipemicin; cytarabine oxphosphate; cytolytic factor; cytostatin; dacliximab; dehydrodidemnin B; deslorelin; dexiphosphamide; dexrazoxane; dexverapamil; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenylspiromustine; docosanol; dolasetron; doxifluridine; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomitin; elemen; emifur; epirubicin; episteride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorucin hydrochloride; forfenimex; formestane; fotemustine; gadolinium texaphyrin; gallium nitrate; halocitabine; ganirelix; gelatinase inhibitors; glutathione inhibitors; hepsulfame; Heregulin; Hexamethylene bisacetamide; Hypericin; Ibandronic acid; Idoxifene; Idramantone; Ilmofosine; Ilomastat; Imidazoacridones; Imiquimod; Immunostimulating peptides; Insulin-like growth factor-I receptor inhibitor; Interferon agonists; Interferons; Interleukins; Iobenguane; Iododoxorubicin; Ipomeanol, 4-; Irinotecan; Iroplast; Irsogladine; Isobengazole; Isohomohalicondrine B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate; Leptolstatin; Leukemia inhibitory factor; Leukocyte alpha-interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribin; lartothecan; lutetium texaphyrin; lysophylline; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; merbarone; metrelin; methioninase; metoclopramide; MTF inhibitor; mifepristone; miltefosine; mirmostim; mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor - saporin; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A + myobacterial cell wall sk; mopidamole; multidrug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer compound; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinalin; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavine; nafterpine; narthragrastim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nizamycin; nitric oxide modulators; nitroxide antioxidant; nitrulline; O6-benzylguanine; octreotide; oxycenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoyl rhizoxin; pamidronic acid; panaxytriol; panomifen; parabactin; pazelliptin; pegaspargase; peldesin; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perphosphamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; pyritrexime; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum triamine complex; sodium porfimer; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin-polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras-arnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptin demethylated; rhenium etidronate Re 186; rhizoxin; ribozymes; RII retinamide; rohitukin; romurtide; roquinimex; rubiginone B1; ruboxil; sentopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; senescence inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; thallimustine; tamoxifen methiodide; tavromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrilium; telomerase inhibitors; temozolomide; tetrachlordecaoxide; tetrazomine; taliblastine; thalidomide; thiocoralin; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinane; thyroid-stimulating hormone; tin ethyl etiopurpurine; titanocene dichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus growth inhibitory factor; urokinase receptor antagonists; Variolinin B; vector system, gene therapy of red blood cells; Velarezole; Veramin; Verdeny; Vinorelbine; Vinxaltin; Vitaxin; Zanotherone; Zilascorb; and Zinostatin Stimulant.

The compounds disclosed and claimed herein may also be used in combination with any of the following treatment methods:

Combination therapy with poly(ADP-ribose) polymerase (PARP) inhibitors, a class of chemotherapeutic agents that target DNA damage repair in cancers (Yuan, et al., Expert Opin Ther Pat, 2017, 27: 363). Such PARP inhibitors include, but are not limited to, olaparib, rupacarib, velaparib, niraparib, talazoparib, pamiparib, iniparib, E7449, and A-966492.

Therapy in combination with inhibitors of signaling pathways and mechanisms that lead to repair of single- and double-strand DNA breaks, such as nuclear factor kappaB signaling (Pilie, et al., Nat Rev Clin Oncol, 2019, 16: 81; Zhang, et al., Chin J Cancer, 2012, 31: 359). Such inhibitors include, but are not limited to, inhibitors of ATM and ATR kinases, checkpoint kinases 1 and 2, DNA-dependent protein kinases, and WEE1 kinases (Pilie, et al., Nat Rev Clin Oncol, 2019, 16: 81).

Therapy in combination with an immunomodulator (Khalil, et al., Nat Rev Clin Oncol, 2016, 13: 394), a cancer vaccine (Hollingsworth, et al., NPJ Vaccines, 2019, 4: 7), an immune checkpoint inhibitor (e.g., PD-1, PD-L1, CTLA-4 inhibitor) (Wei, et al., Cancer Discov, 2018, 8: 1069), a Cyclin D-Kinase 4/6 inhibitor (Goel, et al., Trends Cell Biol, 2018, 28: 911), an antibody capable of binding to a tumor cell and/or metastasis and capable of inducing antibody-dependent cellular cytotoxicity (ADCC) (Kellner, et al., Transfus Med Hemother, 2017, 44: 327), T cell or NK cell recruiter (e.g., bispecific antibodies) (Yu, et al., J Cancer Res Clin Oncol, 2019, 145: 941), cell therapy using expanded autologous or allogeneic immune cells (e.g., chimeric antigen receptor T (CAR-T) cells) (Khalil, et al., Nat Rev Clin Oncol, 2016, 13: 394). Immune checkpoint inhibitors include, but are not limited to, nivolumab, ipilimumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

According to the present invention, the compounds can be administered before, simultaneously with, or after other anti-cancer compounds. The administration schedule can include administration of different agents in turn. In other embodiments, the compounds can be delivered before and during, or during and after, or before and after treatment with other therapies. In some cases, the compound is administered more than 24 hours before administration of another anti-proliferative treatment. In other embodiments, the subject can be administered more than one anti-proliferative therapy. For example, the subject can receive the present compounds in combination with both surgery and at least one other anti-proliferative compound. Alternatively, the compound can be administered in combination with more than one anti-cancer drug.

In one embodiment of the invention, the compounds of the present invention are used to detect cells and tissues overexpressing FAP, wherein such detection is achieved by conjugating a detectable label to the compounds of the present invention, preferably a detectable radionuclide. In a preferred embodiment of the invention, the detected cells and tissues are diseased cells and tissues and/or are either the cause of a disease and/or symptoms of a disease or part of the pathology underlying the disease. In another preferred embodiment of the invention, the diseased cells and tissues cause and/or are part of an oncological indication (e.g. neoplasms, tumors and cancer) or a non-oncological indication (e.g. inflammatory disease, cardiovascular disease, autoimmune disease and fibrotic disease).

In another embodiment of the invention, the compounds of the present invention are used to treat cells and tissues that overexpress FAP. In a preferred embodiment of the invention, the cells and tissues treated are diseased cells and tissues and/or are either the cause of a disease and/or symptoms of a disease or part of the pathology underlying the disease. In a further preferred embodiment of the invention, the diseased cells and tissues cause and/or are part of an oncological indication (e.g., neoplasms, tumors and cancer), and the therapeutic activity is achieved by conjugating a therapeutically active effector to the compounds of the present invention, preferably a therapeutically active radionuclide. In another preferred embodiment of the invention, the diseased cells and tissues cause and/or are part of a non-oncological indication (e.g., an inflammatory disease, a cardiovascular disease, an autoimmune disease and a fibrotic disease), and the therapeutic activity is achieved by inhibiting the enzymatic activity of FAP.

In a further embodiment of the invention, particularly where the disease is a non-oncologic disease or non-oncologic indication (e.g., an inflammatory disease, a cardiovascular disease, an autoimmune disease, and a fibrotic disease), the compounds of the present invention are administered in therapeutically effective amounts; preferably, the compound of the present invention does not contain a therapeutically active nuclide. An effective amount is a dose of the compound sufficient to provide a therapeutically or medically desired result or effect in the subject to which the compound is administered. An effective amount will vary depending on the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concomitant or combination therapy (if any), the particular route of administration, and similar factors within the knowledge and experience of the practitioner. For example, in connection with methods directed to the treatment of subjects having a condition characterized by abnormal cell proliferation, an effective amount for suppressing proliferation will be an amount sufficient to reduce or completely stop the abnormal cell proliferation in order to slow or stop the development or progression of a cell mass, such as, for example, a tumor. As used in embodiments of the invention, "inhibit" includes all of the above.

In other embodiments of the invention, the therapeutically effective amount will be the amount necessary to prolong the dormancy period of micrometastases or to stabilize any residual primary tumor cells after surgical or drug therapy.

In general, when using an unconjugated compound without a therapeutically active radionuclide, the therapeutically effective amount will vary depending on the age, condition, and sex of the subject, as well as the nature and extent of the disease in the subject, all of which can be determined by one of skill in the art. The dosage may be adjusted by the attending physician or veterinarian, especially in the event of any complications. The therapeutically effective amount is typically, but not limited to, from 0.1 mcg/kg to about 2000 mg/kg, or from 1.0 mcg/kg to about 1000 mg/kg, or from about 0.1 mg/kg to about 500 mg/kg, or from about 1.0 mg/kg to about 100 mg/kg, in one or more administrations per day for one or more days. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses, for example, administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the compounds are administered for more than 7 days, more than 10 days, more than 14 days, and more than 20 days. In still other embodiments, the compound is administered for weeks or months. In still other embodiments, the compound is delivered every other day. For example, the agent is delivered every two days, or every three days, or every four days, or every five days, or every six days, or every week, or every month.

In a preferred embodiment of the invention, the compound of the present invention is for use in the treatment and/or prevention of a disease, wherein such treatment is radionuclide therapy.

Preferably, radionuclide therapy uses or is based on various forms of radiation emitted by a radionuclide. Such radiation may, for example, be any of photon radiation, electron radiation, including but not limited to β-particles and Auger electrons, proton radiation, neutron radiation, positron radiation, α-particle radiation or an ion beam. Depending on the type of particles or radiation emitted by said radionuclide, radionuclide therapy may, for example, be differentiated as photon radionuclide therapy, electron radionuclide therapy, proton radionuclide therapy, neutron radionuclide therapy, positron radionuclide therapy, α-particle radionuclide therapy or ion beam radionuclide therapy. All these forms of radionuclide therapy are encompassed by the present invention, and all these forms of radionuclide therapy can be realized with the compound of the invention, preferably provided that the radionuclide is attached to the compound of the invention, more preferably as an effector providing this type of radiation.

Radionuclide therapy works primarily by damaging the DNA of cells. The damage is caused by a beam of photons, electrons, protons, neutrons, positrons, alpha particles or ions that directly or indirectly ionize the atoms that make up the DNA chain. Indirect ionization occurs through the ionization of water to form free radicals, especially hydroxyl radicals, which then damage the DNA.

In the most common forms of radionuclide therapy, most of the radiation exposure comes from free radicals. Because cells have mechanisms to repair DNA damage, DNA breaks on both strands are the most important way to change the characteristics of cells. Because cancer cells are generally undifferentiated and stem cell-like, they reproduce more and have less ability to repair sublethal damage than most healthy differentiated cells. DNA damage is transmitted through cell division, causing cancer cells to accumulate, causing them to die or reproduce more slowly.

Oxygen is a powerful radiosensitizer, increasing the effectiveness of a given dose of radiation by generating DNA-damaging free radicals. Consequently, high-pressure oxygen tanks, blood substitutes containing increased levels of oxygen, hypoxic cell radiosensitizers such as misonidazole and metronidazole, and hypoxic cytotoxins such as tirapazamine can be used.

Other factors considered when choosing a radiation dose include whether the patient is receiving chemotherapy, whether radiation therapy is given before or after surgery, and the success rate of the surgery.

The total radioactive dose may be fractionated, i.e., spread over time in one or more treatments, for several important reasons. Fractionation allows normal cells time to recover, whereas tumor cells tend to recover less efficiently between fractions. Fractionation also allows tumor cells that were in a relatively radioresistant phase of the cell cycle during one treatment to enter a sensitive phase of the cycle before the next fraction is administered. Similarly, tumor cells that have been chronically or acutely hypoxic, and therefore more radioresistant, may reoxygenate between fractions, improving tumor cell killing.

It is well known that different types of cancer respond differently to radiation therapy. A cancer's response to radiation is described by its radiosensitivity. Very radiosensitive cancer cells are quickly destroyed by moderate doses of radiation. These include leukemia, most lymphomas, and germ cell tumors.

In real clinical practice, it is important to distinguish between the radiosensitivity of a particular tumor, which is to some extent a laboratory parameter, and the “curability” of the cancer with an internally delivered dose of radiation. For example, leukemias are generally not curable with radiation therapy because they are spread throughout the body. Lymphoma may be radically curable if localized to one area of the body. Similarly, many common tumors with a moderate radioresponse can be treated with therapeutic doses of radioactivity if they are at an early stage. This applies, for example, to non-melanoma skin cancer, head and neck cancer, non-small cell lung cancer, cervical cancer, anal cancer, prostate cancer.

The response of a tumor to radiation therapy also depends on its size. For complex reasons, very large tumors respond less well to radiation than smaller tumors or microscopic disease. Various strategies are used to overcome this effect. The most common is surgical resection before radiation therapy. This is most often seen in the treatment of breast cancer with wide local excision or mastectomy followed by adjuvant radiation therapy. Another strategy is to shrink the tumor with neoadjuvant chemotherapy before definitive radionuclide therapy. A third strategy is to increase the radiosensitivity of the cancer by administering certain drugs during the course of radiation therapy. Examples of radiosensitizing drugs include, but are not limited to, Cisplatin, Nimorazole, and Cetuximab.

Intraoperative radiotherapy is a special type of radiation therapy that is given immediately after surgery to remove cancer. It has been used for breast cancer (TARGeted Introperative radiotherapy), brain tumors, and rectal cancer.

Radionuclide therapy itself is painless. Many low-dose palliative treatments cause minimal or no side effects. Higher-dose treatments may cause a variety of side effects during treatment (acute side effects), months or years after treatment (long-term side effects), or after repeated treatment (cumulative side effects). The nature, severity, and duration of side effects depend on the organs being irradiated, the treatment itself (type of radionuclide, dose, fractionation, concomitant chemotherapy), and the patient.

Within the scope of the present invention, the method of treating a disease according to the invention may implement each and any of the above strategies, which, as such, are known in the art and which constitute further embodiments of the invention.

Also within the scope of the present invention, the compound of the invention is used in a method for diagnosing a disease as described herein. Such a method preferably comprises the step of administering to a subject in need thereof a diagnostically effective amount of the compound of the invention.

According to the present invention, the imaging method is selected from the group consisting of scintigraphy, Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET).

Scintigraphy is a form of diagnostic test or technique used in nuclear medicine in which radiopharmaceuticals are internalized by cells, tissues, and/or organs, preferably internalized in vivo, and the radiation emitted by said internalized radiopharmaceuticals is detected by external detectors (gamma cameras) to form and display two-dimensional images. In contrast, SPECT and PET form and display three-dimensional images. Because of this, SPECT and PET are classified as separate modalities from scintigraphy, although they also use gamma cameras to detect internal radiation. Scintigraphy is distinct from diagnostic x-rays, in which external radiation is passed through the body to form an image.

Single Photon Emission Tomography (SPECT) is a nuclear imaging technique that uses gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. Before a SPECT scan, the patient is injected with a radioactively labeled chemical that emits gamma rays that can be detected by the scanner. A computer collects the information from the gamma camera and translates it into two-dimensional cross-sections. These cross-sections can be put together to form a three-dimensional image of an organ or tissue. SPECT involves detecting gamma rays emitted individually and sequentially by a radionuclide contained in a radioactively labeled chemical. To produce SPECT images, the gamma camera rotates around the patient. Projections are taken at specific points during the rotation, typically every 3 to 6 degrees. In most cases, a full 360-degree rotation is used to obtain an optimal reconstruction. The time required to obtain each projection also varies, but is typically 15 to 20 seconds. This gives a total scan time of 15-20 minutes. Multi-head gamma cameras are faster. Since SPECT acquisition is very similar to planar gamma camera imaging, the same radiopharmaceuticals can be used.

Positron Emission Tomography (PET) is a noninvasive diagnostic imaging technique for measuring the biochemical status or metabolic activity of cells in the human body. PET is unique in that it produces images of the body’s basic biochemistry or functions. Traditional diagnostic techniques, such as x-rays, CT scans, or MRIs, produce images of the anatomy or structure of the body. The premise of these techniques is that any changes in structure or anatomy associated with disease can be seen. Biochemical processes are also altered by disease and can occur before any major changes in anatomy. PET is an imaging technique that can visualize some of these early biochemical changes. PET scanners rely on radiation emitted from the patient to create images. Each patient is given a small amount of a radioactive pharmaceutical that is either very similar to a natural substance used by the body or that binds specifically to a receptor or molecular structure. When a radioisotope undergoes positron emission decay (also known as positive beta decay), it emits a positron, the antiparticle analogue of an electron. After traveling a few millimeters, the positron encounters an electron and annihilates, creating a pair of annihilation (gamma) photons traveling in opposite directions. These are detected when they hit the scintillator material in the scanner, creating a flash of light that is detected by photomultiplier tubes or silicon avalanche photodiodes. The technique depends on the simultaneous or coincident detection of the photon pair. Photons that do not arrive in pairs, i.e. within a few nanoseconds, are ignored. All coincidences are sent to the image processing unit, where the final image data is produced using image reconstruction routines.

SPECT/CT and PET/CT are a combination of SPECT and PET with computed tomography (CT). The key benefits of combining these modalities is increased reader confidence and accuracy. With traditional PET and SPECT, the limited number of photons emitted from the abnormal area creates a very low-level background, making it difficult to localize the area anatomically. The addition of CT helps to locate the abnormal area anatomically and classify the likelihood that it represents disease.

Within the scope of the present invention, the method for diagnosing a disease according to the present invention may implement each and any of the above strategies, which, as such, are known in the art and which constitute further embodiments of the invention.

The compounds of the present invention are useful for patient stratification, i.e., creating subgroups within a patient population that provide more detailed information about how a patient will respond to a given drug. Stratification can be a critical component in converting a clinical trial from a negative or neutral result to a positive result by identifying a subset of the population that is most likely to respond to a new therapy.

Stratification involves identifying a group of patients with common "biological" characteristics to select optimal patient treatment and achieve the best possible outcome in terms of risk assessment, risk avoidance, and optimal treatment outcome.

The compound of the present invention can be used to assess or detect a particular disease at the earliest possible stage (which is a diagnostic use), the risk of developing a disease (which is a susceptibility/risk use), the progression of a disease, including subtle versus aggressive (which is a prognostic use), and it can be used to predict the response and toxicity to a given treatment (which is a prognostic use).

Also within the scope of the present invention, the compound of the invention is used in a therapeutic method. The concept of theranostics is to combine a therapeutic agent with an appropriate diagnostic test, which can increase the clinical utility of the therapeutic drug. The concept of theranostics is becoming increasingly attractive and is widely considered to be the key to improving the effectiveness of drug treatment, helping physicians identify patients who may benefit from a given therapy and thus avoid unnecessary treatment.

The concept of theranostics is to combine a therapeutic agent with a diagnostic test that allows physicians to identify those patients who will benefit most from a given therapy. In an embodiment of the invention and as preferably used herein, a compound of the invention is used to diagnose a patient, i.e. identify and localize the primary tumor mass as well as potential local and distant metastases. In addition, the tumor volume can be determined, especially using three-dimensional diagnostic methods such as SPECT or PET. Only those patients who have FAP-positive tumor masses and who can therefore benefit from a given therapy are selected for a specific therapy, and therefore unnecessary treatment is avoided. Preferably, such therapy is a FAP-targeted therapy using a compound of the invention. In one particular embodiment, a chemically identical tumor-targeted diagnostic is used, preferably an imaging diagnostic for scintigraphy, PET or SPECT and radiation therapy. Such compounds differ only in the radionuclide and therefore typically have a very similar, if not identical, pharmacokinetic profile. This can be achieved using a chelator and a diagnostic or therapeutic radiometal. Alternatively, this can be achieved using a precursor for a radiolabel and a radiolabel with either a diagnostic or therapeutic radionuclide. In one embodiment of the invention, diagnostic imaging is preferably used by quantifying the radiation of the diagnostic radionuclide and subsequent dosimetry, which is known to those skilled in the art, and predicting drug concentrations in the tumor compared to vulnerable organs with side effects. In this way, a truly individualized drug dosing therapy for the patient is achieved.

In an embodiment of the invention and as preferably used herein, the theranostic method is implemented with only one theranostically active compound, such as a compound of the present invention, labeled with a radionuclide that emits diagnostically detectable radiation (e.g., positrons or gamma rays) as well as therapeutically effective radiation (e.g., electrons or alpha particles).

The invention also provides a method for intraoperative identification/detection of diseased tissues expressing FAP in a subject. Such a method uses a compound of the invention, wherein such a compound of the invention preferably includes a diagnostically active agent as an effector.

According to a further embodiment of the invention, a compound of the invention, particularly if complexed with a radionuclide, may be used as an adjuvant or adjuvant to any other tumor treatment, including surgery as the primary treatment for most isolated solid cancers, radiation therapy including the use of ionizing radiation in an attempt to cure or improve the symptoms of cancer using sealed internal sources in the form of brachytherapy or external sources, chemotherapy such as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and other antineoplastic agents, hormonal agents that modulate the behavior of tumor cells without directly attacking these cells, targeting agents that directly target molecular abnormalities in certain types of cancer including monoclonal antibodies and tyrosine kinase inhibitors, angiogenesis inhibitors, immunotherapy, cancer vaccination, palliative care including actions to reduce physical, emotional, spiritual and psychosocial disorders to improve the patient's quality of life, and alternative therapies, including various groups of health systems, practices and products that are not part of conventional medicine.

In an embodiment of the methods of the invention, the subject is a patient. In an embodiment of the invention, the patient is a subject that has been diagnosed as having or suspected of having, or at risk of developing, a disease or a disease, wherein the disease is a disease described herein, and preferably the disease involves FAP.

The dosages used in the practice of the methods of treatment and diagnosis, respectively, when a radionuclide is used and, more particularly, attached to a compound of the invention or a part thereof, will vary depending on, for example, the particular condition to be treated, for example, the known radiosensitivity of the tumor type, the tumor volume and the desired therapy. Typically, the dose is calculated based on the distribution of radioactivity in each organ and the observed target uptake. The γ-emitting complex can be administered one or more times for diagnostic imaging. For animals, the said dose range can be from 0.1 μg/kg to 5 mg/kg of the compound of the invention complexed with, for example, 1-200 MBq ¹¹¹ In or ⁸⁹ Zr. The β-emitting complex of the compound of the invention can be administered at several time points, for example, over a period of 1 to 3 weeks or longer. For animals, the said dose range may be from 0.1 μg/kg to 5 mg/kg of the compound of the invention complexed with, for example, 1-200 MBq ^{of 90} Y or ¹⁷⁷ Lu. For larger mammals, for example, humans, the said dose range is from 0.1 to 100 μg/kg of the compound of the invention complexed with, for example, 10-400 MBq of ¹¹¹ In or ⁸⁹ Zr. For larger mammals, for example, humans, the said dose range is from 0.1 to 100 μg/kg of the compound of the invention complexed with, for example, 10-5000 MBq ^{of 90} Y or ¹⁷⁷ Lu.

In a further aspect, the present invention relates to a composition and, in particular, to a pharmaceutical composition comprising a compound of the invention.

The pharmaceutical composition of the present invention comprises at least one compound of the invention and optionally one or more carrier substances, excipients and/or adjuvants. The pharmaceutical composition may further comprise, for example, one or more of water, buffers such as, for example, a neutral buffer solution or a phosphate buffer solution, ethanol, mineral oil, vegetable oil, dimethyl sulfoxide, carbohydrates such as, for example, glucose, mannose, sucrose or dextrans, mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, and/or preservatives. In addition, one or more other active ingredients may, but need not, be included in the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention may be formulated for any suitable route of administration, including, for example, topical, such as, for example, transdermal or ocular, oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular, such as, for example, intravenous, intramuscular, intrathecal and intraperitoneal injection, as well as any similar injection or infusion techniques. A preferred route of administration is intravenous administration.

In an embodiment of the invention, the compound of the invention containing the radionuclide is administered by any conventional route, in particular intravenously, for example in the form of solutions or suspensions for injection. The compound of the invention can also be advantageously administered by infusion, for example by infusion over a period of 30 to 60 minutes.

Depending on the tumor site, the compound according to the invention can be administered as close as possible to the tumor site, for example, using a catheter. Such administration can be carried out directly into the tumor tissue, into the surrounding tissue or into the afferent blood vessels. The compound according to the invention can also be administered repeatedly in doses, preferably in divided doses.

According to a preferred embodiment of the invention, the pharmaceutical composition according to the invention comprises a stabilizer, e.g. a free radical scavenger, which inhibits autoradiolysis of the compound according to the invention. Suitable stabilizers include, e.g., serum albumin, ascorbic acid, retinol, gentisic acid or derivatives thereof, or an amino acid infusion solution, e.g., used for parenteral protein nutrition, preferably electrolyte- and glucose-free, e.g., a commercially available amino acid infusion such as Proteinsteril® KE Nephro. Preferred are ascorbic acid and gentisic acid.

The pharmaceutical composition of the invention may contain further additives, for example, an agent for adjusting the pH from 7.2 to 7.4, for example, sodium or ammonium acetate or Na ₂ HPO ₄ . Preferably, the stabilizer is added to the non-radioactive compound of the invention, and the introduction of the radionuclide, for example, complexation with the radionuclide, is carried out in the presence of the stabilizer either at room temperature or, preferably, at a temperature of from 40 to 120°C. Complexation is conveniently carried out under air-free conditions, for example, in an N ₂ or Ar atmosphere. After complexation, an additional stabilizer may be added to the composition.

The elimination of the compound of the invention, especially if the Effector is a radionuclide, occurs primarily via the kidneys. Additional protection of the kidneys from accumulation of radioactivity can be achieved by administering lysine or arginine or an amino acid solution with a high content of lysine and/or arginine, e.g. a commercially available amino acid solution such as Synthamin®-14 or -10, before injection or together with the compound of the invention, especially if the Effector is a radionuclide. Kidney protection can also be achieved by administering plasma expanders such as e.g. gelofusine, instead of or in addition to amino acid infusion. Kidney protection can also be achieved by administering diuretics that provide forced diuresis, which increases the rate of urination. Such diuretics include high-ceiling loop diuretics, thiazides, carbonic anhydrase inhibitors, potassium-sparing diuretics, calcium-sparing diuretics, osmotic diuretics and low-ceiling diuretics. The pharmaceutical composition of the invention may contain, in addition to the compound of the invention, at least one of these additional compounds intended or suitable for kidney protection, preferably for kidney protection in a subject to whom the compound of the invention is administered.

It will be clear to a person skilled in the art that the compound of the invention is disclosed herein for use in various methods. It will also be clear to a person skilled in the art that the composition of the invention and the pharmaceutical composition of the invention can be used equally in said various methods. It will also be clear to a person skilled in the art that the composition of the invention and the pharmaceutical composition are disclosed herein for use in various methods. It will equally be clear to a person skilled in the art that the compound of the invention can be used equally in said various methods.

It will be understood by one skilled in the art that the composition of the invention and the pharmaceutical composition of the invention comprise one or more additional compounds in addition to the compound of the invention. To the extent that such one or more additional compounds are disclosed herein as part of the composition of the invention and/or the pharmaceutical composition of the invention, it will be understood that such one or more additional compounds can be administered separately from the compound of the invention to the subject to be treated or to the subject in accordance with the method of the invention. Such administration of the one or more additional compounds can be performed before, simultaneously with, or after the administration of the compound of the invention. It will also be understood by one skilled in the art that in the method of the invention, one or more additional compounds can be administered to the subject in addition to the compound of the invention. Such administration of one or more additional compounds can be performed before, simultaneously with, or after the administration of the compound of the invention. To the extent that such one or more additional compounds are disclosed herein as being administered as part of the method of the invention, it will be understood that such one or more additional compounds are part of the composition of the invention and/or the pharmaceutical composition of the invention. Within the scope of the present invention, the compound of the invention and one or more additional compounds may be contained in the same or different formulations. Also within the scope of the present invention, the compound of the invention and one or more additional compounds are not contained in the same formulation, but are contained in the same package containing a first formulation containing the compound of the invention and a second formulation containing one or more additional compounds, where the type of formulation may be the same or may be different.

Within the scope of the present invention, more than one type of compound of the invention is contained in the composition of the invention and/or the pharmaceutical composition of the invention. Also within the scope of the present invention, more than one type of compound of the invention is used, preferably administered by the method of the invention.

It will be understood that the composition of the invention and the pharmaceutical composition of the invention can be produced in a conventional manner.

The radioactivity of radiopharmaceuticals decreases over time as a result of radioactive decay. The physical half-life of a radionuclide is often short for diagnostic radiopharmaceuticals. In these cases, final preparation must be carried out shortly before administration to the patient. This applies in particular to positron-emitting tomography radiopharmaceuticals (PET radiopharmaceuticals). This often leads to the use of semi-finished products such as radionuclide generators, radioactive precursors and kits.

Preferably, the kit of the invention comprises, in addition to one or more compounds of the invention, typically at least one of the following: instructions for use, final preparation and/or quality control, one or more optional excipients, one or more additional reagents for the labeling procedure, optionally one or more radionuclides with or without shielded containers, and optionally one or more devices, wherein the device(s) is(are) selected from the group consisting of a labeling device, a purification device, an analytical device, a processing device, a radioprotection device or an administration device.

Shielded containers, known as "pigs" for general handling and transport of radiopharmaceutical containers, come in a variety of configurations for storing radiopharmaceutical containers such as bottles, vials, syringes, etc. One form often includes a removable lid that allows access to the enclosed container containing the radiopharmaceutical. When the pig lid is in place, the radiation is acceptable.

The labeling device is selected from the group of open reactors, closed reactors, microfluidic systems, nanoreactors, cartridges, pressure vessels, vials, temperature-controlled reactors, stirred or shaken reactors, and combinations thereof.

The purification device is preferably selected from the group of columns or devices for ion exchange chromatography, columns or devices for size exclusion chromatography, columns or devices for affinity chromatography, columns or devices for gas or liquid chromatography, columns or devices for solid phase extraction, filter devices, columns or devices for centrifuge bottles.

The analytical device is preferably selected from a group of tests or test devices for determining the identity, radiochemical purity, radionuclide purity, radioactivity content and specific radioactivity of a radiolabeled compound.

The processing device is preferably selected from the group consisting of devices for mixing, diluting, dosing, labeling, injecting and administering radiopharmaceuticals to a subject.

The radioprotective device is used to protect doctors and other personnel from radiation when using therapeutic or diagnostic radionuclides. The radioprotective device is preferably selected from the group consisting of devices with protective barriers made of radiation-absorbing material selected from the group consisting of aluminum, plastic, wood, lead, iron, lead glass, water, rubber, plastic, fabric, devices that ensure adequate distances from radiation sources, devices that reduce the time of exposure to the radionuclide, devices that limit inhalation, ingestion or other methods of getting the radioactive material into the body, and devices that provide a combination of these measures.

The device for administration is preferably selected from the group of syringes, shielded syringes, needles, pumps and infusion devices. Syringe guards are usually hollow cylindrical structures that accommodate the cylindrical body of the syringe and are made of lead or tungsten with a lead glass window that allows the operator to see the syringe plunger and the volume of liquid inside the syringe.

The present invention is further illustrated with reference to the following figures and examples, from which further features, embodiments of the invention and advantages may be taken, wherein

Fig. 1 shows the radiochromatogram ^{of 177} Lu-3BP-3407 in formulation buffer containing 100 mg/ml ascorbate and 5 mg/ml L-methionine, analyzed immediately after synthesis;

Fig. 2 shows the radiochromatogram ^{of 177} Lu-3BP-3407 in formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed six days after synthesis;

Fig. 3 shows the radiochromatogram ^{of 177} Lu-3BP-3554 in formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed immediately after synthesis;

Fig. 4 shows the radiochromatogram ^{of 177} Lu-3BP-3554 in formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed six days after synthesis;

Fig. 5 shows the percentage uptake of the administered dose per gram of tissue (%ID/g) in kidney, liver, blood pool, and HEK-FAP tumor determined by SPECT imaging ^{of 111} In-3BP-3407 at 1 h, 3 h, 6 h, and 24 h after injection in a mouse model;

Fig. 6 shows the percentage uptake of the administered dose per gram of tissue (%ID/g) in kidney, liver, blood pool, and HEK-FAP tumor determined by SPECT imaging ^{of 111} In-3BP-3554 at 1 h, 3 h, 6 h, and 24 h after injection in a mouse model;

Fig. 7 shows the percentage uptake of the administered dose per gram of tissue (%ID/g) in kidney, liver, blood pool, and HEK-FAP tumor determined by SPECT imaging ^{of 111} In-3BP-3554 at 1 h, 3 h, 6 h, and 24 h after injection in a mouse model;

Fig. 8 shows the amino acid sequences of human fibroblast activation protein (FAP), human dipeptidyl peptidase 4 (DDP4), and human prolyl endopeptidase (PREP);

Fig. 9A shows tumor growth over time in HEKFAP tumor-bearing mice treated with vehicle, cold ^nat Lu-3BP-3554, 30 MBq (low dose) ¹⁷⁷ Lu-3BP-3554, and 60 MBq (high dose) 177Lu-3BP-3554;

Fig. 9B shows the percentage change in body weight over time in HEK-FAP tumor-bearing mice treated with vehicle, cold ^nat Lu-3BP-3554, 30 MBq (low dose) ¹⁷⁷ Lu-3BP-3554, and 60 MBq (high dose) ¹⁷⁷ Lu-3BP-3554;

Fig. 10A shows representative SPECT/CT images over time of the biodistribution of 60 MBq ¹⁷⁷ Lu-3BP-3554 in HEK-FAP tumor-bearing mice;

Fig. 10B shows representative SPECT/CT images of the biodistribution of 30 MBq ¹⁷⁷ Lu-3BP-3554 over time in HEK-FAP tumor-bearing mice;

Fig. 11A shows representative SPECT/CT images of four different PDX sarcoma models 3 h after ¹¹¹ In-3BP-3554 administration;

Fig. 11B shows the %ID/g uptake of ¹¹¹ In-3BP-3554 for four different PDX sarcoma models at 3 hours post injection;

Fig. 12A shows tumor growth over time in mice bearing Sarc4809 PDX tumors treated with vehicle, cold ^nat Lu-3BP-3554, 30 MBq ¹⁷⁷ Lu-3BP-3554, or 60 MBq ¹⁷⁷ Lu-3BP-3554; and

Fig. 12B shows changes in body weight over time in mice bearing Sarc4809 PDX sarcoma tumors treated with vehicle, cold ^nat Lu-3BP-3554, 30 MBq ¹⁷⁷ Lu-3BP-3554, or 60 MBq ¹⁷⁷ Lu-3BP-3554.

The following Examples have been included to provide guidance to those of ordinary skill in the art in practicing representative embodiments of the subject matter disclosed herein. In light of the present disclosure and the general state of the art, those skilled in the art will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be made without departing from the scope of the subject matter disclosed herein. The descriptions of the syntheses and specific examples that follow are intended for illustrative purposes only and should not be construed as limiting in any way the preparation of compounds according to the disclosure and by other methods.

EXAMPLES

The abbreviations used in this application, and in particular in the following examples, are as follows:

4PL stands for Four Parameter Logistic Curve Fitting

Å stands for angstrom

ACN stands for acetonitrile

Ahx stands for 6-Aminohexanoic acid

AMC stands for 7-amino-4-methylcoumarin

a.m.u. means atomic mass unit

aq. means water

AUC _inf means area under the curve extrapolated to infinity

BSA stands for bovine serum albumin

C ₀ means the initial concentration of the compound

CAF stands for cancer associated fibroblasts

CL stands for clear

CM stands for ChemMatrix™

CT stands for computed tomography

Cy5 stands for Cyanine-5

DAD stands for Diode Array Detector

DCM stands for dichloromethane

Dde stands for N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)

DEG stands for diethylene glycol dimethacrylate

DIC stands for N,N'-Diisopropylcarbodiimide

DICOM stands for Digital Imaging and Communications in Medicine

DIPEA stands for diisopropylethylamine

DMF stands for N,N-dimethylformamide

DMSO stands for dimethyl sulfoxide

DOTA stands for 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

DOTA(tBu) ₃ -OH means Tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate

DPP stands for dipeptide or peptide azu

EC stands for electron capture

EC ₅₀ means half-maximum excitation concentration

ECACC stands for European Collection of Authenticated Cell Cultures

EDC stands for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EMEM stands for Eagle Minimum Essential Medium

eq or eqv. means equivalent

ESI stands for Electrospray Ionization

_Et2O means Diethyl Ether

EtOAc stands for ethyl acetate

FACS stands for Fluorescence Activated Cell Sorting.

FAP stands for fibroblast activating protein

Fb means background fluorescence intensity

FBS stands for fetal bovine serum

FGF21 stands for fibroblast growth factor 21

FITC stands for 5(6)-fluorescein isothiocyanate

Fmoc stands for 9-Fluorenylmethoxycarbonyl

FRET stands for Fluorescence Resonance Energy Transfer

Ft means fluorescence intensity

Gab stands for gamma-aminobutyric acid

GABA stands for gamma-aminobutyric acid

h means hour(s)

HATU stands for O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HBST stands for Moving Buffer SPR

HEK-FAP stands for human embryonic kidney 293 cells expressing human FAP

HEPES stands for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HFIP stands for hexafluoro-2-isopanol

HOAC stands for acetic acid

HOAt stands for 1-Hydroxy-7-azabenzotriazole

HPLC stands for high performance liquid chromatography

HPLC/MS stands for high performance liquid chromatography/mass spectrometry

IC ₅₀ means half-maximal inhibitory concentration

ID/g means administered dose per gram IS means isomeric transition

iTLC-SG stands for Instant Thin Layer Chromatography on Silica Gel

K2EDTA stands for dipotassium ethylenediaminetetraacetic acid

K _D stands for dissociation constant

kDa means 1000 Daltons

K _i means inhibitor constant

k _off means the rate of dissociation

k _on means speed of association

LC/TOF-MS stands for Liquid Chromatography/Time of Flight/Mass Spectrometry

LC-MS stands for high performance liquid chromatography combined with mass spectrometry

LDH stands for lactate dehydrogenase

Leu means leucine

LiOH stands for lithium hydroxide

M stands for molar or moles per liter

m/z means mass divided by charge

max means maximum

MeOH means Methanol

MeV stands for megaelectronvolt

min means minute(s)

MMP stands for matrix metalloproteinase.

MRM stands for multiple reaction monitoring

MTBE stands for Methyl tert-butyl ether.

Mtt stands for Methyltrityl

MTV means mean tumor volume

MM stands for molecular weight

n/a means not defined

Na ₂ SO ₄ means sodium sulfate

NaCl means sodium chloride

NaHCO ₃ means sodium hydrogen carbonate

NCA stands for non-compartmental analysis

NHS stands for N-Hydroxysuccinimide

NMP stands for 1-methyl-2-pyrrolidone

BDU means without further specification

Oic stands for L-octahydroindole-2-carboxylic acid

r.a. means: for analytical purposes (quality class)

p.i. means after injection

Pbf stands for 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl

PBS stands for phosphate buffered saline

PDX stands for patient-derived xenograft

PET stands for positron emission tomography

pIC50 means the negative logarithm of the IC50 value after conversion to molar

POP stands for prolyl oligopeptidase

ppm means parts per million PREP means prolyl endopeptidase

prep. means preparative PS means polystyrene

Q-TOF stands for quadrupole time of flight

Ref means reference

RFU stands for relative fluorescence unit.

RLB stands for radioligand binding assay

RMCE stands for recombinase-mediated cassette exchange.

RP stands for Reversed Phase

R _t means retention time

RT means room temperature

RU stands for resonant units

SAR stands for structure-activity ratio

sat. means saturated

SCID stands for severe combined immunodeficiency.

SCK stands for single cycle kinetics

sec or s means second

SF stands for spontaneous fission

SPECT stands for single photon emission computed tomography.

SPPS stands for Solid Phase Peptide Synthesis

t _1/2 means the terminal half-life

tBu stands for tert-butyl

TFA stands for trifluoroacetate or trifluoroacetic acid.

TG stands for TentaGel

TGI stands for tumor growth inhibition.

THF stands for Tetrahydrofuran

TIPS stands for triisopropylsilane

TLC stands for thin layer chromatography

TME stands for tumor microenvironment

t _R means retention time

UHPLC stands for Ultra High Performance Liquid Chromatography

UV stands for ultraviolet

V _SS means the steady state distribution volume

V _Z means the distribution volume in the final phase

Example 1: Materials and Methods

The materials and methods, as well as the general methods, are further illustrated by the following examples.

Solvents:

Solvents were used as specified without further purification. Acetonitrile (Super Gradient, HPLC, VWR - analytical grade; PrepSolv, Merck - preparative grade); dichloromethane (synthesis grade, Roth); ethyl acetate (synthesis grade, Roth); N,N-dimethylformamide (peptide synthesis grade, Biosolve); 1-methyl-2-pyrrolidone (peptide grade, IRIS BioTech), 1,4-dioxane (reinst, Roth); methanol (p.a., Merck).

Water: Milli-Q Plus, Millipore, demineralized.

Chemicals:

Chemicals were synthesized according to or analogous to literature procedures or purchased from Sigma-Aldrich-Merck (Deisenhofen, Germany), Bachem (Bubendorf, Switzerland), VWR (Darmstadt, Germany), Novabiochem (Merck Group, Darmstadt, Germany), Acros Organics (distribution company Fisher Scientific GmbH, Schwerte, Germany), Iris Biotech (Marktredwitz, Germany), Amatek Chemical (Jiangsu, China), Roth (Karlsruhe, Deutschland), Molecular Devices (Chicago, USA), Biochrom (Berlin, Germany), Peptech (Cambridge, MA, USA), Synthetech (Albany, OR, USA), Pharmacore (High Point, NC, USA), PCAS Biomatrix Inc (Saint-Jean-sur-Richelieu, Quebec, Canada), Alfa Aesar (Karlsruhe, Germany), Tianjin Nankai Hecheng S&T Co., Ltd (Tianjin, China), CheMatech (Dijon, France) and Anaspec (San Jose, CA, USA) or other companies and were used as intended without further purification. Cells:

HT29 (ECACC Cat. No. 91072201) and WI-38 (ECACC Cat. No. 90020107) were purchased from ECACC and HEK293 cells expressing human FAP (Q12884) were produced by InSCREENeX GmbH (Braunschweig, Germany) using recombinase-mediated cassette exchange (RMCE). The RMCE procedure is described by Nehlsen et al. (Nehlsen, et al., BMC Biotechnol, 2009, 9: 100).

HPLC/MS Analysis

HPLC/MS analyses were performed by injecting 5 µl of sample solution using a two-step gradient for all chromatograms (5-65% B over 12 min then 65-90% over 0.5 min, A: 0.1% TFA in water and B: 0.1% TFA in ACN). RP columns were from Agilent (Type Poroshell 120, 2.7 µm, EC-C18, 50×3.00 mm, 0.8 ml flow, HPLC at room temperature); Mass spectrometer: Agilent 6230 LC/TOF-MS, ESI ionization. MassHunter Qualitative Analysis B.07.00 SP2 software was used. UV detection was performed at λ=230 nm. Retention times (R _t ) are reported in decimal (e.g. 1.9 min = 1 min 54 s) and refer to detection in the UV spectrometer. The 'Find Compounds by Formula' function was used to estimate the observed masses of compounds. In particular, the individual 'neutral mass of a compound (in units of Daltons)' values and the corresponding isotope distribution pattern were used to confirm the identity of the compound. The accuracy of the mass spectrometer was approximately ±5 ppm.

Preparative HPLC:

Preparative HPLC separations were performed on reversed phase columns (Kinetex 5 μ XB-C18 100 Å, 150 × 30 mm from Phenomenex or RLRP-S 8 μ, 100 Å, 150 × 25 mm) as stationary phase. The mobile phases were 0.1% TFA in water (A) and 0.1% TFA in ACN (B), which were mixed in linear binary gradients. The gradients are described as: “10 to 40% B in 30 min”, meaning that the linear gradient from 10% B (and thus 90% A) to 40% B (and thus 60% A) was achieved over 30 min. Flow rates ranged from 30 to 50 mL/min. A typical gradient for the purification of compounds of the invention starts from 5-25% B and ends after 30 min at 35-50% B, and the difference between the percentage of B at the end and the beginning was at least 10%. A commonly used gradient was "15 to 40% B in 30 min."

General procedures for Automatic/Semi-automated Solid Phase Synthesis:

Automated solid-phase synthesis of peptides and polyamides was performed on a Tetras Peptide Synthesizer (Advanced ChemTech) at the 50 μmol and 100 μmol scales. Manual operations were performed in plastic syringes equipped with frits (PE material, Roland Vetter Laborbedarf OHG, Ammerbuch, Germany). The amounts of reagents in the described protocols correspond to the 100 μmol scale unless otherwise stated.

Solid-phase synthesis was performed on polystyrene (cross-linked with 1,4-divinylbenzene (PS) or di(ethylene glycol) dimethacrylate (DEG)), ChemMatrix (CM) or TentaGel (TG) resin. The resin linkers were trityl, van der Wahn and rince amide.

Resin loading:

In the case of the trityl linker, the attachment of the first building block (resin loading) was performed as follows. The resin (polystyrene trityl chloride (PS), initial loading: 1.8 mmol/g) was swollen in DCM (5 mL) for 30 min and then washed with DCM (3 mL, 1 min). The resin was then treated with a mixture of the corresponding building block (0.5 mmol, 5 equiv.) and DIPEA (350 μL, 3.5 mmol, 35 equiv.) in DCM (4 mL) for 1 h. The resin was then washed with methanol (5 mL, 5 min) and DMF (3 mL, 2×1 min).

In the case of Wang linker, preloaded resins (polystyrene (PS) and TentaGel (TG)) were used.

In the case of the Rink amide linker, the attachment of the first resin residue (CM, DEG) was performed using the same procedure as for chain assembly, as described below.

Removing Alloc/Allil protection:

After swelling in DMF, the resin was washed with DMF and DCM. DCM was deoxygenated by passing a stream of nitrogen through the stirred solvent. Anoxic solvent was used to wash the resin three times. Then 2 ml of 2 M barbituric acid in anoxic DCM and 1 ml of 25 μM tetrakis(triphenylphosphine)palladium(0) in anoxic DCM were added to the resin. The resin was stirred for 1 h and then washed with DCM, MeOH, DMF, 5% DIPEA in DMF, 5% dithiocarbamate in DMF, DMF and DCM (each washing step was repeated 3 times with 3 ml, 1 min).

Removing Fmoc protection:

After swelling in DMF, the resin was washed with DMF and then treated with piperidine/DMF (1:4, 3 ml, 2 and 20 min), and then washed with DMF (3 ml, 5×1 min).

Removing Dde protection:

After swelling in DMF, the resin was washed with DMF and then treated with hydrazine hydrate/DMF (2/98, 3 ml, 2 × 10 min), and then washed with DMF (3 ml, 5 × 1 min).

Removing Mtt protection:

After swelling in DCM, the resin was washed with DCM and then treated with HFIP/DCM (7/3, 4-6 mL, 4 h) and washed sequentially with DCM (3 mL, 3×1 min), DMF (3 mL, 3×1 mL), and DIPEA (0.9 M in DMF, 3 mL, 1 min).

Reagent solutions:

Building Blocks (0.3 M in DMF or NMP), DIPEA (0.9 M in DMF), HATU (0.4 M in DMF), Acetic Anhydride (0.75 M in DMF)

Linking: Linking of building blocks/amino acids (chain assembly):

Unless otherwise stated, coupling of building blocks was performed as follows: After subsequent addition of the respective building block solutions (1.7 mL, 5 equiv), DIPEA solution (1.15 mL, 10 equiv), and HATU solution (1.25 mL, 5 equiv), the resin was shaken for 45 min. If necessary, the resin was washed with DMF (3 mL, 1 min) and the coupling step was repeated.

Terminal acetylation:

After adding DIPEA solution (1.75 mL, 16 equiv.) and acetic anhydride solution (1.75 mL, 13 equiv.), the resin was shaken for 10 min. Afterwards, the resin was washed with DMF (3 mL, 6×1 min).

Cleavage method A: Cleavage of protected fragments from hyperacid labile resin:

After completion of the sequence assembly, the resin was given a final wash with DCM (3 mL, 4×1 min) and then dried under vacuum. The resin was then treated with HFIP/DCM (7/1, 4 mL, 4 h) and the pooled solution was evaporated to dryness. The residue was purified by preparative HPLC or used without further purification.

Cleavage method B: Cleavage of unprotected fragments (complete cleavage of resin):

After completion of the sequence assembly, the resin was given a final wash with DCM (3 ml, 4×1 min), dried under vacuum overnight, and treated with TFA, EDT, water, and TIPS (94/2.5/2.5/1) for 2 h (unless otherwise noted). The digestion solution was then poured into a chilled mixture of MTBE and cyclohexane (1/1, 10-fold excess over the volume of the digestion solution), centrifuged at 4°C for 5 min, and the precipitate collected and dried under vacuum. The residue was lyophilized from water/acetonitrile prior to purification or further modification.

Cleavage method C: Cleavage of peptide protective groups in solution

The protected/partially protected compound was dissolved in TFA, water and TIPS (95/2.5/2.5) for 2 h (unless otherwise stated). The digestion solution was then poured into a cooled mixture of MTBE and cyclohexane (1/1, 10-fold excess over the volume of the digestion solution), centrifuged at 4 °C for 5 min, the precipitate was collected and dried under vacuum. The residue was lyophilized from water/acetonitrile before purification or further modification.

More suitable methods for Fmoc solid phase peptide synthesis are described in detail in “Fmoc Solid Phase Peptide Synthesis” Editors W. Chan, P. White, Oxford University Press, USA, 2000. Compounds were named using the MestreNova version 12 Mnova IUPAC Name plugin (Mestrelab Research, S.L.) or AutoNom version 2.2 (Beilstein Informationssysteme Copyright© 1988-1998, Beilstein Institut für Literatur der Organischen Chemie licensed to Beilstein Chemiedaten and Software GmbH, where appropriate.

Getting connections:

Specific embodiments for preparing compounds of the invention are provided in the following examples. Unless otherwise indicated, all starting materials and reagents are of standard commercial purity and are used without further purification or can be readily prepared from such materials by conventional methods. Those skilled in the art of organic synthesis will appreciate in light of the present disclosure that starting materials and reaction conditions can be varied, including additional steps, to prepare compounds encompassed by the present invention.

One general route to synthesizing the compounds of the invention involves

1. Solid Phase Peptide Synthesis (SPPS) of a linear peptide precursor with two thiol moieties.

2. specific thiol-site cyclization of this linear peptide precursor with

a. derivative of bis(bromomethyl)benzene or

b. derivative of tris(bromomethyl)benzene.

3. In the case of cyclization with a tris(bromomethyl)benzene derivative, the intermediate formed in the cyclization reaction was further reacted with a linker, which allowed the attachment of the chelator.

Example 2: Synthesis of Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3554)

The synthesis of the title compound was accomplished either by initially synthesizing the linear peptide precursor on solid phase and subsequent cyclization in solution phase (Example 2a), or in non-aqueous solution (Method A), or in aqueous solution (Method B), or alternatively by performing all steps on solid phase, including cyclization in solid phase (Example 2b).

Example 2a: Synthesis via two alternative cyclization routes in solution Fmoc-Cys(Trt)-OH was loaded onto trityl resin as described in the General Procedures for Automated/Semi-Automated Solid Phase Synthesis at 50 μmol scale. The (Hex-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH) peptide sequence was assembled on this resin according to the General Procedures for Automated/Semi-Automated Solid Phase Synthesis. After performing the steps of Cleavage Method B, the crude peptide was lyophilized and cyclized in solution via two alternative routes.

Cyclization method A:

The crude peptide (based on a resin loading of 50 μmol) was dissolved in 10 mL of a 1:1 mixture of ethanol and acetonitrile. To this mixture were added first 35 μL of DIPEA and then 23.7 mg of 1,3,5-tris(bromomethyl)benzene (66.6 μmol, 1.3 equiv. over the initial resin loading). The solution was stirred for 1 h and then 42.8 mg of cysteamine (555 μmol, 11 equiv. over the initial resin loading) were added. After 1 h, the solvents were removed in vacuo and 25 mL of a 1:1 mixture of acetonitrile and water (containing 50 μL of TFA) was added. The solvents were removed by lyophilization. The residue was purified by HPLC (15 to 45% B over 30 min - Kinetex) to give 17.8 mg (16.4 μmol) of the intermediate Hex-[Cys(tMeBn(H-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (32.8%).

Cyclization method B:

The crude peptide (based on a resin loading of 50 μmol) was dissolved in 60 mL of a 1:1 mixture of ammonium bicarbonate (50 mM, pH 8.5) and acetonitrile. To this mixture was added a solution of 26.8 mg of 1,3,5-tris(bromomethyl)benzene (75 μmol, 1.5 equiv. compared to the initial resin loading) in 0.5 mL of acetonitrile. The solution was stirred for 1 h and then 38.6 mg of cysteamine (500 μmol, 10 equiv. compared to the initial resin loading) was added. After 2 h, 50 μL of TFA was added and the solvent was removed by lyophilization. The residue was purified by HPLC (15 to 45% B over 30 min - Kinetex) to give 19.47 mg (18 μmol) of Hex-[Cys(tMeBn(H-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (35.9%).

Both solution-based cyclization methods operate similarly and achieve comparable yields and similar purities.

To a solution of the intermediate Hex-[Cys(tMeBn(H-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (in this example prepared by cyclization method B) in 300 μL DMSO, 5 μL DIPEA were added to adjust the pH to approximately 7.5-8. Then, 20.5 mg DOTA-NHS (27 μmol, 1.5 equiv. compared to the intermediate peptide) in 200 μL DMSO were added. During the reaction, 5 μL DIPEA was added 3 times to readjust the pH to the initial value, monitored by LC/TOF-MS. After completion of the reaction, the solution was purified by HPLC (15 to 45% B over 30 min - Kinetex) to give 20.44 mg of pure title compound (yield 77.4%). HPLC: R _t = 5.9 min. LC/TOF-MS: exact mass 1469.640 (calculated 1469.639). C ₆₇ H ₉₉ N ₁₃ O ₁₈ S ₃ (MW = 1470.780).

Example 2b: Synthesis including solid-phase cyclization method

For the synthesis of the resin-bound title compound, Fmoc-Cys(Trt)-WANG Tentagel resin was used as a starting material. The sequence (Hex-Cys(Trt)-Pro-Pro-Thr(tBu)-Gln(Trt)-Phe-Cys-OH) of the peptide was assembled on the latter according to the “General Procedures for Automated/Semi-Automated Solid Phase Synthesis” at 1 mmol scale. After completion of the sequence assembly, the resin was washed with DCM (3×1 min). Trityl protecting groups were then selectively removed from the resin by treatment with TFA solution, TIPS and DCM (5/5/90, 5×5 min). The resin was washed with DCM, DMF, 0.9 M DIPEA in DMF, DMF, DCM (3/3/2/3/3) and dried under vacuum. Subsequent cyclization was performed in 200 μmol portions by swelling the resin in DMF and then treating it with a solution of 1,3,5-Tris(bromomethyl)benzene (86 mg, 240 μmol, 1.2 equiv), DIPEA (235 μl, 1 mmol, 5 equiv) in 2 ml DMF at 50°C for 90 min.

The solution was removed, the resin was washed with DMF and then cysteamine solution (154.3 mg, 2 mmol, 10 equiv) was added to the resin. The resin was stirred for another 90 min at 50 °C. After washing the resin with DMF and DMF (3/3), the peptide resin (Hex-[Cys(tMeBn(H-AET))-Pro-Pro-Thr(tBu)-Gln(Trt)-Phe-Cys]-O-WANG-Tentagel) was dried and stored for further derivatization. By this procedure, it may happen that the Trityl group in Glutamine is partially or completely deprotected. In any case, this does not interfere with the optional derivatization of the free amino group of AET.

For final derivatization with DOTA, peptide resin (Hex-[Cys(tMeBn(H-AET))-Pro-Pro-Thr(tBu)-Gln(Trt)-Phe-Cys]-O-WANG-Tentagel) was used at 50 μmol scale. DOTA(tBu) ₃ -OH was coupled according to the "General Procedures for Automated/Semi-Automated Solid Phase Synthesis". After drying, the resin was subjected to "Cleavage Method B". The crude peptide was lyophilized and then purified by preparative HPLC (15 to 45% B in 30 min - Kinetex) to yield 11.0 mg (7.5 μmol) of pure title compound (15%). HPLC: R _t = 5.9 min. LC/TOF-MS: exact mass 1469.640 (calculated 1469.639). C ₆₇ H ₉₉ N ₁₃ O ₁₈ S ₃ (MW=1470.780).

Example 3: Synthesis of Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (3BP-3407)

a) Synthesis of the intermediate compound Hex-[Cys(tMeBn(H-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ by two different cyclization routes.

The sequence (Hex-Cys-Pro-Pro-Thr-Gln-Phe-Cys-Asp-NH ₂ ) of the peptide was assembled according to the “General Procedures

Automated/Semiautomated Solid Phase Synthesis" at 50 μmol scale on Rink amide resin. After the steps of "Cleavage Method B", the crude peptide was lyophilized and cyclized by two alternative routes.

Cyclization method A:

The crude peptide (based on a resin loading of 50 μmol) was dissolved in 10 ml of a 1:1 mixture of ethanol and acetonitrile. To this mixture were added first 30 μl of DIPEA and then 26.8 mg of 1,3,5-tris(bromomethyl)benzene (75 μmol, 1.5 equiv compared to the initial resin loading). After stirring the solution for 45 min, a solution of 43 mg of piperazine (500 μmol, 10 equiv compared to the initial resin loading) in 200 μl of a 1:1 mixture of ethanol/acetonitrile was added. After 1 h, the solvents were removed in vacuo, 25 ml of a 1:1 mixture of acetonitrile and water (containing 50 μl of TFA) were added and the solvents were removed by lyophilization. The residue was purified by HPLC (15 to 40% B over 30 min - Kinetex) to give 15.3 mg (12.7 μmol) of the intermediate peptide Hex-Cys(tMeBn(H-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (25.3%).

Cyclization method B:

The crude peptide (based on a resin loading of 50 μmol) was dissolved in 60 mL of a 1:1 mixture of ammonium bicarbonate (50 mM, pH 8.5) and acetonitrile. To this mixture was added 26.8 mg of 1,3,5-tris(bromomethyl)benzene (75 μmol, 1.5 equiv. compared to the initial resin loading). The solution was stirred for 1 h and 43 mg of piperazine (500 μmol, 10 equiv. compared to the initial resin loading) was added. After 6 h, 100 μL of TFA was added and the solvent was removed by lyophilization. The residue was purified by HPLC (15 to 40% B over 30 min - Kinetex) to yield 17.2 mg (14.2 μmol) of the intermediate peptide Hex-Cys(tMeBn(H-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (28.4%).

Both cyclization methods work in a similar manner and achieve comparable yields and similar purity.

b) Final stages of the synthesis of Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (3BP-3407): DOTA coupling and purification

To a solution of the intermediate (obtained by cyclization route B) in 200 μL DMSO, 2.5 μL DIPEA were added to adjust the pH to approximately 7.5-8. Then, 16.3 mg DOTA-NHS (21.4 μmol, 1.5 equiv. compared to the intermediate peptide) in 100 μL DMSO were added. During the reaction, 2.5 μL DIPEA were added 5 times to readjust the pH to the initial value, monitored by LC/TOF-MS. After completion of the reaction, the solution was purified by HPLC (15 to 40% B over 30 min - Kinetex) to give 19.1 mg (12.0 μmol) of pure title compound (85%). HPLC: R _t = 5.70 min. LC/TOF-MS: exact mass 1592.737 (calculated 1592.737). C ₇₃ H ₁₀₈ N ₁₆ O ₂₀ S ₂ (MW=1593.866).

Example 4: Preparation of DOTA-transition metal complexes of compounds of the invention

A. General procedure for preparing a peptide containing DOTA-transition metal complexes from corresponding peptides containing non-complexed DOTA.

0.1 mM solution of peptide consisting of uncomplexed DOTA, in

• 0.4 M sodium acetate, pH=5 (Buffer A) (in the case of Cu(II), Zn(II), In(III), Lu(III) or Ga(III) complexes) or

• 0.1 M ammonium acetate, pH=8 (Buffer B) (in the case of Eu(III) complexes) was diluted with a solution of 0.1 mM of the corresponding metal salt in water, with the molar ratio of peptide to metal adjusted to 1:3. The solution was stirred

• at 50°C for 20 minutes (also referred to herein as Condition A) (in the case of In(III), Lu(III), Ga(III), Zn(II) or Cu(II) complexes) or

• at room temperature overnight (also referred to herein as Condition B) (in the case of Eu(III) complexes).

The solution was then subjected to

• purification by HPLC (also referred to herein as Purification A) or

• solid phase extraction (also referred to herein as Cleanup B).

For solid phase extraction, 250 mg of Varian Bondesil-ENV was placed in a 15 ml polystyrene syringe previously washed with methanol (1 x 5 ml) and water (2 x 5 ml). The reaction solution was then fed onto the column. Elution was then carried out with water (2 x 5 ml - to remove excess salt), 5 ml of 50% ACN in water as the first fraction and each of the following fractions was eluted with 5 ml of 50% ACN in water containing 0.1% TFA.

In either case (HPLC purification or solid phase extraction), fractions containing pure product were pooled and freeze-dried.

B. Indium complex Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (3BP-3590)

The complex was prepared starting from 25 mg of peptide 3BP-3407 (15.7 μmol) dissolved in Buffer A diluted with _InCl3 × _4H2O solution treated with Condition A. Purification step was performed using “Purification A” (15 to 40% _B over 30 min - RLRP-S) to give 18.24 mg of pure title compound (68.1% _yield ). HPLC: _Rt = 5.6 min. LC/TOF-MS: exact mass 1702.622 (calculated 1702.617 ₎ . _{C73H105InN16O20S2 (} MW=1705.663).

C. Gallium complex Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (3 BP-3592)

The complex was prepared starting from 25 mg of peptide 3BP-3407 (15.7 μmol) dissolved in Buffer A diluted with Ga(NO ₃ ) ₃ ×H ₂ O solution treated with Condition A. Purification step was performed using “Purification A” (15 to 40% B over 30 min - RLRP-S) to afford 16.78 mg of pure title compound (69.3% yield). HPLC: R _t = 5.7 min. LC/TOF-MS: exact mass 1658.664 (calculated 1658.639). C ₇₃ H ₁₀₅ GaN ₁₆ O ₂₀ S ₂ (MW = 1660.568).

D. Lutetium complex Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (3BP-3591)

The complex was prepared starting from 25 mg of peptide 3BP-3407 (15.7 μmol) dissolved in Buffer A diluted with LuCl ₃ solution treated with Condition A. Purification step was performed using “Purification A” (15 to 40% B over 30 min -RLRP-S) to afford 16.66 mg of pure title compound (60.1% yield). HPLC: R _t = 5.6 min. LC/TOF-MS: exact mass 1764.654 (calculated 1764.654). C ₇₃ H ₁₀₅ LuN ₁₆ O ₂₀ S ₂ (MW = 1765.812).

E. Europium complex Hex-[Cys(tMeBn(DOTA-PP))-Pro-Pro-Thr-Gln-Phe-Cys]-Asp-NH ₂ (3BP-3661)

The complex was prepared starting from 9.5 mg of peptide (6 μmol) 3BP-3407 dissolved in Buffer B diluted with _EuCl3 × _6H2O solution which was treated with Condition B. Purification step was performed using “Purification B” to afford _8.24 mg of pure title compound (79.3% yield). HPLC: Rt=5.7 min. LC/TOF-MS: exact _mass 1740.636 (calculated 1740.633). _{C73H105EuN16O20S2 (} MW=1742.809 ₎ .

F. Indium complex Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3623)

The complex was prepared starting from 6 mg of peptide 3BP-3554 (4.1 μmol) dissolved in Buffer A diluted with _InCl3 × _4H2O solution treated with Condition A. Purification step was performed using “Purification B” to afford _5.26 mg _of pure title compound (81% yield). HPLC: _{R t} = 5.8 min. LC/TOF-MS: exact mass 1579.524 (calculated 1579.520). _{C67H96InN13O18S3} (MW=1582.574 ₎ .

G. Lutetium complex Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3624)

The complex was prepared starting from 6 mg of peptide 3BP-3554 (4.1 μmol) dissolved in Buffer A diluted with LuCl ₃ solution treated with Condition A. Purification step was performed using “Purification B” to afford 5.5 mg of pure title compound (82% yield). HPLC: R _t = 5.9 min. LC/TOF-MS: exact mass 1641.560 (calculated 1641.557). C ₆₇ H ₉₆ LuN ₁₃ O ₁₈ S ₃ (MW = 1642.723).

H. Gallium complex Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3949)

The complex was prepared starting from 7.9 mg of peptide 3BP-3554 (5.4 μmol) dissolved in Buffer A diluted with Ga(NO ₃ ) ₃ ×H ₂ O solution treated with Condition A. Purification step was performed using “Purification B” to afford 4.2 mg of pure title compound (51% yield). HPLC: R _t = 6.6 min. LC/TOF-MS: exact mass 1535.543 (calculated 1535.541). C ₆₇ H ₉₆ GaN ₁₃ O ₁₈ S ₃ (MW = 1537.479).

I. Europium complex Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-3662)

The complex was prepared starting from 3.4 mg of peptide 3BP-3554 (2.3 μmol) dissolved in Buffer B diluted with _EuCl3 × _6H2O solution treated with Condition B. Purification step was performed using “Purification B” to afford _3.1 mg of _pure title compound (83% yield). HPLC: R _t = 5.9 min. LC/TOF-MS: _exact mass 1617.541 (calculated 1617.536). _{C67H96EuN13O18S3 (} MW=1619.721).

J. Copper(II) complex Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-4293)

The complex was prepared starting from 18 mg of peptide 3BP-3554 (12.2 μmol) dissolved in Buffer A diluted with Cu(OAc) ₂ solution treated with Condition A. Purification step was performed using “Purification B” to afford 16.5 mg of pure title compound (yield 88%). HPLC: R _t = 6.5 min. LC/TOF-MS: exact mass 1530.553 (calculated 1530.553). C ₆₇ H ₉₇ CuN ₁₃ O ₁₈ S ₃ (MW = 1532.310).

K. Zinc complex Hex-[Cys(tMeBn(DOTA-AET))-Pro-Pro-Thr-Gln-Phe-Cys]-OH (3BP-4343)

The complex was prepared starting from 20 mg of peptide 3BP-3554 (13.6 μmol) dissolved in Buffer A diluted with ZnCl ₂ solution treated with Condition A. Purification step was performed using “Purification B” to afford 16.1 mg of pure title compound (77% yield). HPLC: R _t = 6.4 min. LC/TOF-MS: exact mass 1531.553 (calculated 1531.553). C ₆₇ H ₉₇ N ₁₃ O ₁₈ S ₃ Zn (MW = 1534.160).

Example 5: Stability analysis in plasma

To determine the stability of selected compounds of the present invention in human and mouse plasma, a plasma stability assay was performed. This plasma stability assay measures the degradation of the compounds of the present invention in blood plasma. This is an important characteristic of the compound, since compounds, with the exception of prodrugs, which are rapidly degraded in plasma, usually show low efficacy in vivo. The results show that these compounds are highly stable in human and mouse plasma. The stability is sufficient for diagnostic, therapeutic and therapeutic-diagnostic use of these compounds according to the present invention.

Plasma stability samples were prepared by adding 1 µl of 0.5 mM compound stock in DMSO to 50 µl plasma aliquots (all K2EDTA). After vortexing, samples were incubated in a Thermomixer at 37°C for 0, 4 and 24 h. After incubation, samples were stored on ice until further processing. All samples were prepared in duplicate.

Each sample was supplemented with the appropriate internal standard (1 μl of 0.5 mM stock solution in DMSO). Protein precipitation was performed in two different ways depending on the coupling conditions as listed in Table 5.

A) 250 μl of acetonitrile containing 1% trifluoroacetic acid were added. After incubation at room temperature for 30 min, the precipitate was separated by centrifugation and 150 μl of the supernatant was diluted with 150 μl of 1% aqueous formic acid.

B) 150 μl of zinc sulfate precipitating agent containing 78% 0.1 M zinc sulfate and 22% acetonitrile were added. After incubation at room temperature for 30 min, the precipitate was separated by centrifugation. To 100 μl of the supernatant, 10 μl of 1% formic acid were added, followed by incubation at 60°C for 10 min to complete the formation of the zinc chelate if the compound contains a free DOTA fragment.

Analyte determination in neat sample solutions was performed on an Agilent 1290 UHPLC system coupled to an Agilent 6530 Q-TOF mass spectrometer. Chromatographic separation was performed on a Phenomenex BioZen XB-C18 HPLC column (50×2 mm, 1.7 μm particle size) with gradient elution using a mixture of 0.1% formic acid in water as eluent A and acetonitrile as eluent B (from 2% B to 41% in 7 min, 800 μl/min, 40°C). Mass spectrometric detection was performed in the ESI positive ion mode by scanning the m/z mass range from 50 to 3000 with a sampling rate of 2/sec.

From the raw mass spectrometry data, ion currents were extracted for the di- or tri-charged monoisotopic signal for both the compound and the internal standard.

Quantification was performed by calibrating the external matrix with an internal standard using integrated analyte signals.

In addition, recovery was determined by adding a blank plasma sample that contained only the internal standard after treatment with a defined amount of compound.

Carryover was assessed by analyzing a blank sample (20% acetonitrile) after the highest calibration sample.

The results of this analysis, carried out for some compounds of the present invention, are presented in the following Table 5. The result is reported as "% intact compound remaining after 24 h" and means that of the amount of material at the beginning of the experiment, the indicated percentage is determined to be unchanged material at the end of the experiment according to quantitative LC-MS analysis. Since all compounds remain more than 50% intact after at least 24 h, they are considered sufficiently stable for diagnostic and therapeutic applications.

Example 6: FACS Binding Analysis

To determine the binding of the compounds of the present invention to FAP-expressing cells, a competitive FACS binding assay was established.

FAP-expressing human WI-38 fibroblasts (ECACC) were cultured in EMEM containing 15% fetal bovine serum, 2 mM L-Glutamine, and 1% nonessential amino acids. Cells were detached with Accutase (Biolegend, #BLD-423201) and washed in FACS buffer (PBS containing 1% FBS). Cells were diluted in FACS buffer to a final concentration of 100,000 cells/ml, and 200 μl of the cell suspension was transferred to a U-shaped 96-well plate without binding (Greiner). Cells were washed with ice-cold FACS buffer and incubated with 3 nM Cy5-labeled compound (H-Met-[Cys(3MeBn)-Pro-Pro-Thr-Glu-Phe-Cys]-Asp-His-Phe-Arg-Asp-Ttds-Lys(Cy5SO3)-NH2) in the presence of increasing concentrations of peptides at 4°C for 1 h. Cells were washed twice with FACS buffer and resuspended in 200 μl FACS buffer. Cells were analyzed on an Attune NxT flow cytometer. Mean fluorescence intensities (Cy5 channel) were calculated using Attune NxT software and plotted against peptide concentrations. Four-parameter logistic (4PL) curve fitting and pIC50 calculations were performed using ActivityBase software. The results of this assay, as well as the FAP protease activity assay as described in Example 7, for each compound of the present invention are presented in Table 6 (shown in Example 7). pIC50 category A denotes pIC50 values>8.0, category B denotes pIC50 values from 7.1 to 8.0, category C denotes pIC50 values from 6.1 to 7.0, and category D denotes pIC50 values≤6.0.

Example 7: FAP Protease Activity Assay

To determine the inhibitory activity of the peptides from Example 6, a FRET-based FAP protease activity assay was developed.

Recombinant human FAP (R&D systems, #3715-SE) was diluted in assay buffer (50 mM Tris, 1 M NaCl, 1 mg/ml BSA, pH 7.5) to a concentration of 3.6 nM. 25 µl of FAP solution was mixed with 25 µl of a 3-fold serial dilution of test compounds and incubated for 5 min in a white 96-well ProxiPlate (Perkin Elmer). The FRET peptide HiLyteFluor™ 488 - VS(D-)P SQG K(QXL® 520) - NH2 (Bainbridge, et al., Sci Rep, 2017, 7: 12524) was used as a specific FAP substrate. 25 µl of 30 µM substrate solution diluted in assay buffer were added. All solutions were equilibrated at 37°C before use. Substrate cleavage and fluorescence increase (excitation at 485 nm and emission at 538 nm) were measured kinetically for 5 min at 37°C in a SPECTRAmax M5 plate reader. RFU/sec was calculated using SoftMax Pro software and plotted versus peptide concentration. Four-parameter logistic (4PL) curve fitting and pIC50 calculations were performed using ActivityBase software. The results of this assay for each compound of the present invention are shown in Table 6. pIC50 category A denotes pIC50 values >8.0, category B denotes pIC50 values between 7.1 and 8.0, category C denotes pIC50 values between 6.1 and 7.0, and category D denotes pIC50 values ≤6.0.

As can be seen from Table 6, the compounds of the present invention show unexpectedly better results in both the FACS binding assay and the FAP protease activity assay.

Example 8: Surface Plasmon Resonance Analysis

Surface plasmon resonance studies were performed using the Biacore™ T200 SPR system. Briefly, polarized light is directed onto a gold-labeled sensor surface and minimal reflected light intensity is detected. The angle of reflected light changes as the molecules bind and dissociate. The gold-labeled sensor surface is loaded with anti-FAP antibodies carrying FAP target proteins, resulting in antibody binding not occurring at the FAP substrate binding site. Test compounds are contacted with the loaded surface and the interaction profile with the FAP ligand is recorded in real time on a sensorgram. The association and dissociation of the binding interaction is measured in real time, allowing the calculation of association and dissociation rate constants and corresponding affinity constants. Importantly, background reaction occurs due to refractive index differences between the running and sample buffers as well as non-specific binding of test compounds to the flow cell surface. This background is measured and subtracted by passing the sample into a control flow cell coated with the same density of capture antibody in the absence of immobilized FAP. In addition, a correction is made for the baseline drift of the binding data, which is caused by the slow dissociation of captured FAP from the immobilized antibody. This drift is measured by injecting running buffer through the flow cell with antibody and FAP immobilized on the sensor surface.

Biacore™ CM5 sensor chips were used. Anti-human FAP antibody (MAB3715, R&D systems) was diluted in 10 mM acetate buffer, pH 4.5, to a final concentration of 50 μg/mL. An aliquot of 150 μL was transferred to plastic vials and placed in the sample rack of the Biacore™ T200 instrument. The Amine Coupling Reagent Kit solutions were transferred to plastic vials and placed in the sample rack: 90 μL of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 90 μL of 0.1 M N-hydroxysuccinimide (NHS). 130 µl aliquots of 1 M ethanolamine-HCl, pH 8.5, were transferred to plastic vials and placed in a sample rack. The Biacore™ liquid system was set up as follows: Separate bottles containing distilled water (1 l), Working Buffer (500 ml), and an empty waste bottle were placed on the buffer tray. The preset program for immobilization was used with an immobilization level of 7000 RU. Immobilization was performed at 25°C. The procedure for immobilization of antibodies to FAP was performed as described in Table 7.

Recombinant human FAP was diluted in Working Buffer to a final concentration of 20 μg/mL. A 100 μL aliquot of Human FAP Working Solution was transferred to plastic vials and placed in a sample rack. 0.5 mM Compound Stock Solution was prepared by dissolving each compound in DMSO. For each test compound, Compound Stock Solutions were diluted in Working Buffer (HBST) to 500 nM and further diluted with HBST-DMSO buffer (0.1% DMSO). SPR binding assay for binary complexes was performed in SCK mode at 25 °C. Table 8 describes the protocol for recording and evaluating the binding kinetics. After three SCK measurements, baseline drift was assessed by injecting Working Buffer through the flow cell with immobilized antibody and FAP on the sensor surface.

For each test compound, raw SPR data in the form of resonance units (RU) were plotted as sensorgrams using Biacore™ T200 evaluation software. The signal of the blank sensorgram was subtracted from the signal of the test compound sensorgram (blank-corrected). The blank-corrected sensorgram was corrected for baseline drift by subtracting the sensorgram of the SCK run without the test compound (running buffer only). The association rate (k _on ), dissociation rate (k _off ), dissociation constant (K _D ), and t _1/2 were calculated from the blank-normalized SPR data using the 1:1 Langmuir binding model from Biacore™ T200 evaluation software. Raw data and fit results were imported as text files into IDBS. The pK _D value (negative base 10 logarithm of the dissociation constant) was calculated in the IDBS Excel template.

The results of this analysis for the selection of compounds according to the present invention are presented in Table 9. Category A means pK _D values >8.0, category B means pK _D values from 7.1 to 8.0, category C means pK _D values from 6.1 to 7.0.

Example 9: PREP and DPP4 Protease Activity Assay

To test the selectivity of FAP-binding peptides for both PREP and DPP4, protease activity assays similar to the FAP activity assay described above were performed with the following exceptions.

PREP activity was measured using recombinant human PREP (R&D systems, #4308-SE). 50 μM Z-GP-AMC (Bachem, #4002518) was used as a substrate. DPP4 activity was assayed in DPP assay buffer (25 mM Tris, pH 8.0). Recombinant human DPP4 was purchased from R&D systems (#9168-SE). 20 μM GP-AMC (Santa Cruz Biotechnology, #115035-46-6) was used as a substrate.

The fluorescence of AMC (excitation at 380 nm and emission at 460 nm) after cleavage was measured in kinetic mode for 5 min at 37°C in a SPECTRAmax M5 plate reader. RFU/sec was calculated using SoftMax Pro software and plotted against peptide concentration. Four-parameter logistic (4PL) curve fitting and pIC50 calculations were performed using ActivityBase software. The results of this analysis for some compounds of the present invention are presented in the following Table 10.

Example 10: Specificity Screening

Specificity screening was performed to detect early significant adverse interactions of the compounds of the present invention. Specificity was tested using a standard set of assays (the “SafetyScreen44™ Panel”) comprising 44 selected targets and compounds that bind to them (referred to as “control compounds”, Ctrl. Compounds) as recommended by Bowes et al. (Bowes, et al., Nat Rev Drug Discov, 2012, 11: 909). The control compounds served as positive controls for the respective assays, and inhibition was expected to be detected with these control compounds. However, the compounds of the invention were not expected to exhibit inhibition in these assays. These enzyme binding and inhibition assays were performed by Eurofins Cerep SA (Celle l'Evescault, France).

3BP-3407 and 3BP-3554 were tested at a concentration of 10 μM. Compound binding was calculated as % inhibition of radiolabeled ligand binding specific to each target (% Inhibition of Specific Binding (3BP-3407) or (3BP-3554), respectively). The enzyme inhibition effect of a compound was calculated as % inhibition of control enzyme activity.

Results showing inhibition or stimulation greater than 50% are considered to represent significant effects of the tested compounds. Such effects were not observed at any of the receptors studied, listed in the following Table 11. The results of this analysis are summarized in the following Table 11.

Additionally, protease specificity screening was performed using BPS Biosciences to further determine the specificity of the compounds of the invention (Turk, Nat Rev Drug Discov, 2006, 5: 785; Overall, et al., Nat Rev Cancer, 2006, 6: 227; Anderson, et al., Handb Exp Pharmacol, 2009,189: 85).

3BP-3407 and 3BP-3554 were tested at 1 μM and 10 μM concentrations in duplicate. In the absence of compound, the fluorescence intensity (Ft) in each data set was defined as 100% activity. In the absence of enzyme, the background fluorescence intensity (Fb) in each data set was defined as 0% activity. Percent activity in the presence of each compound was calculated according to the following equation: % activity = (F - Fb) / (Ft - Fb), where F = fluorescence intensity in the presence of compound. Percent inhibition was calculated according to the following formula: % inhibition = 100% - % activity. Results showing inhibition greater than 50% are considered to represent significant effects of the test compound. The results of this analysis are presented in the following Table 12.

Example 11: Labeling of selected compounds with ¹¹¹ In and ¹⁷⁷ Lu

In order to be a diagnostic, therapeutic or therapeutic-diagnostic active agent, the compound must be labeled with a radioactive isotope. The labeling procedure must be suitable to ensure a high radiochemical yield and purity of the radioactively labeled compound of the invention. This example shows that the compounds of the present invention are suitable for radioactive labeling and can be labeled with a high radiochemical yield and purity.

30-100 MBq ¹¹¹ _InCl3 (in 0.02 M HCl) was mixed with 1 nmol compound (200 μM stock solution in 0.1 M HEPES, pH 7) per 30 MBq and buffer (1 M sodium acetate buffer pH 5 or 1 M sodium acetate/ascorbic acid buffer pH 5 containing 25 mg/mL methionine) at a final buffer concentration of 0.1-0.2 M. The mixture was heated to 80°C for 20-30 min. After cooling, DTPA and TWEEN-20 were added to a final concentration of 0.2 mM and 0.1%, respectively.

0.2-2.0 GBq ¹⁷⁷ LuCl ₃ (in 0.04 M HCl) was mixed with 1 nmol compound (200 μM stock solution in 0.1 M HEPES, pH 7) at 45 MBq and buffer (1 M sodium acetate/ascorbic acid buffer pH 5 containing 25 mg/mL methionine) at a final buffer concentration of ~0.4 M. The mixture was heated to 90°C for 20 min.

The labeling efficiency was analyzed by thin layer chromatography (TLC) and HPLC. For TLC analysis, 1-2 μl of the diluted labeling solution was applied to an iTLC-SG chromatography paper strip (Agilent, 7.6 x 2.3 mm) and developed in citrate-dextrose solution (Sigma). The iTLC strip was then cut into 3 pieces and the corresponding radioactivity was measured with a gamma counter. The radioactivity measured at the solvent front represents the free radionuclide and colloids, while the radioactivity in the source represents the radiolabeled compound. For HPLC, 5 μl of the diluted labeling solution was analyzed on Poroshell SB-C18 2.7 μm (Agilent). Eluent A: MeCN, eluent B: _H2O , 0.1% TFA, gradient from 5% B to 70% B over 15 min, flow rate 0.5 ml/min; detector: NaI (Raytest), DAD 230 nm. The peak eluting with dead volume represents the free radionuclide, the peak eluting with a retention time specific for the peptide as determined with unlabeled sample represents the radiolabeled compound.

The yield of radionuclides was ≥90% and the radiochemical purity was ≥76% at the end of the synthesis. Examples of radiochemical purity for ¹¹¹ In-labeled compounds are shown in Table 13. ¹⁷⁷ Lu-labeled compounds in formulations suitable for human use maintained a radiochemical purity of ≥90% for up to 6 days after synthesis (Table 14). Radiochromatograms of selected compounds are shown in Figs. 1-4, with Fig. 1 showing a radiochromatogram ^{of 177} Lu-3BP-3407 in formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed immediately after synthesis, and Fig. 2 shows a radiochromatogram ^{of 177} Lu-3BP-3407 in a formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed six days after synthesis, Fig. 3 shows a radiochromatogram ^{of 177} Lu-3BP-3554 in a formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed immediately after synthesis, and Fig. 4 shows a radiochromatogram ^{of 177} Lu-3BP-3554 in a formulation buffer containing 100 mg/mL ascorbate and 5 mg/mL L-methionine, analyzed six days after synthesis.

Example 12: Imaging and biodistribution studies

Radioactively labeled compounds can be detected by imaging methods such as SPECT and PET. In addition, data obtained by such methods can be confirmed by direct measurement of radioactivity contained in individual organs obtained from an animal injected with a radioactively labeled compound of the present invention. In this way, the biodistribution (measurement of radioactivity in individual organs) of the radioactively labeled compound can be determined and analyzed. This example shows that the compounds of the present invention exhibit biodistribution suitable for diagnostic imaging and therapeutic treatment of tumors.

All animal experiments were performed in accordance with German animal protection laws. Beige male SCID mice (6 to 8 weeks old, Charles River, Sulzfeld, Germany) were inoculated with 5 x 10 ⁶ HEK-FAP (293 human embryonic kidney cells genetically engineered to express high levels of FAP) in one arm. When the tumors reached a size of >150 mm ³ , the mice received ~ 30 MBq of ^111In -labeled compounds of the invention (diluted to 100 μl PBS) administered intravenously via the tail vein. Images were acquired on a NanoSPECT/CT system (Mediso Medical Imaging Systems, Budapest, Hungary) using, for example, the following acquisition and reconstruction parameters (Table 15).

The imaging data were saved as DICOM files and analyzed using VivoQuant™ software (Invicro, Boston, USA). Results are expressed as percentage of the injected dose per gram of tissue (%ID/g). For biodistribution studies, animals were sacrificed by cervical dislocation 24 or 48 h after injection and then necropsied. The various organs and tissues were collected and weighed, and radioactivity was determined by γ-counting. Two animals were used at each time point. Results are expressed as percentage of the injected dose per gram of tissue (%ID/g).

The results of imaging and biodistribution studies for selected compounds are shown in Figs. 5–7.

Example 13: Efficacy Study - HEK-FAP

Radioactively labeled compounds can be used for therapeutic and diagnostic applications in various diseases, especially cancer. This example shows that the compounds of the present invention have antitumor activity suitable for the therapeutic treatment of tumors.

All animal experiments were performed in accordance with German animal protection laws. Female Swiss nude mice (7 to 8 weeks old, Charles River Laboratories, France) were inoculated with 5× ¹⁰⁶ HEK-FAP cells per arm and treatment was administered when the tumors reached a mean tumor volume of 160±44 ^mm3 . Mice were divided into 4 different groups with 10 animals per group: Group 1 - vehicle control, Group 2 - cold compound ^nat Lu-3BP-3554, Group 3 - 30 MBq ¹⁷⁷ Lu-3BP-3554 (low dose) and Group 4 - 60 MBq ¹⁷⁷ Lu-FAP-3554 (high dose). Treatment was administered on Day 0 by intravenous injection into the tail vein at a dose of 4 ml/kg (100 μl/mouse). Tumor volume and body weight were measured on Day 0 (i.e., the first day of tracer administration) and then three times weekly until study completion.

The distribution of tracers in mice injected with ^177Lu -labeled 3BP-3554 was determined by SPECT imaging in three dose groups of mice. Subsequently, CT scanning was performed after SPECT for anatomical information. Imaging was performed at 3 h, 24 h, 48 h, and 120 h post-injection using the NanoSPECT/CT system (Mediso Medical Imaging Systems, Budapest, Hungary) using, for example, the following acquisition and reconstruction parameters (Table 16).

Image data were saved as DICOM files and analyzed using VivoQuant™ software (Invicro, Boston, USA). Results are expressed as percentage of injected dose per gram of tissue (%ID/g).

Tumors in vehicle- and cold ^nat Lu-3BP-3554-treated mice reached mean tumor volumes (MTVs) of 1338±670 ^mm3 and 1392±420 ^mm3 on day 14, respectively (Fig. 9A). Statistically significant (P<0.01) antitumor activity was observed in mice of both treatment groups. Tumor growth inhibition (TGI) on day 14 was 111% and 113% in mice receiving a single dose of 30 or 60 MBq ^{of 177} Lu-3BP-3554, respectively, compared to the vehicle-treated group. MTVs in all ¹⁷⁷ Lu-3BP-3554-treated mice decreased to ≤70 ^mm3 on day 14. Tumors were monitored for regrowth at Day 42 (which represents the end of the study), and three of ten and nine of ten mice treated with 30 or 60 MBq ^177Lu -3BP-3554, respectively, were tumor-free (<10 mm ³ ), suggesting a potential dose-response in this model. No treatment-related body weight loss was observed throughout the study (Fig. 9B). Following a 3-5% body weight loss observed in all groups on Day 2, animal body weight increased over time.

SPECT/CT images of 3 animals from both treatment groups labeled with ^177Lu showed high tumor-to-background contrast at all time points examined (3-120 h post injection (pi)). High tumor retention was observed up to 120 h. The organ with the highest non-target uptake was the kidney with tumor/kidney ratios of 8.6±0.6 and 8.0±1.6 at 3 h post injection in mice receiving 30 or 60 MBq ^177Lu -3BP3554, respectively. These ratios increased with time, reaching the highest value at 120 h with tumor/kidney ratios of 40±7.9 and 32±7.4 in mice receiving 30 or 60 MBq ^177Lu -3BP3554, respectively. A representative SPECT/CT image panel for mouse 5, which was the high-dose animal, is shown in Fig. 10A, and for mouse 1, which was a low dose animal, it is shown in Fig. 10B.

Example 14: Imaging Study - PDX Sarcoma Models

Sarcoma tumors have been reported to express FAP and imaging of four different patient-derived xenograft (PDX) tumor models was performed to assess 3 BP-3554 uptake. The PDX models Sarc4183, Sarc4605, Sarc4809 and Sarc12616 were derived from patients with rhabdomyosarcoma, osteosarcoma, undifferentiated sarcoma and undifferentiated pleiomorphic sarcoma, respectively (Experimental Pharmacology & Oncology Berlin-Buch, Germany). Tumor fragments were transplanted subcutaneously into the left flank of 8-week-old NMRI nu/nu mice (Janvier Labs, France). All animal experiments were performed in accordance with German animal protection laws. At 47 days (Sarc4183, Sarc4809) or 46 days (Sarc4605, Sarc12616) post-transplantation, 2-3 mice per model were imaged 3 hours after a single intravenous injection of 30 MBq ¹¹¹ In-3 BP-3554. Imaging was performed as described in Example 12.

The imaging results with ¹¹¹ In-3BP-3554 showed high tumor uptake at 3 h post-injection and high tumor-to-background contrast. Representative SPECT/CT images are shown in Fig. 11A. Quantification of tumor uptake in two (Sarc4605, Sarc12616) or three (Sarc4183, Sarc4809) PDX-bearing mice, respectively, revealed %ID/g values of 4.9±1.7 (Sarc4183), 5.2±0.8 (Sarc4605), 4.4±0.7 (Sarc4809), and 6.1±0.6 (Sarc12616), as shown in Fig. 11B. These results demonstrate the uptake of ¹¹¹ In-3BP-3554 in all four sarcoma models. Tumor/kidney ratios were 4.7±1.2 (Sarc4183), 3.2±0.4 (Sarc4605), 4.1±0.7 (Sarc4809), and 4.3±1.2 (Sarc12616).

Example 15: Efficacy Study - PDX Sarc4809 Sarcoma Model

The efficacy of ¹⁷⁷ Lu-3BP-3554 was investigated in the PDX tumor model of human sarcoma Sarc4809. This undifferentiated sarcoma model exhibits uptake of ¹¹¹ In-3BP-3554 (Example 14) and has also been shown to express FAP by immunohistochemistry.

All animal experiments were performed in accordance with German animal protection laws. Sarc4809 tumor fragments were transplanted subcutaneously into the left flank of 8-week-old NMRI nu/nu mice (Janvier Labs, France). Treatment was started 23 days after transplantation at an average tumor volume of 187.08±123.8 ^mm3 . Mice were divided into four groups of 10 animals per group: Group 1 - vehicle control, Group 2 - cold compound ^nat Lu-FAP-3554, Group 3 - 30 MBq ¹⁷⁷ Lu-3BP-3554, Group 4 - 60 MBq ¹⁷⁷ Lu-FAP-3554. Treatment was performed on Day 0 by intravenous injection into the tail vein at a dose of 4 ml/kg (100 μl/mouse). Tumor volume and body weight were determined on Day 0 (i.e., the first day of tracer administration) and then three times weekly until completion of the study.

All tumors grew continuously throughout the observation period until day 42. Tumors in mice treated with vehicle and ^nat Lu-3BP-3554 (control groups) reached MTVs of 894±610 ^mm3 and 1225±775 ^mm3 on day 31 (the last day when at least 50% of the mice in a group were still alive), respectively. Tumors in mice treated with a single dose of 30 or 60 MBq ^{of 177} Lu-3BP-3554 reached MTVs of 635±462 and 723±391 ^mm3 on day 31, respectively (Fig. 12A). Statistically significant (P<0.05) antitumor activity was observed in mice in both treatment groups. Tumor growth inhibition (TGI) at day 31 was 61% and 73% in mice treated with a single dose of 30 or 60 MBq ^177Lu -3BP-3554, respectively, compared to the vehicle group. No treatment-related body weight loss (BWL) was observed throughout the study. In all groups, body weight increased during the study observation period (Fig. 12B).

Example 16: Pharmacokinetic studies

The pharmacokinetic behavior of selected compounds was evaluated in mice and rats. This characterization of the pharmacokinetic behavior of a compound provides new insights into the distribution and elimination of the compound, as well as into the calculation of exposure.

Various amounts of compounds were stably formulated in PBS. The formulations were administered intravenously at a dose of 4 nmol/kg, 40 nmol/kg, and 400 nmol/kg in mice and 2 nmol/kg, 20 nmol/kg, and 200 nmol/kg (3BP-3554) or 40 nmol/kg and 400 nmol/kg (3BP-3623) in rats. Assuming an allometric translation coefficient of 12.3 in humans to mice and 6.2 in humans to rats (Nair AB, Jacob S. Journal of Basic and Clinical Pharmacy, 2016, 7(2): 27-31), the doses administered correspond to the human dose range of 0.325 nmol/kg to 32.5 nmol/kg.

Blood samples were collected at different times (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h) from the tail vein (rats) or retrobulbar vein (mice).

After separation of blood cells from plasma by centrifugation, the compounds were quantified in the prepared plasma samples and subjected to a protein precipitation procedure. 150 μl of zinc sulfate precipitating agent containing 78% 0.1 M zinc sulfate and 22% acetonitrile were added. After incubation at room temperature for 30 min, the precipitate was separated by centrifugation. 10 μl of 1% formic acid were added to 100 μl of the supernatant, followed by incubation at 60°C for 10 min to complete the formation of the zinc chelate if the compound contains a free DOTA fragment.

Analyte determination in neat sample solutions was performed on an Agilent 1290 UHPLC system coupled to an Agilent 6470 triple quadrupole mass spectrometer. Chromatographic separation was performed on a Phenomenex BioZen Peptide XB-C18 HPLC column (50×2 mm, 1.7 μm particle size) at 40°C with gradient elution using a mixture of 0.1% formic acid in water as eluent A and acetonitrile as eluent B (isocratic at 5% B for 1 min followed by a linear gradient to 43% B over 4 min, 500 μL/min).

Mass spectrometric detection was performed in positive ion ESI mode using multiple reaction monitoring (MRM).

Quantitative analysis of test objects was performed using the Quantitative Analysis software from the Agilent MassHunter software package. Quadratic regression was performed with a weighting factor of 1/x.

Plasma levels were subjected to non-compartmental analysis (NCA) with the following results: initial compound concentration (Co), volume of distribution at steady state ( _Vss ), volume of distribution at terminal phase ( _Vz ), terminal half-life (t1 _/2 ), clearance (CL), and area under the curve extrapolated to infinity ( _AUCinf ). A summary of the NCA parameters of 3BP-3554 is presented in Table 18 for 3BP-3554 in mouse plasma and in Table 19 for 3BP-3554 in rat plasma, and of the NCA parameters of 3BP-3623 in Table 20 for 3BP-3623 in mouse plasma and in Table 21 for 3BP-3623 in rat plasma.

The results show a distribution mainly in blood and interstitial fluid and a clearance typical for peptides, with terminal half-lives ranging from 23 to 59 minutes in mice and 45 to 71 minutes in rats. Exposure, described by AUC, correlates almost linearly with the administered dose, and clearance is constant for all doses administered in this animal model. These observations indicate the absence of significant nonlinearity in pharmacokinetic behavior that should be taken into account when calculating the dose for the first administration in humans.

The features of the present invention disclosed in the description, claims, sequence list and/or drawings may, either individually or in any combination thereof, be material for implementing the invention in its various forms.

Links

The description of each document cited in this document is incorporated by reference.

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SEQUENCE LIST

<110> 3B Pharmaceuticals GmbH

<120> Compounds comprising a fibroblast activation protein ligand and

use it

<130> D 10026 PCT

<150> EP 19 000 325.1

<151> 2019-07-08

<150> EP 19 198 813.8

<151> 2019-09-20

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 760

<212> PRT

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> Fibroblast activation protein (FAP)

<400> 1

Met Lys Thr Trp Val Lys Ile Val Phe Gly Val Ala Thr Ser Ala Val

1 5 10 15

Leu Ala Leu Leu Val Met Cys Ile Val Leu Arg Pro Ser Arg Val His

20 25 30

Asn Ser Glu Glu Asn Thr Met Arg Ala Leu Thr Leu Lys Asp Ile Leu

35 40 45

Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe Pro Asn Trp Ile Ser Gly

50 55 60

Gln Glu Tyr Leu His Gln Ser Ala Asp Asn Asn Ile Val Leu Tyr Asn

65 70 75 80

Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu Ser Asn Arg Thr Met Lys

85 90 95

Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser Pro Asp Arg Gln Phe Val

100 105 110

Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala

115 120 125

Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly Glu Phe Val Arg Gly Asn

130 135 140

Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser

145 150 155 160

Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro

165 170 175

Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn Gly Arg Glu Asn Lys Ile

180 185 190

Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu Ala Thr

195 200 205

Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly Lys Phe Leu Ala Tyr Ala

210 215 220

Glu Phe Asn Asp Thr Asp Ile Pro Val Ile Ala Tyr Ser Tyr Tyr Gly

225 230 235 240

Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly

245 250 255

Ala Lys Asn Pro Val Val Arg Ile Phe Ile Ile Asp Thr Thr Tyr Pro

260 265 270

Ala Tyr Val Gly Pro Gln Glu Val Pro Val Pro Ala Met Ile Ala Ser

275 280 285

Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Thr Asp Glu Arg Val

290 295 300

Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu Ser Ile

305 310 315 320

Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys Thr Gln

325 330 335

Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val

340 345 350

Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe

355 360 365

Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val

370 375 380

Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Asn Ile

385 390 395 400

Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu

405 410 415

Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Ser Tyr

420 425 430

Pro Pro Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg Cys

435 440 445

Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala Leu

450 455 460

Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser Thr Leu His Asp Gly Arg

465 470 475 480

Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu Asn Lys Glu Leu Glu Asn

485 490 495

Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu Glu Ile Lys Lys Leu Glu

500 505 510

Val Asp Glu Ile Thr Leu Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe

515 520 525

Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro

530 535 540

Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile Ser Tyr

545 550 555 560

Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val Asp Gly Arg Gly

565 570 575

Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr Ala Val Tyr Arg Lys Leu

580 585 590

Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val Arg Lys Phe Ile

595 600 605

Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile Trp Gly Trp Ser

610 615 620

Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu Ala Ser Gly Thr Gly Leu

625 630 635 640

Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr

645 650 655

Ala Ser Val Tyr Thr Glu Arg Phe Met Gly Leu Pro Thr Lys Asp Asp

660 665 670

Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala Glu Tyr

675 680 685

Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp Asp Asn

690 695 700

Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val Asn Ala

705 710 715 720

Gln Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His Gly Leu

725 730 735

Ser Gly Leu Ser Thr Asn His Leu Tyr Thr His Met Thr His Phe Leu

740 745 750

Lys Gln Cys Phe Ser Leu Ser Asp

755 760

<210> 2

<211> 766

<212> PRT

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> Dipeptidyl peptidase 4 (DPP4)

<400> 2

Met Lys Thr Pro Trp Lys Val Leu Leu Gly Leu Leu Gly Ala Ala Ala

1 5 10 15

Leu Val Thr Ile Ile Thr Val Pro Val Val Leu Leu Asn Lys Gly Thr

20 25 30

Asp Asp Ala Thr Ala Asp Ser Arg Lys Thr Tyr Thr Leu Thr Asp Tyr

35 40 45

Leu Lys Asn Thr Tyr Arg Leu Lys Leu Tyr Ser Leu Arg Trp Ile Ser

50 55 60

Asp His Glu Tyr Leu Tyr Lys Gln Glu Asn Asn Ile Leu Val Phe Asn

65 70 75 80

Ala Glu Tyr Gly Asn Ser Ser Val Phe Leu Glu Asn Ser Thr Phe Asp

85 90 95

Glu Phe Gly His Ser Ile Asn Asp Tyr Ser Ile Ser Pro Asp Gly Gln

100 105 110

Phe Ile Leu Leu Glu Tyr Asn Tyr Val Lys Gln Trp Arg His Ser Tyr

115 120 125

Thr Ala Ser Tyr Asp Ile Tyr Asp Leu Asn Lys Arg Gln Leu Ile Thr

130 135 140

Glu Glu Arg Ile Pro Asn Asn Thr Gln Trp Val Thr Trp Ser Pro Val

145 150 155 160

Gly His Lys Leu Ala Tyr Val Trp Asn Asn Asp Ile Tyr Val Lys Ile

165 170 175

Glu Pro Asn Leu Pro Ser Tyr Arg Ile Thr Trp Thr Gly Lys Glu Asp

180 185 190

Ile Ile Tyr Asn Gly Ile Thr Asp Trp Val Tyr Glu Glu Glu Val Phe

195 200 205

Ser Ala Tyr Ser Ala Leu Trp Trp Ser Pro Asn Gly Thr Phe Leu Ala

210 215 220

Tyr Ala Gln Phe Asn Asp Thr Glu Val Pro Leu Ile Glu Tyr Ser Phe

225 230 235 240

Tyr Ser Asp Glu Ser Leu Gln Tyr Pro Lys Thr Val Arg Val Pro Tyr

245 250 255

Pro Lys Ala Gly Ala Val Asn Pro Thr Val Lys Phe Phe Val Val Asn

260 265 270

Thr Asp Ser Leu Ser Ser Val Thr Asn Ala Thr Ser Ile Gln Ile Thr

275 280 285

Ala Pro Ala Ser Met Leu Ile Gly Asp His Tyr Leu Cys Asp Val Thr

290 295 300

Trp Ala Thr Gln Glu Arg Ile Ser Leu Gln Trp Leu Arg Arg Ile Gln

305 310 315 320

Asn Tyr Ser Val Met Asp Ile Cys Asp Tyr Asp Glu Ser Ser Gly Arg

325 330 335

Trp Asn Cys Leu Val Ala Arg Gln His Ile Glu Met Ser Thr Thr Gly

340 345 350

Trp Val Gly Arg Phe Arg Pro Ser Glu Pro His Phe Thr Leu Asp Gly

355 360 365

Asn Ser Phe Tyr Lys Ile Ile Ser Asn Glu Glu Gly Tyr Arg His Ile

370 375 380

Cys Tyr Phe Gln Ile Asp Lys Lys Asp Cys Thr Phe Ile Thr Lys Gly

385 390 395 400

Thr Trp Glu Val Ile Gly Ile Glu Ala Leu Thr Ser Asp Tyr Leu Tyr

405 410 415

Tyr Ile Ser Asn Glu Tyr Lys Gly Met Pro Gly Gly Arg Asn Leu Tyr

420 425 430

Lys Ile Gln Leu Ser Asp Tyr Thr Lys Val Thr Cys Leu Ser Cys Glu

435 440 445

Leu Asn Pro Glu Arg Cys Gln Tyr Tyr Ser Val Ser Phe Ser Lys Glu

450 455 460

Ala Lys Tyr Tyr Gln Leu Arg Cys Ser Gly Pro Gly Leu Pro Leu Tyr

465 470 475 480

Thr Leu His Ser Ser Val Asn Asp Lys Gly Leu Arg Val Leu Glu Asp

485 490 495

Asn Ser Ala Leu Asp Lys Met Leu Gln Asn Val Gln Met Pro Ser Lys

500 505 510

Lys Leu Asp Phe Ile Ile Leu Asn Glu Thr Lys Phe Trp Tyr Gln Met

515 520 525

Ile Leu Pro Pro His Phe Asp Lys Ser Lys Lys Tyr Pro Leu Leu Leu

530 535 540

Asp Val Tyr Ala Gly Pro Cys Ser Gln Lys Ala Asp Thr Val Phe Arg

545 550 555 560

Leu Asn Trp Ala Thr Tyr Leu Ala Ser Thr Glu Asn Ile Ile Val Ala

565 570 575

Ser Phe Asp Gly Arg Gly Ser Gly Tyr Gln Gly Asp Lys Ile Met His

580 585 590

Ala Ile Asn Arg Arg Leu Gly Thr Phe Glu Val Glu Asp Gln Ile Glu

595 600 605

Ala Ala Arg Gln Phe Ser Lys Met Gly Phe Val Asp Asn Lys Arg Ile

610 615 620

Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Thr Ser Met Val Leu

625 630 635 640

Gly Ser Gly Ser Gly Val Phe Lys Cys Gly Ile Ala Val Ala Pro Val

645 650 655

Ser Arg Trp Glu Tyr Tyr Asp Ser Val Tyr Thr Glu Arg Tyr Met Gly

660 665 670

Leu Pro Thr Pro Glu Asp Asn Leu Asp His Tyr Arg Asn Ser Thr Val

675 680 685

Met Ser Arg Ala Glu Asn Phe Lys Gln Val Glu Tyr Leu Leu Ile His

690 695 700

Gly Thr Ala Asp Asp Asn Val His Phe Gln Gln Ser Ala Gln Ile Ser

705 710 715 720

Lys Ala Leu Val Asp Val Gly Val Asp Phe Gln Ala Met Trp Tyr Thr

725 730 735

Asp Glu Asp His Gly Ile Ala Ser Ser Thr Ala His Gln His Ile Tyr

740 745 750

Thr His Met Ser His Phe Ile Lys Gln Cys Phe Ser Leu Pro

755 760 765

<210> 3

<211> 710

<212> PRT

<213> Homo sapiens

<220>

<221>MISC_FEATURE

<223> Prolyl endopeptidase (PREP)

<400> 3

Met Leu Ser Leu Gln Tyr Pro Asp Val Tyr Arg Asp Glu Thr Ala Val

1 5 10 15

Gln Asp Tyr His Gly His Lys Ile Cys Asp Pro Tyr Ala Trp Leu Glu

20 25 30

Asp Pro Asp Ser Glu Gln Thr Lys Ala Phe Val Glu Ala Gln Asn Lys

35 40 45

Ile Thr Val Pro Phe Leu Glu Gln Cys Pro Ile Arg Gly Leu Tyr Lys

50 55 60

Glu Arg Met Thr Glu Leu Tyr Asp Tyr Pro Lys Tyr Ser Cys His Phe

65 70 75 80

Lys Lys Gly Lys Arg Tyr Phe Tyr Phe Tyr Asn Thr Gly Leu Gln Asn

85 90 95

Gln Arg Val Leu Tyr Val Gln Asp Ser Leu Glu Gly Glu Ala Arg Val

100 105 110

Phe Leu Asp Pro Asn Ile Leu Ser Asp Asp Gly Thr Val Ala Leu Arg

115 120 125

Gly Tyr Ala Phe Ser Glu Asp Gly Glu Tyr Phe Ala Tyr Gly Leu Ser

130 135 140

Ala Ser Gly Ser Asp Trp Val Thr Ile Lys Phe Met Lys Val Asp Gly

145 150 155 160

Ala Lys Glu Leu Pro Asp Val Leu Glu Arg Val Lys Phe Ser Cys Met

165 170 175

Ala Trp Thr His Asp Gly Lys Gly Met Phe Tyr Asn Ser Tyr Pro Gln

180 185 190

Gln Asp Gly Lys Ser Asp Gly Thr Glu Thr Ser Thr Asn Leu His Gln

195 200 205

Lys Leu Tyr Tyr His Val Leu Gly Thr Asp Gln Ser Glu Asp Ile Leu

210 215 220

Cys Ala Glu Phe Pro Asp Glu Pro Lys Trp Met Gly Gly Ala Glu Leu

225 230 235 240

Ser Asp Asp Gly Arg Tyr Val Leu Leu Ser Ile Arg Glu Gly Cys Asp

245 250 255

Pro Val Asn Arg Leu Trp Tyr Cys Asp Leu Gln Gln Glu Ser Ser Gly

260 265 270

Ile Ala Gly Ile Leu Lys Trp Val Lys Leu Ile Asp Asn Phe Glu Gly

275 280 285

Glu Tyr Asp Tyr Val Thr Asn Glu Gly Thr Val Phe Thr Phe Lys Thr

290 295 300

Asn Arg Gln Ser Pro Asn Tyr Arg Val Ile Asn Ile Asp Phe Arg Asp

305 310 315 320

Pro Glu Glu Ser Lys Trp Lys Val Leu Val Pro Glu His Glu Lys Asp

325 330 335

Val Leu Glu Trp Ile Ala Cys Val Arg Ser Asn Phe Leu Val Leu Cys

340 345 350

Tyr Leu His Asp Val Lys Asn Ile Leu Gln Leu His Asp Leu Thr Thr

355 360 365

Gly Ala Leu Leu Lys Thr Phe Pro Leu Asp Val Gly Ser Ile Val Gly

370 375 380

Tyr Ser Gly Gln Lys Lys Asp Thr Glu Ile Phe Tyr Gln Phe Thr Ser

385 390 395 400

Phe Leu Ser Pro Gly Ile Ile Tyr His Cys Asp Leu Thr Lys Glu Glu

405 410 415

Leu Glu Pro Arg Val Phe Arg Glu Val Thr Val Lys Gly Ile Asp Ala

420 425 430

Ser Asp Tyr Gln Thr Val Gln Ile Phe Tyr Pro Ser Lys Asp Gly Thr

435 440 445

Lys Ile Pro Met Phe Ile Val His Lys Lys Gly Ile Lys Leu Asp Gly

450 455 460

Ser His Pro Ala Phe Leu Tyr Gly Tyr Gly Gly Phe Asn Ile Ser Ile

465 470 475 480

Thr Pro Asn Tyr Ser Val Ser Arg Leu Ile Phe Val Arg His Met Gly

485 490 495

Gly Ile Leu Ala Val Ala Asn Ile Arg Gly Gly Gly Glu Tyr Gly Glu

500 505 510

Thr Trp His Lys Gly Gly Ile Leu Ala Asn Lys Gln Asn Cys Phe Asp

515 520 525

Asp Phe Gln Cys Ala Ala Glu Tyr Leu Ile Lys Glu Gly Tyr Thr Ser

530 535 540

Pro Lys Arg Leu Thr Ile Asn Gly Gly Ser Asn Gly Gly Leu Leu Val

545 550 555 560

Ala Ala Cys Ala Asn Gln Arg Pro Asp Leu Phe Gly Cys Val Ile Ala

565 570 575

Gln Val Gly Val Met Asp Met Leu Lys Phe His Lys Tyr Thr Ile Gly

580 585 590

His Ala Trp Thr Thr Asp Tyr Gly Cys Ser Asp Ser Lys Gln His Phe

595 600 605

Glu Trp Leu Val Lys Tyr Ser Pro Leu His Asn Val Lys Leu Pro Glu

610 615 620

Ala Asp Asp Ile Gln Tyr Pro Ser Met Leu Leu Leu Thr Ala Asp His

625 630 635 640

Asp Asp Arg Val Val Pro Leu His Ser Leu Lys Phe Ile Ala Thr Leu

645 650 655

Gln Tyr Ile Val Gly Arg Ser Arg Lys Gln Ser Asn Pro Leu Leu Ile

660 665 670

His Val Asp Thr Lys Ala Gly His Gly Ala Gly Lys Pro Thr Ala Lys

675 680 685

Val Ile Glu Glu Val Ser Asp Met Phe Ala Phe Ile Ala Arg Cys Leu

690 695 700

Asn Val Asp Trp Ile Pro

705 710

<---

Claims (103)

1. Compound of formula: or a pharmaceutically acceptable salt thereof. 2. Compound of formula: or a pharmaceutically acceptable salt thereof. 3. Compound of formula: 4. Compound of formula: 5. The compound of the following formula or a pharmaceutically acceptable salt thereof, where the compound contains a therapeutically active nuclide. 6. Compound of formula or a pharmaceutically acceptable salt thereof, where the compound contains a therapeutically active nuclide. 7. The compound of the following formula: where the compound contains a therapeutically active nuclide. 8. The compound of the following formula where the compound contains a therapeutically active nuclide. 9. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 5 to 8, wherein the therapeutically active nuclide is a therapeutically active radionuclide. 10. The compound or pharmaceutically acceptable salt thereof according to claim 9, wherein the therapeutically active radionuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁶ Th, ²²⁷ Th, ¹³¹ I, ²¹¹ At. 11. A compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein the therapeutically active nuclide is a therapeutically active radiometal. 12. A compound or pharmaceutically acceptable salt thereof according to any one of claims 7 to 11, wherein the therapeutically active radionuclide is ¹⁷⁷ Lu. 13. A compound selected from the group comprising: or a pharmaceutically acceptable salt thereof; and or its pharmaceutically acceptable salt, wherein the compound comprises a therapeutically active nuclide, wherein the therapeutically active nuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁸⁶ Re, ¹⁸⁸ Re, ^{212 Pb, 213 Bi} , ²²³ Ra, ²²⁶ Th, ²²⁷ Th, ¹³¹ I, and ²¹¹ At. 14. Compound of formula or a pharmaceutically acceptable salt thereof, wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ¹⁷⁷ Lu. 15. Compound of formula or a pharmaceutically acceptable salt thereof, wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ²²⁵ Ac. 16. Compound of formula: or a pharmaceutically acceptable salt thereof, wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ¹⁷⁷ Lu. 17. Compound of formula or a pharmaceutically acceptable salt thereof, wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ²²⁵ Ac. 18. Compound of formula: wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ¹⁷⁷ Lu. 19. Compound of formula: wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ²²⁵ Ac. 20. Compound of formula: wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ¹⁷⁷ Lu. 21. Compound of formula: wherein the compound comprises a therapeutically active radionuclide, wherein the therapeutically active radionuclide is ²²⁵ Ac. 22. A compound or a pharmaceutically acceptable salt thereof according to claims 9, 10 or 13, wherein the therapeutically active radionuclide is an isotope that emits particles for therapeutic use and has a decay energy from 0.039 to 10 MeV. 23. A compound or a pharmaceutically acceptable salt thereof according to claim 19, wherein the therapeutically active radionuclide has a decay energy from 0.4 to 6.5 MeV. 24. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 5-23, wherein the therapeutically active nuclide or radionuclide is complexed with the chelator 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). 25. A radionuclide chelate complex comprising a compound or a pharmaceutically acceptable salt thereof as defined in any of paragraphs 1-4 and a radionuclide that is complexed with the chelator 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). 26. The radionuclide chelate complex according to item 25, the radionuclide is a therapeutically active radionuclide. 27. The radionuclide chelate complex according to claim 26, wherein the therapeutically active radionuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ¹⁵³ Sm, ¹⁴⁹ Tb, ¹⁶¹ Tb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ^{188 Re, 212 Pb} , ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁶ Th, ²²⁷ Th, ¹³¹ I, ²¹¹ At. 28. A radionuclide chelate complex according to claim 25, wherein the therapeutically active nuclide is a therapeutically active radiometal. 29. The radionuclide chelate complex according to claim 27, wherein the therapeutically active radionuclide is ¹⁷⁷ Lu. 30. A radionuclide chelate complex comprising a compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 4 and a therapeutically active nuclide that is complexed with a chelator of 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), wherein the therapeutically active nuclide is selected from the group consisting of ⁴⁷ Sc, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ^{153 Sm, 149 Tb} , ¹⁶¹ Tb, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹³ Bi, ²²³ Ra, ²²⁶ Th, ^{227 Th, 131 I} and ²¹¹ At. 31. A radionuclide chelate complex according to claim 30, wherein the therapeutically active radionuclide is an isotope that emits particles for therapeutic use and has a decay energy from 0.039 to 10 MeV. 32. A radionuclide chelate complex according to claim 31, wherein the therapeutically active radionuclide has a decay energy from 0.4 to 6.5 MeV. 33. A radionuclide chelate complex comprising a compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-4 and a therapeutically active nuclide that is complexed with the chelator 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), wherein the therapeutically active nuclide is ²²⁵ Ac. 34. A radionuclide chelate complex comprising a compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1-4 and a therapeutically active nuclide that is complexed with the chelator 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), wherein the therapeutically active nuclide is ¹⁷⁷ Lu. 35. A therapeutically active radionuclide chelate complex selected from the group consisting of or its pharmaceutically acceptable salt, or its pharmaceutically acceptable salt, or its pharmaceutically acceptable salt, or its pharmaceutically acceptable salt, or its pharmaceutically acceptable salt, or its pharmaceutically acceptable salt, or a pharmaceutically acceptable salt thereof. 36. A pharmaceutical composition for inhibiting the enzymatic activity of fibroblast activation protein (FAP), comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-24 or a radionuclide chelate complex according to any one of claims 25-35 in an effective amount and a pharmaceutically acceptable excipient. 37. A pharmaceutical composition for treating a disease, where the disease is a neoplasm, cancer or tumor, comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-24 or a radionuclide chelate complex according to any one of claims 25-35 in an effective amount and a pharmaceutically acceptable excipient. 38. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-24, a radionuclide chelate complex according to any one of claims 25-35, or a pharmaceutical composition according to any one of claims 36 or 37 for the treatment of a disease, wherein the disease is a neoplasm. 39. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-24, a radionuclide chelate complex according to any one of claims 25-35, or a pharmaceutical composition according to any one of claims 36 or 37 for the treatment of a disease, wherein the disease is cancer or a tumor. 40. The use according to any one of claims 38 or 39, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP). 41. The use according to any one of claims 38-40, wherein the disease involves tumor-associated fibroblasts. 42. The use according to claim 41, wherein the neoplasm is selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma, and thyroid cancer. 43. The use according to claim 41, wherein the neoplasm is selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, head and neck cancer, lung cancer, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and squamous cell carcinoma. 44. A kit for treating a disease, wherein the disease is a neoplasm, cancer or tumor, comprising a compound, a pharmaceutically acceptable salt according to any one of claims 1-24, a radionuclide chelate complex according to any one of claims 25-35 or a pharmaceutically acceptable composition according to any one of claims 36 or 37, one or more optional excipients and optionally one or more devices, wherein the device(s) is(are) selected from the group consisting of a labeling device, a purification device, a processing device, a radioprotection device, an assay device or an administration device. 45. The kit of claim 44, wherein the disease involves cells exhibiting increased expression of fibroblast activation protein (FAP). 46. The kit according to any one of claims 44, 45, wherein the disease involves tumor-associated fibroblasts. 47. The kit of claim 46, wherein the neoplasm is selected from the group consisting of a solid tumor, an epithelial tumor, bladder cancer, breast cancer, cervical cancer, colorectal cancer, cholangiocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, head and neck cancer, liver cancer, lung cancer, melanoma, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary carcinoma, sarcoma, squamous cell carcinoma, and thyroid cancer. 48. The use according to claim 46, wherein the neoplasm is selected from the group consisting of breast cancer, colorectal cancer, cholangiocarcinoma, head and neck cancer, lung cancer, mesothelioma, neuroendocrine tumors and carcinomas, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma and squamous cell carcinoma.