CXCR4-ligand and its precursor for diagnostic and therapeutic use

Abstract

The present invention relates to a compound of formula (I): TIFF2024506700000214.tif26149 [In the formula, a is 0 or 1, b is 0 or 1, c is 0 or 1, and d is 0 or 1, provided that at least one of c and d is 1, e is an integer of 1 to 4, and R CP is the binding motif that binds this compound to the CXCR4 receptor, and R L1 is H or alkyl, and R L2 is -NH 2 and -NH-C(=X)-NH 2 X is selected from NH and O; R L3 is -CH 2 -NH 2 or -CH 2 -(1H-imidazol-4-yl), and R L4 Ha-NH 2 and X 1 is a coupling group, R S is a divalent spacer group, and R A is a functional group that includes a moiety having diagnostic or therapeutic utility, or a salt thereof. The compounds of the present invention are suitable for use in the treatment, prevention, and / or diagnosis of diseases or disorders that can be treated or prevented by blocking the CXCR4 receptor, or diseases or disorders associated with increased or abnormal expression of the CXCR4 receptor.

Description

[Technical Field]

[0001] This invention relates to a compound that can bind with high affinity to the seven-transmembrane G protein-coupled chemokine receptor subtype (CXCR4) and is therefore considered a CXCR4 ligand. It can preferably act as an agonist, or as an antagonist, inverse agonist, or partial agonist. The compound is suitable for use in diagnostic and therapeutic applications. [Overview of the project]

[0002] When stroma cell-derived factor 1 (SDF-1) (hereinafter referred to as CXC motif chemokine 12 (CXCL12)) binds to CXCR4 (1), it activates the downstream protein kinase B (AKT) / mitogen-activated protein kinase (MAPK) signaling pathway, leading to altered gene expression, actin polymerization, cytoskeletal rearrangement, and cell migration. The physiological functions of the CXCL12 / CXCR4 axis include embryogenesis, immune response, hematopoiesis, brain development, and angiogenesis (2-6). In addition to CXCR4's basic involvement in physiological processes, elevated CXCR4 expression is associated with various malignancies. CXCR4 is the first to be identified as a co-receptor, mediating the entry of HIV-1 into T cells (3). CXCR4 is involved in B cell transport and tissue localization in patients with chronic leukemia (7), as well as in the regulation of organ-specific metastasis in various breast cancer models (8). Therefore, CXCR4 overexpression is known in more than 20 human tumor types, including hematopoietic malignancies, brain neoplasms, gastrointestinal cancers, and other cancer types (2, 8-10). There is growing evidence that the CXCL12 / CXCR4 axis functions as a crucial communication bridge between tumor cells and stromal cells, creating a tolerant microenvironment (11). Thus, because abnormal expression of CXCR4 potently promotes the growth, migration, and invasion of various cancer types, the cytokine CXCL12 and its receptor CXCR4 are promising and viable targets for therapeutic strategies (12).

[0003] Several peptidogenic and non-peptidogenic CXCR4 antagonists targeting the CXCL12 / CXCR4 axis have been developed due to their potential for pharmaceutical applications. The most well-established example is bisicram AMD3100 (Plerixafor®, tetrazacyclotetradeca-1-ylmethyl)phenyl]methyl}-1,4,8,11-tetrazacyclotetradecane), which has been approved by the FDA for the treatment of non-Hodgkin lymphoma and multiple myeloma. Further peptidogenic CXCR4 antagonists, such as T140 and its derivatives, which are side-chain cyclized peptides containing one or two cyclization sites, have been developed (13-15). A less cytotoxic and biologically stable derivative of T140 is TN14003 (16). The introduction of a 4-fluorobenzoyl group constituted a novel pharmacophore in TF14016, a T140-type CXCR4 antagonist, resulting in sub-nanomolar binding affinity (16). This peptide CXCR4 antagonist further enhances CXCR4 expression in vivo. 18 F-series or 68 Ga-based positron emission tomography (PET) imaging has been used (17-19). T140-based CXCR4 antagonists are already used in the prevention and / or treatment of cancer and rheumatoid arthritis (US8410059B2, US8765683B2).

[0004] LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-D-Arg-2-Nal-Gly-D-Glu]-Lys(iPr)-NH2), a potent CXCR4 antagonist, has demonstrated favorable antitumor activity in solid tumor models and breast cancer metastasis models and is currently in Phase II clinical trials (NCT01391130 and NCT1439568)(20).

[0005] Furthermore, three cyclic pentapeptides (peptides R, I, and S) based on the N-terminal sequence of CXCL12 significantly inhibit the subcutaneous growth of renal cancer cells. Renal cancer cells also cause lung metastasis and primary tumor growth (21). In addition, lactam cyclized heptapeptides have been reported to be potent CXCR4 antagonists useful in the treatment of cancer, rheumatoid arthritis, pulmonary fibrosis, and HIV infection (WO2008 / 150689A1).

[0006] The cyclic pentapeptide (hereinafter referred to as Fc-131) based on cyclo(D-Tyr 1 -Arg 2 -Arg 3 -Nal 4 -Gly 5 ) derived from T140 is used for cancer treatment and anti-inflammation (22). All SAR studies including alanine scanning, N-methyl amino acid scanning, optimization of amino acid residues, and design of retro-inverso sequence peptides were unable to improve the binding affinity or anti-HIV activity compared to Fc-131 (23-25), demonstrating the highly optimized binding scaffold of Fc-131. However, the introduction of an amidine type dipeptide equivalent led to a novel lead structure of cyclic pentapeptides targeting CXCR4 (WO2012 / 118124A1). In addition, N-methylation of the peptide bond of Fc-131 significantly affected its activity, resulting in Fc-122, a cyclic pentapeptide-based CXCR4 antagonist showing a significant enhancement of CXCR4 antagonist activity (26-28).

[0007] Within the scope of the development of molecular imaging probes for CXCR4, N-methylation techniques were used to enhance the binding affinity, while all side chains of Fc-131 were tested for their feasibility of exchange. Arg 2 was replaced with D-ornithine, followed by methylation of the N-terminus, resulting in CPCR4 (cyclo(D-Tyr 1 -D-[NMe]Orn 2 -Arg 3 -Nal 4 -Gly 5The following was obtained, which shows good binding affinity to CXCR4. This read structure served as an anchor point for further modification (29) (WO2007 / 096662). Dimer derivatives of CPCR4 were described, but their application to in vivo diagnostics was hindered by increased accumulation of CXCR4 ligand in the liver (WO2009 / 027706) (29, 30).

[0008] Recently, minimalist methods have been used, and in them, the technetium chelator hydrazino-nicotinic acid is used in D-Orn 2 Direct binding to the side chain resulted in a CXCR4 SPECT imaging agent (31). This compound is currently being investigated in the first demonstration of a conceptual study in humans (32).

[0009] Nevertheless, further structural modifications are still possible, for example, cyclo(D-Tyr 1 -D-[NMe]Orn 2 -Arg 3 -Nal 4 -Gly 5 ) of [NMe]Orn 2 The introduction of an aromatic spacer bonded to the side chain facilitated the introduction of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a labeling moiety (WO2011 / 131735) for pharmaceutical applications (33-35).

[0010] In most cases, additional structural changes to detectable labels (36) result in a significant loss of binding affinity. 1 -D-[NMe](DOTA)-Orn 2 Arg 3 -Nal 4 -Gly 5 An additional modification (introduction of (3-iodo)tyrosine) to the cyclic pentapeptide scaffold of pentixafor was described, which significantly increased the binding affinity to hCXCR4. Thus, the obtained 177 Lu-pentixather and 90Y-pentixate has been used as a treatment for CXCR4-related malignancies (WO2015 / 185162)(37).

[0011] Building on the success of these clinically applied peptides, further optimizations were attempted to enhance the affinity and versatility of Peptiscope. Replacing short aromatic spacers with tailor-made peptidolytic linker units ultimately resulted in a CXCR4 binding motif with enhanced affinity and significantly increased internalization rates due to the semi-agonistic nature of the newly induced peptide scaffold (38). These modifications did not significantly impair the affinity of the resulting peptides. 18 It has become possible to introduce various functional modes such as mas3-type technetium chelators or AmBF3 as F-labeling units (WO2020 / 053255).

[0012] The diagnostic and therapeutic potential of CXCR4 ligands has been demonstrated in a variety of cases, for example, for the treatment of HIV infection and cancer, or for the visualization of CXCR4 expression in patients. In particular, cyclic pentapeptides are optimized for complete interaction of compounds with the CXCR4 binding cavity and for minor modifications, such as the introduction of a labeling moiety or labeling residue to affect the pharmacokinetics of the CXCR4 ligand, resulting in a considerable loss of high affinity (36), and therefore must be structurally optimized for each desired application. Consequently, the design and development of novel CXCR4-targeted compounds require meticulous structure-activity relationship (SAR) studies.

[0013] For this reason, there is a need for a universally applicable ligand design that ensures high affinity for the human CXCR4 receptor while simultaneously enabling the binding of a wide variety of functional groups that have diagnostic or therapeutic utility. This invention provides such novel ligand compounds and their use in medical and scientific applications. The compounds of this invention can bind to the human CXCR4 receptor with high affinity and to the mouse receptor with medium affinity, and are therefore suitable as CXCR4 ligands. These ligands can function as agonists, inverse agonists, partial agonists, or antagonists. The linker structure in the ligand compounds provided by this invention remarkably results in the high adaptability of the compounds to the binding of various functional moieties while simultaneously preserving or even enhancing the affinity for CXCR4. Furthermore, based on favorable in vitro properties such as higher affinity and enhanced internalization, higher and more consistent tumor uptake is often achieved. Therefore, the compounds of this invention are particularly suitable for medical applications such as preclinical and clinical imaging, as well as therapeutic applications such as internal radiotherapy.

[0014] Given the above background, the present invention relates to a CXC chemokine receptor type 4 ligand compound, or more simply, a CXCR4 receptor ligand compound, of formula (I):

[0015] [ka]

[0016] [In the formula, a is 0 or 1, preferably 0. b is either 0 or 1, c is 0 or 1, and d is 0 or 1, provided that at least one of c and d is 1. e is an integer between 1 and 4, preferably between 2 and 4. R CP Equation (II):

[0017] [ka]

[0018] (Here, in equation (II), R B1 is H or I, preferably H. R B2 It is an alkanediyl chain, The dashed line is R CP (This shows a bond that attaches the group to the remainder of the compound in formula (I).) It is a cyclopeptide group, R L1 is H or alkyl, R L2 is a substituted alkyl group that is substituted with at least one group selected from -NH2 and -NH-C(=X)-NH2, where X is selected from NH and O. R L3 It is -CH2-NH2 or -CH2-(1H-imidazole-4-yl), R L4 It is -NH2, X 1 is a coupling group, R S is a divalent spacer group, and R A This is a functional group that includes a portion that has diagnostic or therapeutic utility. The present invention provides compounds or salts thereof. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows the MIP (micro-intensity) of both CT and SPECT scans 1 hour after injection of 99mTc-CXCR4-6 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 2]Figure 2 shows the MIP (micro-injection) of both CT and SPECT scans 1 hour after injection of 99mTc-CXCR4-9 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 3] Figure 3 shows the MIP (micro-intensity) of both CT and SPECT scans 1 hour after injection of 99mTc-CXCR4-11 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 4] Figure 4 shows the MIP (Multiple Intake) of both CT and SPECT scans 1 hour after injection of 99mTc-CXCR4-12 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 5] Figure 5 shows the MIP (Multiple Intake) of both CT and SPECT scans 1 hour after injection of 99mTc-CXCR4-13 in female Jurkat tumor-bearing mice. No competing substances, 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 6] Figure 6 shows the MIP (micro-injection) of both CT and SPECT scans 1 hour after injection of 99mTc-CXCR4-14 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 7] Figure 7 shows the MIP (Multiple Intake) of both CT and SPECT scans 2 hours after injection of 99mTc-CXCR4-14 in female Jurkat tumor-bearing mice. No competing substances, 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 8]Figure 8 shows maximum projection (MIP) images obtained from SPECT and PET imaging of a female patient with multiple myeloma. A) PET MIP of [18F]FDG (1 hour post-injection, 189 MBq of [18F]FDG), B) SPECT MIP of [99mTc]CXCR4-Tc-14 (3 hours post-injection, 604 MBq of [99mTc]CXCR4-Tc-14). Linear arrows indicate tumor lesions, and dotted arrows indicate physiological uptake of [18F]FDG in the heart and physiological uptake of [99mTc]CXCR4-Tc-14 in the spleen. [Figure 9] Figure 9A) shows an axial SPECT / CT image 3 hours after injection of 604 MBq of [99mTc]CXCR4-Tc-14 in a patient with multiple myeloma, and Figure 9B) shows an axial PET / CT image 1 hour after injection of 189 MBq of [18F]FDG in the same patient, where straight arrows indicate the bladder and dashed arrows indicate osteolytic lesions. [Figure 10] Figure 10 shows the in vivo distribution of [99mTc]CXCR4-Tc-14 between 5 minutes and 21 hours after injection in the same patient. Figure 10A) shows MIP images obtained from SPECT imaging at 5 minutes, 60 minutes, 120 minutes, 5 hours, and 21 hours after injection of 604 MBq of [99mTc]CXCR4-Tc-14 in a female patient with multiple myeloma, and Figure 10B) shows the calculated specific dose [μGy / MBq] for selected organs and tissues in the same patient. [Figure 11] Figure 11 shows the MIP (micro-intensity) of both CT and SPECT scans 1 hour after injection of 177Lu-CXCR4-DOTA-1 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 2–10% iD / mL. White arrows indicate the organ of interest: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 12] Figure 12 shows the MIP of both CT and SPECT scans 1 hour after injection of 177Lu-CXCR4-DOTA-2 in female Jurkat tumor-bearing mice. No competing substances, 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 13] Figure 13 shows the MIP (micro-intensity) of both CT and SPECT scans 1 hour after injection of 177Lu-CXCR4-DOTA-3 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 14] Figure 14 shows the MIP (micro-intensity) of both CT and SPECT scans 1 hour after injection of 177Lu-CXCR4-DOTA-4 in female Jurkat tumor-bearing mice. With and without (left) and with (right, 100 nmol AMD3100), 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 15] Figure 15 shows the MIP of both CT and SPECT scans 6 hours after 177Lu-CXCR4-DOTA-4 injection in female Jurkat tumor-bearing mice. No competing substances, 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. [Figure 16] Figure 16 shows the in vivo distribution profile 1 hour after injection of [18F, natGa]CXCR4-SiFA-07 in female CB-17SCID mice bearing Jurkat tumors. Different amounts of radioligand, i.e., 36 pmol, 1,000 pmol, and 2,000 pmol, were applied. Data are expressed as %iD / g values, and for the 36 pmol experiment, the values ​​are the mean ± SD of 5 animals. One animal was used for each of the other two experiments. [Figure 17] Figure 17 shows the results of flow cytometry analysis of cell viability using Raji cells (left) and U2932 cells (right). Cells were incubated with CXCR4-MMAE-02 at 0 (blank), 10, 20, 40, and 100 nM for 0, 24, 48, 72, and 96 hours, then stained with propidium iodide and subjected to flow cytometry analysis. Cell viability is defined as the number of surviving cells relative to the total number of cells. [Figure 18]Figure 18 shows the results of flow cytometry analysis of cell viability using Raji cells and U2932 cells. Cells were incubated with CXCR4-MMAE-02 at concentrations of 0 (blank), 10, 20, 40, and 100 nM for 96 hours, then stained with propidium iodide, and flow cytometry analysis of dead cells was performed. [Figure 19] Figure 19 shows the results of cell cycle profiling experiments using U2932 cells. Cells were incubated with 0 (blank), 10, 20, 40, and 100 nM CXCR4-MMAE-02 for 72 hours, then fixed, permeabilized, and stained with propidium iodide. The percentage of cells in the G0 / G1, S, or G2 / M phases was determined. [Figure 20] Figure 20 shows the in vivo distribution profile 1 hour after injection of [177Lu]CXCR4-OI-03 in female CB-17SCID mice bearing Jurkat tumors. Different amounts of radioligand, namely 59 pmol and 1,000 pmol, were applied. Data are expressed as %iD / g values, with the 59 pmol experiment using the mean ± SD of 5 animals, and the other experiment using 1 animal. [Modes for carrying out the invention]

[0020] In the CXCR4 receptor ligand compound of the present invention, the binding motif R CP and functional group R A These are joined by a linker with the following structure:

[0021] [ka]

[0022] This linker, which forms part of the compound of formula (I), is R L3 , R L4 as, or R L3 and R L4The linker structure is characterized by the presence of small substituents and functional groups that typically carry a positive charge under physiological conditions. In relation to the present invention, this linker structure is particularly beneficial for the functional group R A It was found that the high affinity of CXCR4 is maintained in the compounds of the present invention with little to no adverse effects. Therefore, by relying on the combination of binding motifs and linkers contained in the compounds of the present invention, there is no concern regarding the significant loss of affinity caused by the binding motif, and a wide variety of CXCR4 receptor ligands with diverse functional groups can be provided.

[0023] In a more relevant aspect, the present invention provides a therapeutic or diagnostic composition comprising a CXCR4 receptor ligand compound of formula (I) or a salt thereof. As stated above, salts of the compound of formula (I), typically pharmaceutically acceptable salts, are included in the present invention. Therefore, unless otherwise stated, any reference to the compounds according to the present invention herein includes both the compound of formula (I), the preferred embodiments of this formula disclosed herein, and its salts. Furthermore, any racemic, enantiomer, or diastereomer of the compound of formula (I) is included unless the specific stereochemistry of the compound under consideration is indicated in a particular context.

[0024] As explained by the reference to the compound of formula (I) as a CXCR4 receptor ligand compound, this compound can act as a ligand compound that binds to the CXCR4 receptor. Accordingly, the compound of formula (I) and its salts have a cyclopeptide group R which may be referred herein as the "binding motif" of the CXCR4 receptor ligand compound of formula (I). CP It contains the cyclopeptide group R. CP This is because it ensures a binding interaction between the compound or a salt thereof according to the present invention and the CXCR4 receptor, thereby serving as an affinity anchor for the compound to the CXCR4 receptor.

[0025] The binding motif of a CXCR4 receptor ligand compound is preferably capable of specifically binding to the CXCR4 receptor. In this regard, specifically binding preferably means that the binding motif of the CXCR4 receptor ligand compound does not bind to or substantially binds to other proteins other than the CXCR4 receptor, in particular, does not bind to or substantially binds to other members of the CXC chemokine receptor family. The term "substantially unbound" is preferably used, for example, in IC 50 The value indicates that the binding affinity of the binding motif to CXCR4 is at least 100 times, preferably at least 1000 times, and most preferably at least 10000 times stronger than the binding affinity to other proteins, particularly other members of the CXC chemokine receptor family.

[0026] Cyclopeptide group R as the binding motif in formula (I) CP Equation (II):

[0027] [ka]

[0028] [In the formula, R B1 is H or I, preferably H, and R B2 [It is an alkanediyl chain.] It is the basis of.

[0029] As experienced readers will understand, the bond shown by the dashed line in equation (II) does not have a methyl group at the end opposite the nitrogen atom, R CP The group is the remainder of the compound of formula (I), i.e., in this case, R of formula (I). CP This represents the bond that is attached to the bonding point. In other words, the bond shown by the dashed line in formula (II) is the bond between the nitrogen atom of the -NH- group shown in formula (II) and the R shown on the left side of formula (I) that is present in the compound according to the present invention. CPThis represents a covalent bond between the carbon atom of the carbonyl group to which it is bonded. Thus, the amide bond is represented by the R shown in formula (II). CP It is provided using the -NH- group and the carbonyl group shown in formula (I).

[0030] Under equation (II), -R B2 - is an alkanediyl chain, preferably a C2-C6 alkanediyl chain, more preferably a C2-C3 alkanediyl chain, and most preferably a -CH2-CH2-CH2-. Therefore, R CP Preferably, formula (IIa):

[0031] [ka]

[0032] It is the basis of, and in the formula, R B1 This is defined for formula (II) and includes any preferred embodiment of this variable as further defined herein, where the dashed line is R CP This shows a bond that attaches the group to the remainder of the compound of formula (I).

[0033] R CP Equation (IIb):

[0034] [ka]

[0035] It is even more preferable that it be the base of, in the formula R B1 This is defined for formula (II) and includes any preferred embodiment of this variable as further defined herein, where the dashed line is R CP This shows a bond that attaches the group to the remainder of the compound of formula (I).

[0036] The variable a in formula (I) is either 0 or 1, so the -CH2- group bonded to the phenylene ring in the linker structure of formula (I) is an optional group that may or may not be present. Naturally, if it is not present, i.e., a is 0, the -CH2- group is replaced by a direct bond between the atoms adjacent to this group in formula (I).

[0037] R in equation (I) L1 The group is H or C1-C6 alkyl, more preferably H or C1-C3 alkyl, even more preferably H or methyl, and most preferably methyl. R in equation (I) L2 R is a substituted alkyl group substituted with at least one group selected from -NH2 and -NH-C(=X)-NH2. X is selected from NH and O. The alkyl moiety of the substituted alkyl group is preferably C1-C6 alkyl, more preferably C1-C4 alkyl, and even more preferably C2-C4 alkyl. The alkyl moiety is preferably a linear alkyl moiety. The alkyl moiety has at least one, preferably exactly one, substituent selected from -NH2 and -NH-C(=X)-NH2 groups. X is preferably NH, in which case the -NH-C(=X)-NH2 group is a guanidino group. Accordingly, R L2 Preferably, -(CH2) A -NH2 and -(CH2) A -NH-C(=NH)-NH2 is a group selected from NH-C(=NH)-NH2, where A is an integer from 1 to 6, preferably 1 to 4, more preferably 2 to 4. Most preferably, R L2 This is -(CH2)3-NH-C(=NH)-NH2.

[0038] Formula (I) and R of its preferred embodiment as defined herein L3 The group is -CH2-NH2 or -CH2-(1H-imidazole-4-yl), preferably -CH2-NH2. The -CH2-(1H-imidazole-4-yl) group is represented by the following formula:

[0039] [ka]

[0040] It can be expressed as follows, where the dashed line indicates a bond that connects this group to the remainder of the compound of formula (I). Formula (I) and R of its preferred embodiment as defined herein L4 It is -NH2.

[0041] The variable e in formula (I) and its preferred embodiment as defined herein is an integer between 1 and 4, preferably between 2 and 4, and more preferably 2. X in equation (I) 1 X is a coupling group. As experienced readers will understand, the coupling group X 1 R S or R A The remainder of the compound of formula (I) contains X 1 Base and R S Between the base, or X 1 Base and R A A functional group that enables coupling via a covalent bond formed between it and another group. The coupling group can consist of one or more atoms. Preferred coupling group X 1 The coupling group X is selected from -NH-, -C(O)-, -O-, and -S-. Typically, the coupling group X 1 R S or R A It is covalently bonded to further complementary coupling groups contained therein, and as a result the two coupling groups combine to form an amide bond -C(O)-NH-, an ester bond -C(O)-O-, or the following formula

[0042] [ka]

[0043] It forms a bonding unit such as a thiosuccinimidyl group, which can be represented as (in the formula, each dashed line indicates a bond that connects this group to an adjacent atom or group in the compound of formula (I)). For example, coupling group X 1 =-NH- can form an amide bond -NH-C(O)- with the complementary group -C(O)- contained in R S or R A . The coupling group X 1 =-C(O)- can form an amide bond -C(O)-NH- with the complementary group -NH- contained in R S or R A , or can form an ester bond -C(O)-O- with the complementary group -O- contained in R S or R A . The coupling group X 1 =-O- can form an ester bond -O-C(O)- with the complementary group -C(O)- contained in R S or R A , or can form an ether bond -O- with the carbon atom contained in R S or R A . The coupling group X 1 =-S- can form a thioester bond -S-C(O)- with the complementary group -C(O)- contained in R S or R A , or can form a thioether bond -S- with the carbon atom contained in R S or R A . According to a preferred embodiment, X 1 is a sulfur atom -S-, which forms a covalent bond with the complementary succinimidyl group contained in R S or R A . It will be understood that the latter combination can be conveniently realized by reacting a compound having a thiol group with a compound containing a maleimidyl group.

[0044] The variable c in formula (I) is either 0 or 1, whereby the substituent R contained within the brackets [... ] having the exponent c L3The group having c may or may not be present. Naturally, if it is not present, i.e., c is 0, the group is substituted for a direct bond. Preferably, c is 1. Similarly, the variable d in formula (I) is either 0 or 1, thereby the substituent R contained within the parentheses [···] with exponent d L4 The group having may or may not be present. Naturally, if it is not present, i.e., if d is 0, the group is directly substituted for the bond. However, in the compounds according to the present invention, at least one of c and d must be 1.

[0045] Therefore, compounds of formula (I) are (Ia), (Ib), or (Ic):

[0046] [ka]

[0047] It is preferable that the formula has the variable R CP a, R L1 , R L2 , R L3 , R S , b, R L4 , e, X 1 and R A This is defined for formula (I) and includes any preferred embodiments of these variables as further defined herein.

[0048] Among these preferred formulas of the compound of formula (I), formula (Ia) has further priority:

[0049] [ka]

[0050] It is given to. The compound of formula (I) is formula (Iaa):

[0051] [ka]

[0052] It is particularly preferable that the compound be of the form R, in the formula, CP a, R L1 , R L2 , R L3 , R S , b and R A This is defined for formula (I) and includes any preferred embodiments of these variables as further defined herein.

[0053] Formula (I) and R of its preferred embodiment as defined herein S is a divalent spacer group. The variable b is 0 or 1, thereby the spacer group R S is a base of arbitrary choice that may or may not exist. Naturally, if it does not exist, i.e., if e is 0, then R S The base is R in equation (I). S It is replaced by a direct bond between adjacent atoms. S The group preferably contains a linear chain of 3 to 10 carbon atoms, preferably 4 to 6 carbon atoms. This linear chain of carbon atoms consists of an NH group and an R group. A Between the base (when b is 1 and d is 0) or X 1 Base and R A It spans between the group (when b is 1 and d is 1). In addition to a linear chain of 3 to 10 carbon atoms, preferably 4 to 6 carbon atoms, R S The carbon atom chain consists of an NH group and R A Based on, or X 1 base and R A The base may include one or two, preferably two, coupling groups that enable each to be bonded. Typically, a spacer group R S It is an unbranched chain and does not contain charged groups.

[0054] Comfortable, R S The group includes a linear alkanediyl chain having 3 to 10 carbon atoms, preferably 4 to 6 carbon atoms. This alkanediyl chain has an NH group and R ABetween the base (when b is 1 and d is 0) or X 1 Base and R A It spans between the group (when b is 1 and d is 1). In addition to a linear alkanediyl chain having 3 to 10 carbon atoms, preferably 4 to 6 carbon atoms, R 6L The alkanediyl chain has an NH group and R A base, or X 1 base and R A The base may include one or two, preferably two, coupling groups that enable each to be bonded.

[0055] Therefore, R S The base is, formula -X 2 -(CH2) B -X 3 - [In the formula, X 2 If d is 0, the NH group of formula (I) is used, or if d is 1, the X group of formula (I) is used. 1 It is a coupling group that bonds to a base, B is an integer between 3 and 10, preferably between 4 and 6, and X 3 R A It is a coupling group that binds. It is preferable that it be the base of.

[0056] Coupling group X 2 and X 3 Regarding the above coupling group X 1 Similar considerations apply, as in the case of coupling group X. 2 R S However, X 2 The NH group is connected to the X group via a covalent bond formed between the group and the NH group. 2 Base and X 1 X through the covalent bond formed between the group and the group 1 A coupling group X is a functional group that allows bonding to either of the following: 2It can consist of one or more atoms. Preferred coupling groups are selected from -NH-, -C(O)-, -O-, and -S-. 2 When it binds to NH, it is preferably -C(O)-, and this results in X 2 and X 1 This forms an amide bond -C(O)-NH-. 2 is X 1 When bonding, coupling group X 2 and X 1 These typically combine to form an amide bond -C(O)-NH-, an ester bond -C(O)-O-, or a thiosuccinimidyl group.

[0057] [ka]

[0058] These are complementary coupling groups that form bonding units such as those mentioned above. Particularly preferred R S The base is the formula -C(O)-(CH2) B It is an -NH- group, B is as defined above, and the N-terminal bond is R A It is connected.

[0059] Accordingly, the preferred combination of variables for the compound in formula (I) is R CP R inside B2 is -CH2-CH2-CH2-, and R L1 is methyl, and R L2 is -(CH2)3-NH-C(=NH)-NH2, c is 1, and the remaining variables are defined for formulas (I) and (II), and will be understood to include any preferred embodiments of these variables as further defined herein. A more preferred combination of variables for the compound of formula (I) is R CP R inside B2 is -CH2-CH2-CH2-, and R L1 is methyl, and R L2is -(CH2)3-NH-C(=NH)-NH2, c is 1, d is 0, b is 0 or 1, R S If present, -C(O)-(CH2) B It is -NH- (in which case B is as described above, and the N-terminal bond is R A The remaining variables (combined with ) are defined for formulas (I) and (II) and include any preferred embodiments of these variables as further defined herein.

[0060] Therefore, a particularly preferred compound of formula (I) is the following formula (Iab) or the following formula (Iac)

[0061] [ka]

[0062] [In the formula, R B1 a, R S and R A [These are defined for formulas (I) and (II), respectively, and include any preferred embodiments of these variables as further defined herein.] It holds.

[0063] R AThe portion having diagnostic or therapeutic utility is a functional group, such as a labeling group. As will be understood by those skilled in the art, the portion having diagnostic utility is a group or a precursor of such a group that facilitates the detection of the ligand compound according to the present invention after administration to a patient or after the ligand compound according to the present invention has been brought into contact with a physiological sample in vitro or ex vivo. Examples of such precursors are groups that may have detectable radioactive elements but do not yet contain such elements, such as a SiFA moiety or a chelated moiety. Preferably, the portion having diagnostic utility is a group or a precursor of such a group that enables the compound according to the present invention to be detected and localized in the patient's body after administration to the patient. Compounds of the present invention containing a portion having diagnostic utility can function in particular as tracers of CXCR4 due to their affinity for CXCR4. The portion having therapeutic utility is a group or a precursor of such a group that enables the compound according to the present invention to treat or prevent a disease or disorder, in particular a disease or disorder that can be treated or prevented by blocking the CXCR4 receptor or a disease or disorder associated with increased or abnormal expression of CXCR4, after administration to a patient. Examples of such precursors are groups that may contain radioactive elements with therapeutic effects but do not yet contain such elements, such as chelated moieties.

[0064] R A The base includes a portion having diagnostic or therapeutic utility. Preferably, it includes one or two such portions. A combination of two portions may be useful, for example, to provide the compound of the present invention having both diagnostic and therapeutic utility.

[0065] Preferably, the compound of formula (I) and R of its preferred embodiment as defined herein. A The following parts have diagnostic or therapeutic utility: (i) Chelate portion, (ii) Chelated radioactive or non-radioactive cations or anions, preferably chelated radioactive or non-radioactive cations, together with the chelated moiety (i), (iii) A silicon fluoride acceptor (SiFA) moiety containing a silicon atom and a fluorine atom, wherein the fluorine atom is directly bonded to the silicon atom via a covalent bond, and the SiFA moiety 19 F 18 Isotope exchange by F 18 It is possible to label it with F, or 18 The silicon fluoride acceptor (SiFA) moiety labeled with F, (iv) Cytotoxic portion, and (v) Fluorescent part It includes at least one of the above, or consists of them.

[0066] Comfortable, R A This includes or consists of one of parts (i) to (v), or a combination of one part selected from the chelated part (i) and chelate (ii) and one SiFA part (iii).

[0067] The chelates of (i) and (ii) are suitable for forming chelates with radioactive or non-radioactive cations or anions, preferably radioactive cations. Suitable chelating agents that provide chelates for various cations and anions are well known in the art and can be used in connection with the present invention. Suitable metal chelating agents or cation chelating agents, such as macrocyclic or acyclic compounds, that provide chelates are available from several manufacturers. It will be understood that many chelating agents can be used commercially with little difficulty by those skilled in the art. It will also be understood that the suitability of a chelate to form a chelate with a given anion or cation requires that the chelate is capable of providing a chelate ligand in a chelate complex containing the anion or cation under consideration, but it is not necessary that the chelate is solely responsible for forming a ligand for the anion or cation in the chelate complex. Therefore, a chelate according to option (ii) above may include a chelated cation or anion, a chelated moiety (i) as a chelating ligand, and additional ligands that coordinate with the chelated cation or anion.

[0068] For example, the chelated portion is A macrocyclic ring structure having 8 to 20 ring atoms (of which 2 or more, preferably 3 or more, are selected from oxygen atoms, sulfur atoms, and nitrogen atoms), and Acyclic open-chain chelate structure having 8 to 20 main chain atoms (of which 2 or more, preferably 3 or more, are heteroatoms selected from oxygen, sulfur, and nitrogen atoms) It can include at least one of the following.

[0069] Preferably, the chelated portion described in (i) or (ii) above is 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co, 52Faith、 56 It、 57 It、 nat Cu、 62 Cu、 64 Cu、 67 Cu、 66 Ga、 nat Ga、 68 Ga、 67 Ga、 89 Zr、 90 Y、 86 Y、 94m Tc、 99m Tc、 97 Ru、 105 Rh、 109 Pd、 111 Ag、 110m In、 111 In、 113m In、 114m In、 117m Sn、 121 Sn、 127 For, 142 Pr、 143 Pr、 147 Nd、 149 Gd、 149 Pm、 151 Pm、 149 Tb、 152 Tb、 155 Tb、 153 Sm、 156 Eu、 157 Gd、 161 Tb、 164 Tb、 161 Ho、 166 Ho、 157 Dy、 165 Dy、 166 Dy、 160 Er、 165 Er、 169 Er、 171 Er、 166 Yb、 169 Yb、 175 Yb、 167 Tm、 172 Tm、 nat Lu、 177 Lu、 186 Re, 188 Re, 188 W、 191 Pt、 195m Pt、 194 Is、197 Hg, 198 Au, 199 Au, nat Pb, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 and 227 From the Th cation, and 18 F-[AlF] 2+ etc. 18 This chelate portion is suitable as a chelate ligand for a cation selected from cationic molecules containing F.

[0070] Therefore, preferred chelating agents that can be used to provide the chelated moiety of (i) or (ii) above are bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradeca-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-amine] Nopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutanediamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabiscle[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanediic acid (DOTAGA or DOTA-G) A) N,N'-Dipyridoxylethylenediamine-N,N'-Diacetate-5,5'-Bis(phosphat) (DPDP), Diethylenetriaminepentaacetic acid (DTPA), Ethylenediamine-N,N'-Tetraacetic acid (EDTA), Ethylene glycol-O,O-Bis(2-aminoethyl)-N,N,N',N'-Tetraacetic acid (EGTA), N,N-Bis(hydroxybenzyl)-Ethylenediamine-N,N'-Diacetic acid (HBED), Hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-Nito Robenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 1,4,7-triazacyclononane-1-succinate-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononane triacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] Hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), terpyridine-bis(methyleneamine)tetraacetic acid (TMT), 1,4,7,10-tetraazacyclotridecane-N,N',N'',N'''-tetraacetic acid (TRITA), and triethylenetetraaminehexaacetic acid (TTHA), N,N'-bis[(6-carboxy-2-pyridyl)meth [H2 macropa (H2macropa)), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-ylmethyl)-carbamoyl]-ethyl}heptanedioate bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-ylmethyl)amide](THP), 1,4, 7-Triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid (TRAP), 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (DO3AM), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene(2-carboxyethylphosphinic acid)] The following are selected from (DOTPI), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid, mercaptoacetyl-tricerine (mas3), hydrazinonicotinic acid (HYNIC), and 3-(2-aminoethylamino)-2-[(2-aminoethylamino)methyl]propanoic acid (N4 chelator, 6-carboxy-1,4,8,11-tetraazaundecane). A more preferred example is a modified mercaptoacetylserine chelating agent (modified mas3) in which one or more serine residues are substituted with another amino acid containing a hydrophilic side chain.

[0071] Among these preferred chelating agents that can be used to provide the chelated portion of (i) or (ii) above, particularly preferred are chelating agents selected from mas3, modified mas3, HYNIC, N4 chelator, DOTA, and DOTAGA.

[0072] As will be understood by experienced readers, the preferred and particularly preferred chelating agents listed above can easily provide a chelated moiety in the compound according to the present invention by providing a bonding unit that uses a functional group contained in the chelating agent to bond the chelated moiety to the remainder of the compound. Examples of such bonding units include, for example, amide bonds (-C(O)-NH-) or ester bonds (-C(O)-O-) that can be provided using a carboxyl group or an amino group that can be contained as a functional group in the chelating agent.

[0073] Accordingly, the chelated cations mentioned in (ii) above are preferably, 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co, 52 Fe, 56 Ni, 57 Ni, nat Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, nat Ga, 68 Ga, 67 Ga, 89 Zr, 90 Y, 86 Y, 94m Tc, 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 147 Nd, 149 Gd, 149 PM, 151 PM, 149 Tb,152 Tb, 155 Tb, 153 Sm, 156 EU, 157 Gd, 161 Tb, 164 Tb, 161 Ho, 166 Ho, 157 Dy, 165 Dy, 166 Dy, 160 Er, 165 Er, 169 Er, 171 Er, 166 Yb, 169 Yb, 175 Yb, 167 Tm, 172 Tm, nat Lu, 177 Lu, 186 Re, 188 Re, 188 W, 191 Pt, 195m Pt, 194 Ir, 197 Hg, 198 Au, 199 Au, nat Pb, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 and 227 From the Th cation, and 18 F-[AlF] 2+ etc. 18 A cationic molecule containing F is selected. The chelate formed with such a cation is, in addition to the chelated moiety provided by the compound of formula (I) or its salt, one or more additional ligands that coordinate with the chelated cation and are not part of the compound of formula (I) or its salt, e.g., 99m The chelate containing Tc(V)-oxocore may also contain oxo-ligands, etc.

[0074] R A A particularly preferred base is, 99mTc, 177 Lu, 67 Ga and 68 It is a group containing a chelate that can form a chelate with a cation selected from Ga cations. Similarly, R A The preferred base is, 99m Tc, 177 Lu, 67 Ga and 68 This group contains a chelate with a chelated cation selected from Ga cations.

[0075] As described above, the structure of the linker that forms part of the compound of the present invention retains the high affinity of CXCR4 while incorporating a wide variety of functional groups R A This makes it possible to use the functional group R containing the chelates mentioned in options (i) and (ii) above. A In this case, the favorable effect of the linker is R A However, groups other than those consisting of DOTA or DOTAGA residues, or generally M 3+ This is particularly evident in the case of compounds of formula (I) and their salts, which are groups other than those consisting of chelator residues of (i.e., trivalent metal cations).

[0076] The silicon fluoride acceptor (SiFA) portion corresponding to option (iii) above is preferably the base of formula (S-1):

[0077] [ka]

[0078] [In the formula, R S1 and R S2 These are independently linear or branched C3-C10 alkyl groups, preferably R S1 and R S2 is independently selected from isopropyl and tert-butyl, more preferably R S1 and R S2Both are tert-butyl compounds, and the dotted bond indicates the bonding of the group of formula (S-1) to the remainder of the compound of formula (I). Preferably, the group of formula (S-1) is bonded to the phenyl ring as a substituent.

[0079] More preferably, the SiFA portion comprises a base of formula (S-2) and a base of formula (S-3).

[0080] [ka]

[0081] [In the formula, n is 1, 2, or 3, preferably 1, R S1 and R S2 These are independently linear or branched C3-C10 alkyl groups, preferably R S1 and R S2 is independently selected from isopropyl and tert-butyl, more preferably R S1 and R S2 Both are tert-butyl, and the bond indicated by the dashed line is a group selected from [bonding the group of formula (S-2) or the group of formula (S-3) to the remainder of the compound of formula (I)]. A suitable counterion for the positively charged quaternary nitrogen atom shown in formula (S-3), having two methyl substituents, includes an anion (since the anion is described herein with respect to the salt formed of the compound of formula (I)), and may include, for example, a trifluoroacetate anion or an acetate anion.

[0082] As will be understood by experienced readers, the bonds at the carbonyl groups indicated by dashed lines in formulas (S-2) and (S-3) represent bonds that do not have a methyl group at the opposite end of the carbonyl group and bond the SiFA portion to the remainder of the compound of formula (I). In other words, the bonds indicated by dashed lines represent covalent bonds that exist between the carbon atoms of the carbonyl groups shown in formulas (S-2) and (S-3) and the atoms or groups adjacent to the (S-2) or (S-3) group in the compounds according to the present invention. For example, amide bonds (-C(O)-NH-) or ester bonds (-C(O)-O-), preferably amide bonds, are provided using the carbonyl groups shown in formulas (S-2) and (S-3) and the -NH- or -O- groups adjacent to these groups. For example, such -NH- or -O- groups are R A It may be included in the linker-forming portion.

[0083] As will become clearer, the fluorine atoms contained in equations (S-1) to (S-3) are 18 F atom, or 19 F 18 Isotope exchange by F 18 It can be replaced in order to provide F 19 It could even be an F atom.

[0084] The cytotoxic moiety of option (iv) above may be provided by a cytotoxic compound, such as an auristatin analog, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or a residue such as PF-06380101. The residue may be provided using a functional group contained in the cytotoxic compound to form a binding unit that attaches the cytotoxic moiety to the remainder of the compound according to the present invention. Optionally, a metabolically cleavable linker may be further provided as a linker that attaches the cytotoxic moiety to the remainder of the compound according to the present invention.

[0085] The fluorescent portion of option (v) above may be provided, for example, by a residue of a fluorescent dye. Such fluorescent dyes are known in the art and include, for example, Cy5- and Cy7-type cyanine dyes. The residue may be provided using a functional group contained in the fluorescent dye to provide a binding unit for attaching the cytotoxic portion to the remainder of the compound according to the present invention.

[0086] In addition to the parts that have the diagnostic or therapeutic utility described above, R A It may include one or more further parts that serve different purposes. For example, R A It may include a divalent or higher valence linker that allows one or more portions having diagnostic or therapeutic utility to be bonded to the remainder of the compound. As another example, R to adjust the hydrophilic / hydrophobic properties of the compound according to the present invention A Examples of parts included in the structure include parts having one or more polar groups.

[0087] Those skilled in the art will understand, R A R A However, the -NH- group (in the compound of formula (I), when d is 0 and b is 0), -X 1 -Based on (in the compound of formula (I), d is 1 and b is 0), or -R S The compound contains a coupling group that allows for covalent bonding at its terminology (when b is 1 in the compound of formula (I)). Suitable coupling groups can be selected and provided based on established principles of synthetic chemistry. For example, the coupling group is R A It may be included in the linker by optional selection, or R A It can be a part of the component that has diagnostic or therapeutic utility. For example, in the compound of formula (I), if d is 0 and b is 0, R AIt is preferable that the compound contains a coupling group -C(O)- to bond to -NH- in order to provide an amide bond. Such a coupling group can be easily derived, for example, from a carboxyl group. Similarly, in the compound of formula (I), b is 1 and R S The formula is -C(O)-(CH2) B If it is an -NH- group, R A It is preferable that the compound contains a coupling group -C(O)- to bond to -NH- in order to provide an amide bond. As another example, in the compound of formula (I), d is 1, b is 0, and X 1 If R is -S-, A The compound may include a succinimidyl group as a coupling group to bond to the -S- to obtain a thiosuccinimidyl group. Such a coupling group can be easily derived, for example, from a maleimidyl group.

[0088] Accordingly, R A Particularly preferred embodiments can be exemplified below. - R A This is a chelating moiety provided by a mercaptoacetyltricerine (mas3) chelating agent, which is bonded to the remainder of the compound of formula (I) via an amide bond. - R A This comprises a chelated moiety provided by a mercaptoacetyltricerine (mas3) chelating agent, which is bonded to the remainder of the compound of formula (I) via an amide bond, 99m It is a chelate containing chelated cations such as Tc cations. - R A This is a chelated moiety provided by a modified mercaptoacetyltricerine (mas3) chelating agent bonded to the remainder of the compound of formula (I) via an amide bond, in which case one or more serine residues are substituted by another amino acid residue having a hydrophilic side chain, such as citrulline, or by an amino acid residue having a glycosylated side chain. - R AThis is a chelating moiety provided by a modified mercaptoacetyltricerine (mas3) chelating agent bonded to the remainder of the compound of formula (I) via an amide bond (in this case, one or more serine residues are substituted by another amino acid residue having a hydrophilic side chain, such as citrulline, or by an amino acid residue having a glycosylated side chain), 99m It is a chelate containing chelated cations such as Tc cations. - R A This is a chelating moiety provided by a hydrazinonicotinic acid (HYNIC) chelating agent, which is bonded to the remainder of the compound of formula (I) via an amide bond. - R A This comprises a chelated moiety provided by a hydrazinonicotinic acid (HYNIC) chelating agent, which is bonded to the remainder of the compound of formula (I) via an amide bond, 99m It is a chelate containing chelated cations such as Tc cations. - R A is a chelating moiety provided by the 3-(2-aminoethylamino)-2-[(2-aminoethylamino)methyl]propanoic acid (N4) chelating agent, which is bonded to the remainder of the compound of formula (I) via an amide bond, or R A This includes a chelated moiety provided by an N4 chelating agent bonded to the remainder of the compound of formula (I) via an amide bond, - R A This comprises a chelated moiety provided by a 3-(2-aminoethylamino)-2-[(2-aminoethylamino)methyl]propanoic acid (N4) chelating agent bonded to the remainder of the compound of formula (I) via an amide bond, 99m It is a chelate containing chelated cations such as Tc cations, or R A This comprises a chelated moiety provided by an N4 chelating agent bonded to the remainder of the compound of formula (I) via an amide bond, 99m A chelate containing chelated cations such as Tc cations, - R AThis includes a SiFA moiety and a chelated moiety, for example, a chelated moiety provided by a DOTA or DOTAGA chelating agent bonded to the remainder of the compound of formula (I) via an amide bond. - R A This comprises a SiFA moiety and a chelated moiety, for example, a chelated moiety provided by a DOTA or DOTAGA chelating agent, and a chelated cation, for example, 177 Lu cation, 68 Ga cation or 69 A chelate containing a Ga cation is included. - R A This includes a cytotoxic portion provided by MMAE, or - R A It includes a fluorescent moiety provided by the fluorescent dye Cy5.5.

[0089] R S The existence of (i.e., the choice of b in equation (I) is 1) is particularly important for R A If the group has a high molecular weight, R A If the group contains a large group near the bonding site to the remainder of the compound of formula (I), or R A If the group contains a negatively charged functional group (not considering functional groups whose negative charge is neutralized by the formation of a chelate complex), additional benefits can be obtained in light of the high affinity of CXCR4 in the compounds of the present invention. Therefore, as a direction, an optional spacer group R S The existence of R satisfies two of the following three requirements. A It is expected that this will further improve the affinity for compounds containing the group. i)R A Its molecular weight exceeds 300 g / mol. ii) If the compound of formula (I) is maintained in solution at a neutral pH (without considering the charged groups in the chelating agent that are neutralized by the formation of a chelating complex), R A It has a charge of <-1, iii) R A R A Near the bonding site to the remainder of the compound in formula (I) (e.g., R AThe atom to which it is bonded contains a phenyl ring or a larger aromatic group (less than 7 covalent bonds (CC, CO-, or CN bonds) away from the atom to which it is bonded).

[0090] As described above, the ligand compounds according to the present invention include the compound of formula (I) and its salts, preferably pharmaceutically acceptable salts. Such salts may be formed, for example, by protonation of an atom having a lone pair of electrons that is easily protonated, such as a nitrogen atom, with an inorganic or organic acid, or by separating a proton from an acidic group such as a carboxylic acid group, for example, by neutralization with a base. Other charged groups that may be present in the compounds according to the present invention include persistently charged groups, such as quaternary ammonium cations or charged chelate complexes substituted with four organyl groups.

[0091] Examples of anions that can exist in salt form of the compounds of the present invention include, if the salt form includes the positively charged form of the compound of formula (I), for example, chloride ions, bromide ions, iodide ions, sulfate ions, nitrate ions, phosphate ions (e.g., phosphates, hydrogen phosphates, or dihydrogen phosphates), carbonate ions, bicarbonate ions or perchlorate ions, acetate ions, trifluoroacetate ions, propionate ions, butyrate ions, pentanoate ions, hexanoate ions, heptanoate ions, octanoate ions, cyclopentanepropionate ions, and undecanoate ions. Examples of anions include lactate ions, maleate ions, oxalate ions, fumarate ions, tartrate ions, malate ions, citrate ions, nicotinate ions, benzoate ions, salicylate ions or ascorbic acid ions, sulfonate ions, such as methanesulfonate ions, ethanesulfonate ions, 2-hydroxyethanesulfonate ions, benzenesulfonate ions, p-toluenesulfonate ions (tosylate ions), 2-naphthalenesulfonate ions, 3-phenylsulfonate ions, or camphorsulfonate ions. Since trifluoroacetic acid is frequently used in peptide synthesis, trifluoroacetate salts are typical salts provided when compounds containing peptide structures are formed. Such trifluoroacetate salts may be converted to acetate salts during their workup. Examples of cations that can exist in salt form of the compound of the present invention include, when the salt form includes a negatively charged form of the compound of formula (I), cations selected from, for example, alkali metal cations such as lithium ions, sodium ions or potassium ions, alkaline earth metal cations such as calcium ions or magnesium ions, and ammonium ions (including ammonium ions substituted with organic groups).

[0092] The ligand compound according to the present invention preferably has an IC of 100 nM or less, more preferably 10 nM or less, and even more preferably 5 nM or less. 50 The affinity, which is reflected in the value, indicates that it can bind to human CXCR4.

[0093] Examples of compounds according to the present invention include the following: The following formula:

[0094] [ka]

[0095] Compounds in which the mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0096] [ka]

[0097] Compounds in which the mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0098] [ka]

[0099] Compounds in which the mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0100] [ka]

[0101] Compounds in which the mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0102] [ka]

[0103] Compounds in which the modified mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0104] [ka]

[0105] Compounds in which the modified mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0106] [ka]

[0107] Compounds in which the modified mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0108] [ka]

[0109] Compounds in which the modified mas3 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0110] [ka]

[0111] Compounds in which the HYNIC chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0112] [ka]

[0113] Compounds in which the N4 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0114] [ka]

[0115] Compounds in which the N4 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0116] [ka]

[0117] Compounds in which the N4 chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0118] [ka]

[0119] Compounds in which the DOTA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0120] [ka]

[0121] Compounds in which the DOTA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0122] [ka]

[0123] Compounds in which the DOTA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0124] [ka]

[0125] Compounds in which the DOTA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0126] [ka]

[0127] Compounds in which the DOTA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0128] [ka]

[0129] Compounds in which the DOTAGA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0130] [ka]

[0131] Compounds in which any of the DOTAGA chelate moieties shown in the formula form a chelate with a chelated radioactive or non-radioactive cation, or salts of any of these, The following formula:

[0132] [ka]

[0133] Compounds in which the DOTAGA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0134] [ka]

[0135] Compounds in which the DOTAGA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0136] [ka]

[0137] Compounds in which the DOTAGA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0138] [ka]

[0139] Compounds in which the DOTA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0140] [ka]

[0141] Compounds or salts thereof The following formula:

[0142] [ka]

[0143] Compounds or salts thereof The following formula:

[0144] [ka]

[0145] Compounds or salts thereof The following formula:

[0146] [ka]

[0147] Compounds in which the DOTAGA chelate moiety shown in the formula forms a chelate with chelated radioactive or non-radioactive cations, or salts of any of these, The following formula:

[0148] [ka]

[0149] Compounds or salts thereof The following formula:

[0150] [ka]

[0151] Compounds or salts thereof, and The following formula:

[0152] [ka]

[0153] A compound in which the DOTA chelate moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these.

[0154] In further embodiments, the present invention provides pharmaceutical compositions (also called therapeutic compositions) comprising or comprising one or more types, preferably one type, of ligand compounds according to the present invention, i.e., compounds of formula (I) (including any preferred embodiments thereof as described herein), or salts thereof. In related embodiments, ligand compounds according to the present invention are provided for use in therapy or as pharmaceuticals. Accordingly, ligand compounds of the present invention can be used in therapeutic methods, the methods may include a step of administering the ligand compound to a subject. The subject may be human or animal, preferably human. According to these medical embodiments of the present invention, the functional group R A It should be understood that, in general, it includes a portion that has therapeutic utility, such as a group having a radioactive element with therapeutic effects, or a cytotoxic portion.

[0155] The above-described treatments or therapeutic methods generally aim to treat or prevent diseases or disorders of the human or animal body that can be treated or prevented by blocking the CXCR4 receptor, or diseases or disorders associated with increased or abnormal expression of the CXCR4 receptor, such as cancer, cardiovascular disorders, or inflammatory disorders. Therefore, from the viewpoint of therapeutic application, the compounds of the present invention are preferably provided for use in the treatment or prevention of cancer, inflammatory disorders, or cardiovascular disorders, such as atherosclerosis, myocardial infarction, or stroke.

[0156] R in equation (I) ADue to the versatility provided by the present invention in terms of the selection of bases, the compounds of the present invention can be conveniently adapted to various therapeutic methods. For example, compounds according to the present invention that include a chelateable moiety capable of forming a chelate with a radioactive component, such as a radioactive metal cation, or compounds containing such a chelate, can be used for radiotherapy, particularly targeted radioligand therapy (RLT). In the case of radiotherapy, the chelated radioactive cation is preferably a gamma or beta emitter, because they can emit a dose of radiation that weakens or destroys specific target cells at the target site. Examples of gamma or beta emitters are: 177 Lu, 89 Zr and 186 Re. As another example, compounds according to the present invention, which include a cytotoxic moiety, may be provided for use in therapies involving the chemotherapeutic destruction of cells, such as cancer cells.

[0157] In another embodiment, the present invention provides diagnostic compositions comprising or comprising one or more types, preferably one type, of ligand compounds according to the present invention, namely, compounds of formula (I) (including any preferred embodiments thereof as described herein), or salts thereof. In a related embodiment, ligand compounds according to the present invention are provided for use in a method of in vivo diagnosis of a disease or disorder. Accordingly, ligand compounds according to the present invention can be used in a diagnostic method, which may include the steps of administering the ligand compound to a subject and detecting the ligand compound in the subject, or monitoring the distribution of the ligand compound in the subject to detect or monitor a disease to be diagnosed. The subject may be human or animal, preferably human. Alternatively, the diagnostic method may also include the step of adding the ligand compound in vitro or ex vivo to a sample, e.g., a physiological sample obtained from a subject, and detecting the ligand compound in the sample. According to these diagnostic embodiments of the present invention, the functional group R AIt should be understood that, in general, these include parts that have diagnostic utility, such as a group containing a detectable radioactive element, or a fluorescent part.

[0158] The diagnostic methods described above generally aim to identify diseases or disorders in humans or animals that can be treated or prevented by blocking the CXCR4 receptor, or diseases or disorders associated with increased expression of the CXCR4 receptor, such as cancer, cardiovascular disorders, or inflammatory disorders. Therefore, from the viewpoint of diagnostic application, the compounds of the present invention are preferably provided for use in methods of in vivo diagnosis of disorders such as cancer, cardiovascular disorders, or inflammatory disorders, such as atherosclerosis, myocardial infarction, or stroke.

[0159] R in equation (I) A Due to the versatility provided by the present invention in terms of group selection, the compounds of the present invention can be conveniently adapted to various diagnostic methods. For example, compounds according to the present invention that include a chelateable moiety capable of forming a chelate with a radioactive component, such as a radioactive metal cation, compounds containing such a chelate, or 18 Compounds containing fluorine atoms can be used for nuclear imaging. For example, the compounds according to the present invention can be used as positron emitters, e.g., chelated compounds. 64 Cu cation, chelated 68 Ga cation, or bonded in the SiFA moiety 18 If the compound contains an F atom, it can be used for diagnostic purposes by positron emission tomography (PET) imaging. Other compounds according to the present invention, for example, chelated compounds 99m Compounds containing a Tc cation can be used for diagnosis by single-photon emission computed tomography (SPECT) imaging. As a further example, the compound according to the present invention containing a SiFA moiety is 19The F compound can be used for diagnosis by MRI, or the compound according to the present invention, which includes a fluorescent moiety, can be used in diagnostic methods involving optical imaging.

[0160] It will be understood that the suitability for therapeutic and diagnostic applications is not mutually exclusive. That is, the compounds according to the present invention may be suitable for both applications, and therefore, the functional group R A The compound may include a portion having both diagnostic and therapeutic utility, or a portion having diagnostic utility and a portion having therapeutic utility as separate portions. For example, the compound according to the present invention may include radioactive species active in radiotherapy and diagnostic imaging. Furthermore, the compound according to the present invention includes radioactive hybrid compounds that include both a SiFA moiety and a chelate moiety suitable for forming chelates with therapeutically active radioactive cations. Such a radioactive hybrid compound may include a SiFA moiety 18 If it has an F atom and the chelate portion forms a chelate with a non-radioactive (cold) cation, it may be used for diagnostic purposes, and the SiFA portion 19 If a molecule has an F atom and the chelate is formed with the corresponding radioactive (hot) cation, it may be used for therapeutic purposes. Advantageously, if the radioactive and non-radioactive cations are different isotopes of the same chemical species, the pharmacokinetic properties of the diagnostic and therapeutic variations of the radiohybrid compound remain the same. Exemplary radiohybrid ligand compounds for use in diagnosis and therapy are: 18 F atom / nat Ga cation combination compounds and 19 F atom / 68 Ga cation combination compound, 18 F atom / nat Y cation combination compound and 19 F atom / 90 Y cation combination compound, or 18 F atom / nat Compounds that combine Lu cations and 19 F atom / 177 This is a compound that combines Lu cations.

[0161] A pharmaceutical or diagnostic composition may further comprise one or more pharmaceutically acceptable carriers, excipients, and / or diluents. Examples of suitable pharmaceutical carriers, excipients, and / or diluents are well known in the art and include phosphate-buffered saline, water, emulsions, e.g., oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These compositions can be administered to a subject in an appropriate dose. Administration of a suitable composition can be achieved in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal, or intrabronchial administration. Such administration is particularly preferred by injection and / or delivery. The composition may also be administered directly to the target site. The administration plan will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, overall health, and other drugs administered concomitantly. Pharmacologically active substances may be present in amounts between 0.1 ng and 10 mg / kg body weight per dose, for example, but doses below or above this exemplary range are conceivable, especially considering the factors mentioned above.

[0162] The following sections summarize aspects of the present invention. It will be understood that these sections are closely related to the descriptive sections above, and that the information provided in these sections can supplement the descriptive sections above, and vice versa.

[0163] 1. Equation (I):

[0164] [ka]

[0165] [In the formula, a is 0 or 1, preferably 0. b is either 0 or 1, c is 0 or 1, and d is 0 or 1, provided that at least one of c and d is 1. e is an integer between 1 and 4, preferably between 2 and 4. R CP Equation (II):

[0166] [ka]

[0167] (Here, in equation (II), R B1 is H or I, preferably H. R B2 It is an alkanediyl chain, The dashed line is R CP (This shows a bond that attaches the group to the remainder of the compound in formula (I).) It is a cyclopeptide group, R L1 is H or alkyl, R L2 is a substituted alkyl group that is substituted with at least one group selected from -NH2 and -NH-C(=X)-NH2, where X is selected from NH and O. R L3 It is -CH2-NH2 or -CH2-(1H-imidazole-4-yl), R L4 It is -NH2, X 1 is a coupling group, R S is a divalent spacer group, and R A This is a functional group that includes a portion that has diagnostic or therapeutic utility. Compounds or salts thereof.

[0168] 2. R in equation (I) CP Equation (IIa):

[0169] [ka]

[0170] [In the formula, R B1 This is defined in item 1, and the dashed line is R CP [This shows a bond that attaches the group to the remainder of the compound of formula (I)] The compound or salt listed in item 1, which is the base of.

[0171] 3. R ​​in equation (I) L1 The compound or salt described in item 1 or 2, wherein the group is H or C1-C6 alkyl, more preferably H or C1-C3 alkyl. 4. R in equation (I) L1 The compound or salt listed in item 3, whose group is methyl.

[0172] 5. R in equation (I) L2 The compound or salt described in any of items 1 to 4 is a C1-C6 alkyl, more preferably a C1-C4 alkyl, even more preferably a C2-C4 alkyl, having one substituent selected from -NH2 and -NH-C(=X)-NH2 groups, where X is NH or O.

[0173] 6. R in equation (I) L2 is, -(CH2) A -NH2 and -(CH2) A A compound or salt according to item 5, wherein A is an integer selected from -NH-C(=NH)-NH2, and A is an integer from 1 to 6, preferably 1 to 4, more preferably 2 to 4.

[0174] 7. R in equation (I) L2 The compound or salt described in item 6 is -(CH2)3-NH-C(=NH)-NH2. 8. R in equation (I) L3 is a compound or salt listed in any of items 1-7, which is -CH2-NH2.

[0175] 9. A compound or salt listed in any of items 1-8, where e in formula (I) is 2. 10. X in equation (I) 1 A compound or salt listed in any of items 1-9, which is -S-.

[0176] 11. A compound or salt described in any of items 1 to 10, wherein c in formula (I) is 1 and d in formula (I) is 0, or c in formula (I) is 0 and d in formula (I) is 1.

[0177] 12. A compound or salt listed in any of items 1-10, wherein c in formula (I) is 1 and d in formula (I) is 0. 13. A compound or salt listed in any of items 1-12, where b in formula (I) is 0.

[0178] 14. R in equation (I) S is -C(O)-(CH2) B The bond is -NH-, where B is an integer from 3 to 10, preferably from 4 to 6, and the N-terminal bond is R A A compound or salt listed in any of items 1 to 12 that is bonded to it.

[0179] 15. R in equation (I) A This refers to a compound or salt described in any of items 1 to 14, comprising one or two parts that have diagnostic or therapeutic utility. 16. R in equation (I) A The portion included in which there is diagnostic or therapeutic utility is (i) Chelate portion, (ii) Chelated radioactive or non-radioactive cations or anions, preferably chelated radioactive or non-radioactive cations, together with the chelated moiety (i), (iii) A silicon fluoride acceptor (SiFA) moiety containing a silicon atom and a fluorine atom, wherein the fluorine atom is directly bonded to the silicon atom via a covalent bond, and the SiFA moiety 19 F 18 Isotope exchange by F 18It is possible to label it with F, or 18 The silicon fluoride acceptor (SiFA) moiety labeled with F, (iv) Cytotoxic portion, and (v) Fluorescent part A compound or salt listed in any of items 1-15, selected from the following.

[0180] 17. R in equation (I) A The compound or salt described in item 16, comprising one of parts (i) to (v), or a combination of one part selected from chelate part (i) and chelate (ii) and one SiFA part (iii).

[0181] The chelated portions described in 18.(i) and (ii) are 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co, 52 Fe, 56 Ni, 57 Ni, nat Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, nat Ga, 68 Ga, 67 Ga, 89 Zr, 90 Y, 86 Y, 94m Tc, 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 147 Nd, 149 Gd, 149 PM, 151PM, 149 Tb, 152 Tb, 155 Tb, 153 Sm, 156 EU, 157 Gd, 161 Tb, 164 Tb, 161 Ho, 166 Ho, 157 Dy, 165 Dy, 166 Dy, 160 Er, 165 Er, 169 Er, 171 Er, 166 Yb, 169 Yb, 175 Yb, 167 Tm, 172 Tm, nat Lu, 177 Lu, 186 Re, 188 Re, 188 W, 191 Pt, 195m Pt, 194 Ir, 197 Hg, 198 Au, 199 Au, nat Pb, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 and 227 From the Th cation, and 18 F-[AlF] 2+ etc. 18 A compound or salt described in item 16 or 17, which is a chelate moiety suitable as a chelate ligand for a cation selected from cationic molecules containing F.

[0182] 19. The chelates described in (i) and (ii) are bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradeca-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4- Oxobutanoyl]amino]pentyl]-N-hydroxybutanediamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabiscle[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 2-[1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA or DOTA-GA), N,N'-dipyridoxine Silethylenediamine-N,N'-diacetate-5,5'-bis(phosphat) (DPDP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl) -1,4,7,10-Tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 1,4,7-Triazacyclononane-1-succinate-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononane triacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] Hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), terpyridine-bis(methyleneamine)tetraacetic acid (TMT), 1,4,7,10-tetraazacyclotridecane-N,N',N'',N'''-tetraacetic acid (TRITA), and triethylenetetraaminehexaacetic acid (TTHA), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6(H2 Macropa), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-ylmethyl)-carbamoyl]-ethyl}heptanedioate bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridine-2-ylmethyl)amide](THP), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinate (TRAP), 2-(4,7, 10-Tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid (DO3AM), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis[methylene(2-carboxyethylphosphinic acid)](DOTPI), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid, mercaptoacetyl-tricerine (mas3), hydrazinonicoty Compounds or salts described in any of items 16-18, provided by a chelating agent selected from HYNIC acid and 3-(2-aminoethylamino)-2-[(2-aminoethylamino)methyl]propanoic acid (N4 chelator, 6-carboxy-1,4,8,11-tetraazaundecane), or by a modified mercaptoacetylserine chelating agent in which one or more serine residues are substituted with another amino acid containing a hydrophilic side chain.

[0183] 20.R A teeth, 99m Tc, 177 Lu and 68 It contains a chelate moiety that can form a chelate with a cation selected from Ga cations, or R A teeth,99m Tc, 177 Lu and 68 A compound or salt described in any of items 16-19, comprising a chelate having a chelated cation selected from Ga cations.

[0184] 21. Part (iii) of SiFA is the basis of equation (S-1):

[0185] [ka]

[0186] [In the formula, R S1 and R S2 These are independently linear or branched C3-C10 alkyl groups, preferably R S1 and R S2 is independently selected from isopropyl and tert-butyl, more preferably R S1 and R S2 Both are tert-butyl compounds, and the dashed line indicates that the group of formula (S-1) is bonded to the remainder of the compound of formula (I). A compound or salt listed in any of items 16-20, including those listed in item 16-20.

[0187] 22. The SiFA part (iii) is the base of formula (S-2) and the base of formula (S-3)

[0188] [ka]

[0189] [In the formula, n is 1, 2, or 3, preferably 1, R S1 and R S2 These are independently linear or branched C3-C10 alkyl groups, preferably R S1 and R S2 is independently selected from isopropyl and tert-butyl, more preferably R S1 and R S2Both are tert-butyl compounds, and the dotted bond indicates that the group of formula (S-2) or the group of formula (S-3) is attached to the remainder of the compound of formula (I). A compound or salt listed in item 21, selected from the following.

[0190] 23. The cytotoxic moiety (iv) is provided by residues of an auristatin analog selected from monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), or by residues of PF-06380101, the compound or salt described in any of items 16-22.

[0191] 24. The fluorescent moiety (v) is provided by a residue of a fluorescent dye, preferably a Cy5- or Cy7- cyanine dye, and is a compound or salt of any of the items 16 to 23. 25. A pharmaceutical composition containing or comprising any of the compounds or salts described in items 1 to 24.

[0192] 26. A compound or salt listed in any of items 1 to 24 for use as a pharmaceutical agent. 27. Compounds or salts described in any of items 1 to 24, for use in the treatment or prevention of diseases or disorders that can be treated or prevented by blocking the CXCR4 receptor, or diseases or disorders associated with increased or abnormal expression of the CXCR4 receptor.

[0193] 28. Compounds or salts described in any of items 1-24 and 27, for use in the treatment or prevention of cancer, cardiovascular disorders or inflammatory disorders. 29. A diagnostic composition comprising, or consisting of, any of the compounds or salts described in items 1 to 24.

[0194] 30. A compound or salt described in any of items 1 to 24, for use in a method of in vivo diagnosis of a disease or disorder. 31. Compounds or salts described in any of items 1 to 24, for use in a method of in vivo diagnosis of a disease or disorder that can be treated or prevented by blocking the CXCR4 receptor, or a disease or disorder associated with increased or abnormal expression of the CXCR4 receptor.

[0195] 32. Compounds or salts described in any of items 1-24 and 31 for use in a method of in vivo diagnosis of cancer, cardiovascular disease, or inflammatory disease. This specification references several documents, including patent applications and manufacturer manuals. While the disclosures of these documents are not considered relevant to the patentability of the present invention, their entirety is incorporated herein by reference. More specifically, all reference documents are incorporated by reference to the same extent as if each individual document were explicitly and individually indicated to be incorporated by reference. References 1. Zlotnik A, Yoshie O. Chemokines. Immunity. 2000;12:121-127. doi:10.1016 / s1074-7613(00)80165-x. 2. Domanska UM, Kruizinga RC, Nagengast WB, et al. A review on CXCR4 / CXCL12 axis in oncology: No place to hide. Eur J Cancer. 2013;49:219-230. doi:10.1016 / j.ejca.2012.05.005. 3. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996;272:872-877. doi:10.1126 / science.272.5263.872. 4. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF / SDF-1. Nature. 1996;382:635-638. doi:10.1038 / 382635a0. 5. Loetscher P, Moser B, Baggiolini M. Chemokines and Their Receptors in Lymphocyte Traffic and HIV Infection. In: Vol. 74: Elsevier; 1999:127-180. Advances in Immunology. 6. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med. 1997;185:111-120. doi:10.1084 / jem.185.1.111. 7. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood. 1999;94:3658-3667. 8. Muller A, Homey B, Soto H, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001;410:50-56. doi:10.1038 / 35065016. 9. Burger JA, Kipps TJ. CXCR4: A key receptor in the crosstalk between tumor cells and their microenvironment. Blood. 2006;107:1761-1767. doi:10.1182 / blood-2005-08-3182. 10. Chatterjee S, Behnam Azad B, Nimmagadda S. The intricate role of CXCR4 in cancer. Adv Cancer Res. 2014;124:31-82. doi:10.1016 / B978-0-12-411638-2.00002-1. 11. Guo F, Wang Y, Liu J, Mok SC, Xue F, Zhang W. CXCL12 / CXCR4: A symbiotic bridge linking cancer cells and their stromal neighbors in oncogenic communication networks. Oncogene. 2016;35:816-826. doi:10.1038 / onc.2015.139. 12. Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1 / CXCL12 secretion. Cell. 2005;121:335-348. doi:10.1016 / j.cell.2005.02.034. 13. Tamamura H, Xu Y, Hattori T, et al. A low-molecular-weight inhibitor against the chemokine receptor CXCR4: A strong anti-HIV peptide T140. Biochem Biophys Res Commun. 1998;253:877-882. doi:10.1006 / bbrc.1998.9871. 14. Tamamura H, Kuroda M, Masuda M, et al. A comparative study of the solution structures of tachyplesin I and a novel anti-HIV synthetic peptide, T22 ([Tyr5,12, Lys7]-polyphemusin II), determined by nuclear magnetic resonance. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1993;1163:209-216. doi:10.1016 / 0167-4838(93)90183-R. 15. Tamamura H, Waki M, Imai M, et al. Downsizing of an HIV-cell fusion inhibitor, T22 ([Tyr 5, 12 , Lys 7 ]-Polyphemusin II), with the maintenance of anti-HIV activity and solution structure 1. Bioorganic & Medicinal Chemistry. 1998;6:473-479. doi:10.1016 / s0968-0896(97)10055-4. 16. Tamamura H, Hiramatsu K, Mizumoto M, et al. Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists. Org Biomol Chem. 2003;1:3663-3669. doi:10.1039 / b306613b. 17. George GPC, Stevens E, Aberg O, et al. Preclinical evaluation of a CXCR4-specific (68)Ga-labelled TN14003 derivative for cancer PET imaging. Bioorganic & Medicinal Chemistry. 2014;22:796-803. doi:10.1016 / j.bmc.2013.12.012. 18. Jacobson O, Weiss ID, Kiesewetter DO, Farber JM, Chen X. PET of tumor CXCR4 expression with 4-18F-T140. J Nucl Med. 2010;51:1796-1804. doi:10.2967 / jnumed.110.079418. 19. Yan X, Niu G, Wang Z, et al. Al18FNOTA-T140 Peptide for Noninvasive Visualization of CXCR4 Expression. Mol Imaging Biol. 2016;18:135-142. doi:10.1007 / s11307-015-0872-2. 20. Peng S-B, Zhang X, Paul D, et al. 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Science. 2010;330:1066-1071. doi:10.1126 / science.1194396. 29. Demmer O, Dijkgraaf I, Schumacher U, et al. Design, synthesis, and functionalization of dimeric peptides targeting chemokine receptor CXCR4. J Med Chem. 2011;54:7648-7662. doi:10.1021 / jm2009716. 30. Tanaka T, Nomura W, Narumi T, Masuda A, Tamamura H. Bivalent ligands of CXCR4 with rigid linkers for elucidation of the dimerization state in cells. J Am Chem Soc. 2010;132:15899-15901. doi:10.1021 / ja107447w. 31. Avila-Sanchez M, Ferro-Flores G, Jimenez-Mancilla N, et al. Synthesis and preclinical evaluation of the 99mTc- / 177Lu-CXCR4-L theranostic pair for in vivo chemokine-4 receptor-specific targeting. J Radioanal Nucl Chem. 2020;324:21-32. doi:10.1007 / s10967-020-07043-6. 32. Vallejo-Armenta P, Santos-Cuevas C, Soto-Andonaegui J, et al. 99mTc-CXCR4-L for Imaging of the Chemokine-4 Receptor Associated with Brain Tumor Invasiveness: Biokinetics, Radiation Dosimetry, and Proof of Concept in Humans. Contrast Media Mol Imaging. 2020;2020:2525037. doi:10.1155 / 2020 / 2525037. 33. Demmer O, Dijkgraaf I, Schottelius M, Wester H-J, Kessler H. Introduction of functional groups into peptides via N-alkylation. Org Lett. 2008;10:2015-2018. doi:10.1021 / ol800654n. 34. Demmer O, Gourni E, Schumacher U, Kessler H, Wester H-J. PET imaging of CXCR4 receptors in cancer by a new optimized ligand. ChemMedChem. 2011;6:1789-1791. doi:10.1002 / cmdc.201100320. 35. Gourni E, Demmer O, Schottelius M, et al. PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent. J Nucl Med. 2011;52:1803-1810. doi:10.2967 / jnumed.111.098798. 36. Poschenrieder A, Schottelius M, Schwaiger M, Kessler H, Wester H-J. The influence of different metal-chelate conjugates of pentixafor on the CXCR4 affinity. EJNMMI Res. 2016;6:36. doi:10.1186 / s13550-016-0193-8. 37. Herrmann K, Schottelius M, Lapa C, et al. First-in-Human Experience of CXCR4-Directed Endoradiotherapy with 177Lu- and 90Y-Labeled Pentixather in Advanced-Stage Multiple Myeloma with Extensive Intra- and Extramedullary Disease. J Nucl Med. 2016;57:248-251. doi:10.2967 / jnumed.115.167361. 38. Osl T, Schmidt A, Schwaiger M, Schottelius M, Wester H-J. A new class of PentixaFor- and PentixaTher-based theranostic agents with enhanced CXCR4-targeting efficiency. Theranostics. 2020;10:8264-8280. doi:10.7150 / thno.45537. 39. Schottelius M, Konrad M, Osl T, Poschenrieder A, Wester H-J. An optimized strategy for the mild and efficient solution phase iodination of tyrosine residues in bioactive peptides. Tetrahedron Letters. 2015;56:6602-6605. doi:10.1016 / j.tetlet.2015.10.032. 40. Demmer O, Frank AO, Hagn F, et al. Erhohte CXCR4-Affinitat und Anti-HIV-Aktivitat eines Peptoids durch Konformationsfixierung. Angew. Chem. 2012;124:8234-8237. doi:10.1002 / ange.201202090. 41. Chatterjee J, Gilon C, Hoffman A, Kessler H. N-methylation of peptides: A new perspective in medicinal chemistry. Acc Chem Res. 2008;41:1331-1342. doi:10.1021 / ar8000603. 42. Weineisen M, Simecek J, Schottelius M, Schwaiger M, Wester H-J. Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI Res. 2014;4:63. doi:10.1186 / s13550-014-0063-1. 43. Iovkova L, Wangler B, Schirrmacher E, et al. para-Functionalized aryl-di-tert-butylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals. Chemistry. 2009;15:2140-2147. doi:10.1002 / chem.200802266. 44. Kostikov AP, Iovkova L, Chin J, et al. N-(4-(di-tert-butyl[18F]fluorosilyl)benzyl)-2-hydroxy-N,N-dimethylethylammonium bromide ([18F]SiFAN+Br-): A novel lead compound for the development of hydrophilic SiFA-based prosthetic groups for 18F-labeling. Journal of Fluorine Chemistry. 2011;132:27-34. doi:10.1016 / j.jfluchem.2010.10.008. 45. Abrams MJ, Juweid M, tenKate CI, et al. Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats. J Nucl Med. 1990;31:2022-2028. 46. Joyard Y, Bischoff L, Levacher V, Papamicael C, Vera P, Bohn P. Synthesis and Stability Evaluation of New HYNIC Derivatives as Ligands for Technetium-99m. LOC. 2014;11:208-214. doi:10.2174 / 15701786113106660087. 47. Schottelius M, Schwaiger M, Wester H-J. Rapid and high-yield solution-phase synthesis of DOTA-Tyr3-octreotide and DOTA-Tyr3-octreotate using unprotected DOTA. Tetrahedron Letters. 2003;44:2393-2396. doi:10.1016 / S0040-4039(03)00221-1. 48. Schottelius M, Osl T, Poschenrieder A, et al. 177Lupentixather: Comprehensive Preclinical Characterization of a First CXCR4-directed Endoradiotherapeutic Agent. Theranostics. 2017;7:2350-2362. doi:10.7150 / thno.19119. 49. Robu S, Schottelius M, Eiber M, et al. Preclinical Evaluation and First Patient Application of 99mTc-PSMA-I&S for SPECT Imaging and Radioguided Surgery in Prostate Cancer. J Nucl Med. 2017;58:235-242. doi:10.2967 / jnumed.116.178939. 50. Kuzmanovska S, Vaskova O, Zdraveska Kocovska M. “In-house” preparation of 99mTc-EDDA / HYNIC-TOC, a specific targeting agent for somatostatin receptor scintigraphy. Maced. Pharm. Bull. 2011;57:65-70. doi:10.33320 / maced.pharm.bull.2011.57.007. 51. Wangler C, Niedermoser S, Chin J, et al. One-step (18)F-labeling of peptides for positron emission tomography imaging using the SiFA methodology. Nat Protoc. 2012;7:1946-1955. doi:10.1038 / nprot.2012.109. 52. Othman MFB, Mitry NR, Lewington VJ, Blower PJ, Terry SYA. Re-assessing gallium-67 as a therapeutic radionuclide. Nucl Med Biol. 2017;46:12-18. doi:10.1016 / j.nucmedbio.2016.10.008. 53. Yamazaki K, Kanaoka M. Computational prediction of the plasma protein-binding percent of diverse pharmaceutical compounds. J Pharm Sci. 2004;93:1480-1494. doi:10.1002 / jps.20059. List of abbreviations used %iD / g: Percentage of injection amount per gram 2-CTC 2-chlorotrityl chloride AA amino acids Abz para-aminobenzoic acid ACN Acetonitrile AKT Protein Kinase B Ambz para-aminomethylbenzoic acid Boc tert-butyloxycarbonyl CDI (Carbonyldiimidazole) CPCR4 Cyclo(D-Tyr-D-[NMe]Orn-Arg-2-Nal-Gly) CT (Computed Tomography) CXCR4 CXC chemokine receptor type 4 dap 2,3-diaminopropionic acid DBU 1,8-Diazabicyclo[5.4.0]Undeca-7-Ene DCE Dichloroethane DCM Dichloromethane Dde N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) DIAD Diisopropyl Azodicarboxylate DIPEA N,N-diisopropylethylamine DMAP 4-(dimethylamino)-pyridine DMEM Dulbecco's Modified Eagle Medium DMF (N,N-dimethylformamide) DMG (Dimethylglycine) DMSO (Dimethyl Sulfoxide) DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DOTA-GA 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanoic acid DPPA (Diphenylphosphoryl Azide) EDDA (Ethylenediaminediacetic acid) EDTA (Ethylenediaminetetraacetic acid) Et2O Diethyl ether HCl ethyl acetate EtOH Ethanol EUE (R)-5-(tert-butoxy)-4-(3-((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-oxopentanoic acid FBS (fetal bovine serum), fetal bovine serum HATU [O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluolphosphat] HBSS Hanks buffer solution HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol HIV (Human Immunodeficiency Virus) HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole HPLC (High-Pressure Liquid Chromatography) HSA Human Serum Albumin HYNIC (Hydradinonicotinic Acid) MAPK Mitogen-Activated Protein Kinase mas3 mercaptoacetyl-tricerine MMAE Monomethyl Auristatin NEA (Non-Essential Amino Acids) NIS N-iodosuccinimide NMP N-methyl-2-pyrrolidone OI Optical Imaging O-NBS-Cl 2-nitrobenzenesulfonyl chloride after pi injection Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl PBS (phosphate-buffered saline) PCC (Pyridinium Chlorochromate) PenStrep Penicillin-Streptomycin Mixture PET positron emission tomography rt room temperature RP-HPLC (Reverse-Phase High-Pressure Liquid Chromatography) RPMI (Rosewell Park Memorial Institute) SAR Structure-Activity Relationship sat. saturation SCID Severe Combined Immunodeficiency SDF-1 (Stroma cell-derived factor 1) SiFA Silicon Fluoride Acceptor SiFA-BA SiFA-benzoic acid SiFA-Br SiFA-bromide SPECT single-photon emission tomography SPPS Solid-Phase Peptide Synthesis tbd Undecided TBDMSCl tert-butyldimethylsilyl chloride TBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate tBu tert-butyl TEA (Triethylamine) TFA (Trifluoroacetic Acid) THF (Tetrahydrofuran) TIPS Triisopropylsilane TLC (Thin-Layer Chromatography) t R retention time Trt Trichl [Examples]

[0196] I. Materials and Methods 1. Overview 1.1 Reagents and Solvents The purchased reagents were used without further purification. Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogs were purchased from Bachem (Bubendorf, Switzerland), Iris Biotech GmbH (Marktredwitz, Germany), Carbolution Chemicals GmbH (St. Ingbert, Germany), and Merck Millipore (Darmstadt, Germany). 2-chlorotrityl chloride (2-CTC) resin was obtained from Iris Biotech GmbH (Marktredwitz, Germany) or CEM (Matthews, USA). Reagents for peptide synthesis were purchased from Iris Biotech GmbH (Marktredwitz, Germany), Sigma-Aldrich (Munich, Germany), and Molekula GmBH (Garching, Germany). Solvents and reagents for organic synthesis were purchased from either Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany), or VWR (Darmstadt, Germany).

[0197] Chematech (Dijon, France) delivered the DOTA-GA chelator used, and the DOTA derivative was supplied by Macrocyclics (Plano, USA). Cy5-carboxylic acid was obtained from Lumiprobe (Hunt Valley, USA). Cytotoxic MMAE derivatives were purchased from Creative Biolabs (Shirley, USA).

[0198] Biochemical products, such as DMEM (Ham F-12, stable Gln-containing) and RPMI1640 (Gln-containing) media, fetal bovine serum (FBS superior), phosphate-buffered saline (PBS Dulbecco's, Ca 2+ Mg 2+ (Not included), trypsin / EDTA (0.05% / 0.02% in PBS, Ca 2+ Mg 2+ (Not included) and Hanks buffer solution (HBSS, 0.35 g / L NaHCO3 and Ca 2+ Mg2 The ingredients (containing) were obtained from Biochrom GmbH (Berlin, Germany) or Sigma-Aldrich (Munich, Germany).

[0199] Water for RP-HPLC solvent was obtained from the on-site Millipore system at Thermo Fischer Scientific Inc. (Waltham, MA, USA). Tracepure water for labeling experiments was obtained from Merck Millipore (Darmstadt, Germany). 1.2 Radioisotopes 125 The labeling in I is made by Hartmann Analytik GmbH (Braunschweig, Germany) in NaOH [ 125 The procedure was performed using [I]NaI solution (40 mM, 74 TBq / mmol).

[0200] [ 99m Tc]-Pertecnetate was obtained by elution using a GE Healthcare (Munich, Germany) Drytech™ technetium generator with physiological NaCl solution (0.9% v / v). This generator was provided by Klinikum Rechts der Isar (Technical University Munich, Munich, Germany).

[0201] [ 177 Solution of LuCl3 (HCl (0.04M); S A The 3 TBq / mg and 740 MBq / mL solutions were supplied by ITM GmbH (Garching, Germany) and were used directly in the labeling experiment.

[0202] Target underwater [ 18 F]-Fluoride was provided by Klinikum Rechts der Isar (Technical University Munich, Munich, Germany).

[0203] For radioactive synthesis [ 68 Ga[GaCl3] manufactured by iThemba LABS (Cape Town, South Africa) 68 Ge / 68 The galvanic acid was obtained by elution using an aqueous HCl solution (1.00 M) in a Ga generator. The synthesis was performed using an automated GallElut system manufactured by Scintomics GmbH (Fuerstenfeldbruck, Germany). + I did it with the system.

[0204] [ 67 Ga]Citrate Ga was delivered by Mallinckrodt Pharmaceuticals (Dublin, Ireland) and used before radiosynthesis. 67 Converted to GaCl3. 1.3 Instruments and Analysis Solid-phase peptide synthesis (SPPS) was performed manually using an Intelli-Mixer syringe shaker manufactured by Neolab (Heidelberg, Germany).

[0205] The eluents for all RP-HPLC operations were water (solvent A) and acetonitrile (solvent B), both containing 0.1 vol% trifluoroacetic acid. For semi-preparative RP-HPLC runs, solvent B was used with 5 vol% H2O. Analytical and semi-preparative reverse-phase high-pressure chromatography (RP-HPLC) runs were performed using a Shimadzu gradient system from Shimadzu Deutschland GmbH (Neufahrn, Germany), equipped with SPD-20A UV / Vis detectors (λ=220 nm, 254 nm), respectively. A Multokrom100C18 column (125 × 4.6 mm, 5 μm particle size) provided by CS GmbH (Langerwehe, Germany) was used for analytical RP-HPLC runs at a flow rate of 1 mL / min. Specific gradients and corresponding retention times were used. R Both are cited in the text. Semi-preparative HPLC purification was performed using a Multokrom100 RP18 column (250 × 10 mm, 5 μm particle size) manufactured by CS GmbH (Langerwehe, Germany) at a constant flow rate of 5 mL / min.

[0206] Analytical and semi-preparative radioactive RP-HPLC were performed using a Multokrom100C18 (5 μm, 125 × 4.0 mm) column from CS GmbH (Langerwehe, Germany). Radioactivity was detected by connecting the outlet of a UV photometer to a NaI(Tl) well scintillation counter from EG&G Ortec (Munich, Germany).

[0207] Radioactive probes, such as test vials of mouse organs or cells, are manufactured by Perkin Elmer (Waltham, MA, USA) under the WIZARD brand. 2 Measured with (registered trademark) 2480 automatic gamma counter.

[0208] Radioactive TLC measurements were performed using a Scan-RAM™ device manufactured by LabLogic Systems Ltd. (Broomhill, UK). The resulting chromatograms were analyzed using Laura™ software, also manufactured by LabLogic Systems Ltd. (Broomhill, UK).

[0209] RP-HPLC chromatograms were evaluated using LabSolution software from Shimadzu Corporation (Kyoto, Japan). Mass spectra for characterizing organic materials are obtained using an expression from Advion Ltd. (Harlow, UK). L The spectra were acquired using a CMS quadrupole mass spectrometer. NMR spectra were recorded at 300K using a Bruker AVHD-300 or AVHD-400 spectrometer from Bruker Corporation (Billerica, USA).

[0210] The pH value was measured using a SevenEasy pH meter manufactured by Mettler Toledo (Giessen, Germany). Purification by flash chromatography was performed using a Biotage (Uppsala, Sweden) Isolera® Prime System with a Biotage 09474 Rev.E Bio pump. 18 Cartridge (12g, 93Å pore size, 382m) 2 Using the ( / g surface) method, a linear gradient was applied between solvent B (ACN, 0.1 vol% TFA, 2 vol% H2O) in solvent A (H2O, 0.1 vol% TFA).

[0211] The peptides were freeze-dried using a Christ (Osterode am Harz, Germany) Alpha1-2 LDplus freeze-dryer and a Vacubrand GmbH (Wertheim, Germany) RZ-2 vacuum pump.

[0212] I C 50 The values ​​were calculated using GraphPad Prism6, manufactured by GraphPad Software Inc. (San Diego, USA). 2.Synthesis 2.1 Solid-phase peptide synthesis following the Fmoc strategy Loading of GP1:2-CTC resin Loading of Fmoc-protective amino acids (AA) into 2-CTC resin was performed by stirring a suspension of 2-CTC resin (1.6 mmol / g) and Fmoc-AA-OH (1.5 equivalents) in DMF with DIPEA (3.0 equivalents) at room temperature for 2-5 hours. The remaining trityl chloride was capped by adding methanol (5 mL / g resin) and incubated for 15 minutes. The resin was then filtered, washed with DMF (5 × 5 mL / g resin) and methanol (3 × 5 mL / g resin), and dried under vacuum. The final loading of Fmoc-AA-OH into the resin was determined by the following formula.

[0213]

number

[0214] GP2: Peptide coupling on resin Each side chain-protected Fmoc-AA-OH (1.5 equivalents) was dissolved in DMF (8 mL / g resin) and pre-activated by adding TBTU (1.5 equivalents), HOBt (1.5 equivalents), and DIPEA (3 equivalents). For condensation of low-reactivity amino acids or peptide fragments, HATU (1.5 equivalents) and HOAt (1.5 equivalents) were used instead of TBTU and HOBt. After activation for 15 minutes, the solution was added to the free amine peptide 2-CTC-AA-NH2 bound to the resin and shaken at room temperature for 2 hours. For dap(Boc)-OH, dap(Dde)-OH, and cys(Trt)-OH, and fragments having these amino acids at the C-terminus, the pre-activation was shortened to 2-5 minutes, and 2,4,6-collidine was used as a base. For peptide fragments, the reaction time was often extended up to 48 hours at room temperature. Next, the resin was washed with DMF (6 × 5 mL / g resin), deprotected with Fmoc, and then coupled with the following amino acids in the same manner. GP3: Fmoc deprotection on resin The Fmoc-peptide bound to the resin was treated with 20% piperidine in DMF (v / v, 8 mL / g resin) for 5 minutes, followed by 15 minutes. The resin was then thoroughly washed with DMF (8 × 5 mL / g resin). GP4: Dde-deprotection on resin: A Dde-protected peptide (1.0 equivalent) was treated with a solution of 2% hydrazine monohydrate in DMF (v / v, 5 mL / g resin) and shaken for 15 minutes. For this Fmoc- group, Dde-deprotection was carried out at room temperature for 3 hours by adding a solution of imidazole (0.46 g), hydroxylamine hydrochloride (0.63 g) in NMP (2.5 mL) and DMF (0.5 mL). After deprotection, the resin was washed with DMF (6 × 5 mL / g resin). GP5: Deprotection of tBu / Boc / Pbf / Trt The tBu / Boc / Pbf protecting group was removed by dissolving the crude product in TFA and stirring at room temperature for 90 minutes. For the removal of the Trt protecting group, TIPS was added to the mixture. After removing the TFA under a nitrogen stream, the residue was dissolved in a mixture of t-butanol and water. After lyophilization, the crude deprotected peptide was obtained. GP6: N-acetylation Acetylation of the amine functional group was achieved by reacting each peptide with a mixture of DIPEA (5.00 equivalents) and Ac2O (5.00 equivalents) in DMF for 2 hours. Iodination of GP7:CPCR4 Iodination of CPCR4 tyrosine was performed according to the published procedure (39). Briefly, the completely deprotected and purified peptide was dissolved in ACN / H2O (1 / 1(V / V), 1.00 mM) and 0.30-0.50 equivalents of NIS were added. After 5 minutes at room temperature, the reaction mixture was subjected to HPLC purification. GP8: Condensation of fragments in solution The coupling of the CPCR4 binding motif to the functional fragment was performed in DMF using small molar amounts of synthetic fragment (1.10–1.30 equivalents) and HOAt / HATU as the coupling reagent. 2,4,6-collidine was used as the base when the activated amino acid was dap; DIPEA was used in all other cases. GP9: Cleavage of peptides from resins Preservation of acid-unstable protecting groups The peptides bound to the resin were treated with a mixture of DCM / HFIP (4 / 1(v / v), 8 mL / g resin) and shaken for 60 minutes. The solution containing the fully protected peptides was filtered off, and the resin was treated with another portion of the cutting solution for 60 minutes. Both fractions were combined, and the solvent was removed under reduced pressure. After lyophilization, crude, fully protected peptides were obtained. Cleavage of acid-unstable protecting groups The fully protected, resin-bound peptides were treated with a mixture of TFA / TIPS / H2O(95 / 2.5 / 2.5(v / v / v)) and shaken for 30 minutes. The solution was filtered off, and the resin was treated similarly for another 30 minutes. Both filtrates were combined and concentrated under a stream of nitrogen. The residue was dissolved in a mixture of t-butanol and water, and then freeze-dried to obtain the crude peptide. GP10: Sulfhydryl-maleimide coupling Peptides containing cysteine ​​or homocysteine ​​were coupled to a maleimide functional group by the following procedure: A fully unprotected HS-peptide was dissolved in DMF (1.00 mg / mL) and added to a substance containing maleimide in DMF (1.10 mg / mL). DIPEA (0.50 μL / mg peptide) was added, and the reaction mixture was stirred at room temperature for 2 hours. Completion of the reaction was confirmed by RP-HPLC, and the mixture was purified by semi-preparative RP-HPLC. 2.2 NAT GA / NAT LU complex formation Peptides containing DOTA and peptides containing DOTA-GA nat Ga and nat For complex formation with Lu, fully deprotected and purified peptides were used. The peptides were dissolved in DMSO, a DMSO / H2O mixture, or H2O to an ideal concentration of 1 mM. The required amount of peptide (30-500 nmol) was placed in an Eppendorf tube and then in H2O. nat GaNO3 or nat LuCl3 (3.00 equivalents each) was added. The vials were heated to 95°C for 30 minutes, and quantitative conversion was checked by RP-HPLC and ESI-MS. If no traceable extract was found, the reaction mixture was used without further purification. 2.3 Composition of Components 2.3.1 Synthesis of cyclo(D-Tyr-D-[NMe]Orn-Arg-2-Nal-Gly) (=CPCR4)

[0215] [ka]

[0216] The synthesis of the CXCR4-binding motif CPCR4 was carried out in the same manner as described above (40). Briefly, Fmoc-Gly-OH was immobilized on 2-CTC resin, and Fmoc-2-Nal-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-D-Orn(Boc)-OH were coupled according to GP2 and GP3. The N-terminus was then deprotected with Fmoc and newly protected by reacting O-NBS-Cl (4.00 equivalents) and 2,4,6-collidine (10.0 equivalents) in NMP for 15 minutes. N-terminal methylation was achieved using either Mitsunobu conditions (Ph3P (5.00 equivalents), MeOH (10.0 equivalents), DIAD (5.00 equivalents) in THF, 10 min) or dimethyl sulfate (Me2SO4 (10.0 equivalents), DBU (3.00 equivalents) in NMP, 2 × 2 min) (41). Deprotection of the methylated end was achieved by incubating the peptide in DBU (5.00 equivalents) for 5 minutes, followed by the addition of mercaptoethanol (10.00 equivalents). After 30 minutes, the resin was thoroughly washed. The subsequent Fmoc-D-Tyr(tBu)-OH coupling was achieved using HOAt and HATU as coupling reagents. After final Fmoc deprotection, the peptide was cleaved from the resin while retaining the acid-unstable protecting group (GP9). Cyclization was carried out using DPPA (3.00 equivalents) and NaHCO3 (5.00 equivalents) in a 1 mM solution of the peptide in DMF. After the completion of the reaction, which was monitored by RP-HPLC, the product solution was concentrated under reduced pressure. The resulting crude product was completely deprotected by treatment with TFA and then precipitated in Et2O. The peptide was purified by flash chromatography to obtain an off-white solid. CPCR4:RP-HPLC (10-95% B in 15 minutes):t R = 6.49 minutes. Monoisotopic mass calculation value (C 36 H 47 N9O6): 701.36, Measured value: 701.8 [M+H] + , 351.4[M+2H] 2+ . 2.3.2 Synthesis of (R)-5-(tert-butoxy)-4-(3-((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-oxopentanoic acid (=D-(tBu)e(OH)uDe(tBu)2)[3]

[0217] [ka]

[0218] di-tert-butyl-(1H-imidazole-1-carbonyl)-D-glutamate[1] The synthesis was carried out according to the published procedure (42) with minor modifications. Briefly, D-glu(OtBu)-OtBu (1.00 equivalent) was dissolved in DCM and treated with TEA (2.50 equivalents) and DMAP (0.04 equivalents) on ice. CDI (1.10 equivalents) was added, and the mixture was stirred overnight without further cooling. The reaction was stopped by the addition of saturated NaHCO3, and the organic layer was washed twice with H2O and brine, respectively. The solvent was evaporated to obtain the crude product [1] as a colorless oil (91% yield). The product was used in the next reaction without further purification. Di-tert-butyl-(1H-imidazole-1-carbonyl)-D-glutamate[1]:RP-HPLC:(10-90% B at 15 mins):t R = 14.50 minutes. Monoisotopic mass calculation value (C 17 H 27 N3O5): 353.2, Measured value: 376.3 [M+Na] + . 5-benzyl-1-(tert-butyl)-(((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-d-glutamate[2] The extract [1] (1.00 equivalent) was dissolved in DCE and cooled on ice, after which TEA (2.00 equivalent) and D-glu(OBn)-OtBu (1.00 equivalent) were added. The mixture was heated overnight at 40°C. The solvent was then concentrated under vacuum, and the crude product was subjected to silica flash chromatography using siRNA / n-hexane / TEA (500 / 500 / 0.8 (v / v / v)). After removing the solvent under reduced pressure, the desired product [2] was obtained as a colorless oil (84% yield). 5-benzyl-1-(tert-butyl)-(((R)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-d-glutamate[2]:RP-HPLC:(10-90% B at 20 mins):t R = 17.43 minutes. Monoisotopic mass calculation value (C 30 H 46 N2O9): 578.3, Measured value: 601.5 [M+Na] + , 523.3[M-tBu+H] + , 467.3[M-2tBu+H] + , 411.3[M-3tBu+H] + . (tBu)e(OH)ue(tBu)2[3] A benzyl-protected extract [2] (1.00 equivalent) was dissolved in EtOH, and palladium (10% on activated carbon, 0.10 equivalent) was added. The deprotection reaction was promoted by maintaining an H2 atmosphere at room temperature overnight. The catalyst was filtered off by passing the mixture through a Celite pad. The solvent of the resulting clear solution was evaporated under reduced pressure to obtain the desired product [3] as a solidified colorless oil (82% yield). e(tBu)ue(tBu)2[3]:RP-HPLC (10-90% B in 15 minutes):t R = 12.00 minutes. Monoisotopic mass calculation value (C 23 H 49 N2O9): 488.3, ​​Measured value: 489.4 [M+H] + , 516.4[M+Na] + . 2.3.3 Fmoc-D-Hcy(glycosyl / lactosyl)-OH For the incorporation of the sugar moiety into Fmoc-D-Hcy(Trt)-OH, the respective peracetylation precursors (β-D-glucose pentacetate / β-D-lactose octaacetate) were used. The sugar (1.00 equivalent) and the protecting amino acid (1.20 equivalent) were added to a round-bottom flask in an argon stream, and DCM was performed. abs It was dissolved in [a solution]. TIPS (1.30 equivalents) was added as a scavenging agent, and SnCl4 (2.40 equivalents, 1.00 M in DCM) was added dropwise to the reaction mixture. Initially yellow, the mixture became colorless, and after stirring overnight at room temperature, a precipitate formed. The mixture was diluted with DCM and acidified with HCl (1.00 M). The organic layer was extracted twice with HCl and H2O, respectively, and dehydrated with Na2SO4. The solvent was evaporated under vacuum, and the crude product was purified by flash chromatography.

[0219] [ka]

[0220] Fmoc-D-HCy-(β-D-Gluc(OAc)4)-OH:HPLC (30-80% B at 15 min):t R = 9.90 minutes. Monoisotopic mass calculation value (C 33 H 37 NO 13 S): 687.20, Measured value: 688.2 [M+H] + . Fmoc-D-HCy-(β-D-Lac(OAc)7)-OH:HPLC (30~80%B in 15 minutes):t R = 10.70 minutes. Monoisotopic mass calculation value (C 45 H 53 NO 21 S): 975.28, Measured value: 976.0 [M+H] + . 2.3.4 4-(di-tert-butylfluorosilyl)benzoic acid (=SiFA-BA, [8])

[0221] [ka]

[0222] The synthesis of the silicon fluoride acceptor moiety [8] was carried out by following the published procedure (43) with some modifications. The reaction was carried out in a dry flask under an argon atmosphere. Starting with 4-bromobenzyl alcohol, the desired product was obtained after five reaction steps. ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane[4] 4-Bromobenzyl alcohol (1.00 equivalent) in DMF abs. The mixture was dissolved in (15 mL / g extract), and imidazole (1.20 equivalents) and TBDMSCl (1.20 equivalents) were added while vigorously stirring. The reaction mixture was stirred overnight at room temperature, poured into cold H2O, and the aqueous phase was extracted five times with Et2O. The organic phases were combined and washed twice with saturated NaHCO3 and brine.

[0223] [ka]

[0224] The solution was dehydrated with MgSO4 and concentrated under vacuum. The crude product was purified by silica flash chromatography using 5% siRNA (v / v) in petroleum ether. After removing the solvent under reduced pressure, the protected alcohol [4] was obtained as a colorless oil (95% yield). ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane[4]:RP-HPLC:(50-100% B at 15 mins):t R =15.0 minutes. 1 H-NMR (400 MHz, CDCl3, 300 K): δ = 0.10 (6H, s, SiMe2tBu), 0.95 (9H, s, SiMe2tBu), 4.69 (2H, s, CH2OSi), 7.21 (2H, d), 7.46 (2H, d) ppm. Di-tert-butyl(4-((tert-butyldimethylsilyloxy)methyl)phenyl)fluorosilane[5]

[0225] [ka]

[0226] [4] (1.00 equivalent) THF abs. The solution was dissolved in (10 mL / g extract), cooled to -78°C, and then tBuLi (2.40 equivalents) was added in pentane (c=1.70 mol / L). After stirring at -78°C for 30 minutes, the reaction mixture was converted to THF with di-tert-butyldifluorosilane (1.20 equivalents). abs. The solution was added dropwise at -78°C to a 10 mL / g solution. The solution was stirred overnight, warmed to room temperature, and then brine was added. The crude product was extracted three times with Et2O, the combined organic phase was dehydrated with MgSO4, and the solvent was evaporated under vacuum to obtain a yellowish oily substance (yield 95%). The crude product [5] was used without further purification. Di-tert-butyl(4-((tert-butyldimethylsilyloxy)methyl)phenyl)fluorosilane[5]:RP-HPLC:(50-100%B at 20 mins):t R =19.0 minutes. 4-(di-tert-butylfluorosilanyl)benzyl alcohol[6]

[0227] [ka]

[0228] Deprotection of [5] was achieved by suspending the compound in MeOH (25 mL / g extract) and using a catalytic amount of concentrated HCl (0.25 mL / g). The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure. The residue was dissolved in Et2O (20 mL / g extract) and washed with saturated NaHCO3 solution. The aqueous layer was then extracted three times with Et2O, and the combined organic phase was dehydrated with MgSO4. The solvent was evaporated under vacuum to obtain a yellowish oily substance (98% yield). The crude product [6] was used without further purification. 4-(di-tert-butylfluorosilanyl)benzyl alcohol[6]:RP-HPLC:(50-100% B at 15 mins):t R =8.2 minutes. 4-(di-tert-butylfluorosilyl)benzaldehyde[7]

[0229] [ka]

[0230] Oxidation to aldehyde was carried out using Corey-Suggs conditions. The extract [6] (1.00 equivalent) was condensed in DCM. abs. Dissolve in (15 mL / g extract) and add PCC (2.50 equivalents) to DCM abs. The mixture was added dropwise to an ice-cold suspension in (20 mL / g PCC). After stirring at 0°C for 30 minutes, followed by 2.5 hours at room temperature, Et2O was added, and the supernatant was decanted from solid. The black residue was washed with Et2O, and the combined organic phase was filtered through a silica gel pad (10 cm / g product). The solvent was evaporated under vacuum to obtain [7] as a yellowish oily substance (yield 96%). 4-(di-tert-butylfluorosilyl)benzaldehyde[7]:RP-HPLC:(50-100% B at 15 mins):t R =10.5 minutes. 4-(di-tert-butylfluorosilyl)benzoic acid (=SiFA-BA)[8]

[0231] [ka]

[0232] Aldehyde [7] (1.00 equivalent) was dissolved in tBuOH (23 mL / g extract) and DCM (2.5 mL / g extract), and NaH2PO4 × H2O (1.25 M, pH=4.0~4.5, 15 mL / g extract) and KMnO4 aqueous solution (1 M, 23 mL / g extract) were added. After stirring for 25 minutes, the mixture was cooled to 5°C. The reaction was stopped by adding KMnO4 (1.00 equivalent) and immediately after by adding saturated NaHCO3. The mixture was dehydrated with MgSO4 and the solvent was evaporated under reduced pressure. The crude product [8] was purified by recrystallization from Et2O / n-hexane (1 / 3, v / v) to obtain a colorless solid (yield 60%). 4-(di-tert-butylfluorosilyl)benzoic acid (=SiFA-BA[8]):RP-HPLC:(50-100% B at 15 mins):t R = 8.5 minutes. Monoisotopic mass calculation value (C 15 H 23 FO2Si): ​​282.4; Measured value: m / z = 281.1 [MH] - , 235.1[M-COOH] - . 2.3.5 (4-bromomethylphenyl)-di-tert-butyl-fluorosilane (=SiFA-Br[9])

[0233] [ka]

[0234] The synthesis of [9] was carried out according to reference (44). Briefly, SiFA benzyl alcohol [6] was dissolved in DCM and the solution was cooled to 0°C. CBr4 (1.10 equivalents) was added, followed by the addition of PPh3 in small amounts over 30 minutes. The mixture was stirred at room temperature for 2 hours, the solvent was removed under vacuum, and the residue was washed with n-hexane. The precipitate was filtered off and the liquid was concentrated under vacuum. Subsequent silica flash chromatography, using n-pentane as the mobile phase, yielded the desired product [9] as a colorless oil (yield 32–39%). (4-bromomethylphenyl)-di-tert-butyl-fluorosilane (=SiFA-Br[9]): RP-HPLC: (50-100% B at 15 mins): t R = 15.10 minutes. Monoisotopic mass calculation value (C 15 H 24 BrFSi): 330.08; Measured value: m / z = 331.3 [MH] - . 1H NMR (300 MHz, CDCl3, 293 K) δ 7.63 - 7.54 (m, 2H, 2x-CH-), 7.40 (d, J = 8.0 Hz, 2H, 2x-CH-), 4.50 (s, 2H, -CH2-), 1.06 (d, J = 1.2 Hz, 18H, 2xtBu). 13 C NMR (75 MHz, CDCl3, 293 K) δ 139.50 (-CH-), 135.00 (-CH-), 134.95 (-CH-), 134.84 (-CH-), 134.66 (-CH-), 128.72 (-CH-), 33.88 (-CH2-), 27.88 (6xtBu C), 20.92 (Tertiary C), 20.75 (Tertiary C). 2.3.6 Synthesis of Linker Structure 2.3.6.1 Abz-type linkers and Ambz-type linkers

[0235] [ka]

[0236] The entire -Ambz- and -Abz- linkers were synthesized in the same manner (see GP1, GP2, and GP3), the difference being the coupling of Fmoc-D-Ala-OH. For the -Abz- linkers, considering the lower reactivity of the -Abz- amine functional group compared to -Ambz-, HOAt and HATU were used, and the reaction time was simultaneously extended to 4 hours. After inserting the second or third amino acid, each linker was cleaved from the solid support while retaining the protecting group (see GP9). For acetylated linker units, additional Fmoc deprotection and subsequent acetylation as described (see GP6) were performed on the solid support. HO-Abz-ar(Pbf)-Fmoc

[0237] [ka]

[0238] HO-Abz-ar(Pbf)-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 15.33 minutes. Monoisotopic mass calculation value (C 44 H 50 N6O9S): 838.34, Measured value: 839.1 [M+H] + . HO-Abz-ar(Pbf)-dap(Boc)-Fmoc

[0239] [ka]

[0240] HO-Abz-ar(Pbf)-dap(Boc)-Fmoc:RP-HPLC (50-95% B in 15 minutes):t R = 13.52 minutes. Monoisotopic mass calculation value (C 52 H 64 N8O 12 S): 1024.44, Measured value: 1025.2 [M+H] + . HO-Abz-a-cit-dap(Boc)-Fmoc

[0241] [ka]

[0242] HO-Abz-a-cit-dap(Boc)-Fmoc:RP-HPLC (10-95% B in 15 minutes):t R = 10.86 minutes. Monoisotopic mass calculation value (C 39 H 47 N7O 10 ): 773.34, Measured value: 774.5 [M+H] + . HO-Abz-ar(Pbf)-h(Trt)-Fmoc

[0243] [ka]

[0244] HO-Abz-ar(Pbf)-h(Trt)-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 17.15 minutes. Monoisotopic mass calculation value (C 69 H 71 N9O 10 S): 1217.50, Measured value: 1218.0 [M+H] + , 977.3[M-Trt+H] + . HO-Abz-ar(Pbf)-c(Trt)-Fmoc

[0245] [ka]

[0246] HO-Abz-ar(Pbf)-c(Trt)-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 17.77 minutes. Monoisotopic mass calculation value (C 66 H 69 N7O 10 S2): 1183.45, Measured value: 1184.4 [M+H] + . HO-Abz-ar(Pbf)-Hcy(Trt)-Fmoc

[0247] [ka]

[0248] HO-Abz-ar(Pbf)-Hcy(Trt)-Fmoc:RP-HPLC(10~90%B in 15 minutes):t R = 17.79 minutes. Monoisotopic mass calculation value (C 67 H 71 N7O 10 S2): 1197.47, Measured value: 1198.2 [M+H] + . HO-Abz-ar(Pbf)-f-Fmoc

[0249] [ka]

[0250] HO-Abz-ar(Pbf)-f-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 16.30 minutes. Monoisotopic mass calculation value (C 53 H 59 N7O 10 S): 985.40, Measured value: 986.8 [M+H] + . HO-Abz-ar(Pbf)-r(Pbf)-Fmoc

[0251] [ka]

[0252] HO-Abz-ar(Pbf)-r(Pbf)-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 16.82 minutes. Monoisotopic mass calculation value (C 63 H 78 N 10 O 13 S2): 1246.52, Measured value: 1248.6 [M+H] + . HO-Ambz-ar(Pbf)-Fmoc

[0253] [ka]

[0254] HO-Ambz-ar(Pbf)-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 12.51 minutes. Monoisotopic mass calculation value (C 45 H 52 N6O9S): 852.35, Measured value: 601.4 [M-Pbf+H] + . HO-Ambz-ar(Pbf)-dap(Boc)-Fmoc

[0255] [ka]

[0256] HO-Ambz-ar(Pbf)-dap(Boc)-Fmoc:RP-HPLC (30-90% B in 15 minutes):t R = 13.70 minutes. Monoisotopic mass calculation value (C 53 H 66 N8O 12 S): 1038.45, Measured value: 1039.6 [M+H] + . HO-Ambz-ar(Pbf)-h(Trt)-Fmoc

[0257] [ka]

[0258] HO-Ambz-ar(Pbf)-h(Trt)-Fmoc:RP-HPLC (10-90% B in 15 minutes):t R = 17.95 minutes. Monoisotopic mass calculation value (C 70 H 73 N9O 10 S): 1231.52, Measured value: 1233.1 [M+H] + . HO-Abz-ar(Pbf)-dap(Boc)-Ac

[0259] [ka]

[0260] HO-Abz-ar(Pbf)-dap(Boc)-Ac:RP-HPLC(10~95%B in 15 min):t R = 10.43 minutes. Monoisotopic mass calculation value (C 39 H 56 N8O 11 S): 844.38, Measured value: 845.2 [M+H] + . HO-Abz-ar(Pbf)-4-APipAc-Ac

[0261] [ka]

[0262] HO-Abz-ar(Pbf)-4-APipAc-Ac:RP-HPLC (10~95%B in 15 min):t R = 8.13 minutes. Monoisotopic mass calculation value (C 38 H 54 N8O9S): 798.37, Measured value: 799.7 [M+H] + , 400.1[M+2H] 2+ . HO-Abz-a-cit-dap(Boc)-Ac

[0263] [ka]

[0264] HO-Abz-a-cit-dap(Boc)-Ac:RP-HPLC (10~95%B in 15 minutes):t R = 6.61 minutes. Monoisotopic mass calculation value (C 26 H 39 N7O9): 593.28, Measured value: 594.3 [M+H] + . HO-Abz-a-4-APipAc-dap(Boc)-Ac

[0265] [ka]

[0266] HO-Abz-a-4-APipAc-dap(Boc)-Ac:RP-HPLC (10~95%B in 15 minutes):t R = 6.62 minutes. Monoisotopic mass calculation value (C 27 H 40 N6O8): 576.29, Measured value: 577.2 [M+H] + . AHX-series linker

[0267] [ka]

[0268] The synthesis of Ahx-type peptide linkers was carried out by standard SPPS according to GP1, GP2, GP3, and GP4. Briefly, Ahx was immobilized on a 2-CTC resin, Fmoc was deprotected, and it was coupled with Fmoc-D-dap(Dde)-OH. The side chains were deprotected using imidazole and hydroxylamine, and then coupled with the desired chelator, i.e., DOTA(tBu)3, R-DOTAGA(tBu)4, or N4(Boc)4. Subsequent Fmoc deprotection opened up the possibility of further derivatization. The Ahx-type linker structure was further derivatized. See 2.3.8.2 for details. 2.3.7 CPCR4-linker conjugate The CPCR4-linker conjugate was synthesized by fragment condensation between CPCR4 and each Fmoc-protected linker unit under GP8 execution. The reaction mixture was concentrated under vacuum and then Fmoc-deprotected by dissolution in 20 vol% piperidine in DMF. The desired product was obtained by subsequent semi-preparative RP-HPLC. CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2

[0269] [ka]

[0270] The synthesis of CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 was facilitated by fragment condensation according to GP8. CPCR4 (see 2.3.1) was condensed with the linker HO-Abz-ar(Pbf)-dap(Boc)-Fmoc (see 2.3.6), and the resulting peptide was deprotected with Fmoc. The crude product was purified by semi-preparative RP-HPLC. CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2:RP-HPLC (10-95% B at 15 mins):t R = 8.83 minutes. Monoisotopic mass calculation value (C 73 H 99 N 17 O 15 S): 1485.72, Measured value: 744.6 [M+2H] 2+ . CPCR4-Abz-a-cit-dap(Boc)-NH2

[0271] [ka]

[0272] The synthesis of CPCR4-Abz-a-cit-dap(Boc)-NH2 was carried out by fragment condensation according to GP8. CPCR4 (see 2.3.1) was condensed with the linker HO-Abz-a-cit-dap(Boc)-Fmoc (see 2.3.6), and the resulting peptide was deprotected with Fmoc. The crude product was purified by semi-preparative RP-HPLC. CPCR4-Abz-a-cit-dap(Boc)-NH2:RP-HPLC (10-95% B at 15 mins):t R = 7.03 minutes. Monoisotopic mass calculation value (C 60 H 82 N 16 O 13 ): 1234.62, Measured value: 618.2 [M+2H] 2+ . CPCR4-Ambz-ar(Pbf)-dap(Boc)-NH2

[0273] [ka]

[0274] CPCR4-Ambz-ar(Pbf)-dap(Boc)-NH2 was synthesized in the same manner as CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2, by condensation of CPCR4 (see 2.3.1) with the linker HO-Ambz-ar(Pbf)-dap(Boc)-Fmoc (see 2.3.6), followed by Fmoc deprotection and purification. CPCR4-Ambz-ar(Pbf)-dap(Boc)-NH2:RP-HPLC (10-90% B at 15 mins):t R = 8.97 minutes. Monoisotopic mass calculation value (C 74 H 101 N 17 O 15 S): 1499.74, Measured value: 750.2 [M+2H] 2+ . CPCR4-Ambz-ar(Pbf)-NH2

[0275] [ka]

[0276] The synthesis of CPCR4-Ambz-ar(Pbf)-NH2 was achieved by condensation of CPCR4 (see 2.3.1) with the linker HO-Ambz-ar(Pbf)-Fmoc (see 2.3.6) (GP8). The desired product was obtained by Fmoc deprotection and purification by semi-preparative RP-HPLC. CPCR4-Ambz-ar(Pbf)-NH2:RP-HPLC (10-90% B at 15 mins): R = 7.94 minutes. Monoisotopic mass calculation value (C 86 H 87 N 15 O 12 S): 1313.64, Measured value: 658.2 [M+2H] 2+ . CPCR4-Ambz-ar(Pbf)-h(Trt)-NH2

[0277] [ka]

[0278] CPCR4-Ambz-ar(Pbf)-NH2 was synthesized by condensation of CPCR4 (see 2.3.1) with the linker HO-Ambz-ar(Pbf)-h(Trt)-Fmoc (see 2.3.6) (GP8). The desired product was obtained by Fmoc deprotection and purification by semi-preparative RP-HPLC. CPCR4-Ambz-ar(Pbf)-h(Trt)-NH2:RP-HPLC (10-90% B at 15 mins):t R = 13.43 minutes. Monoisotopic mass calculation value (C 91 H 108 N 18 O 13 S): 1692.81, Measured value: 848.3 [M+2H] 2+ . 2.3.8 Synthesis of Cherators 2.3.8.1 Chelaters derived from modified mas3 Multiple chelators derived from mas3 were all synthesized according to standard Fmoc peptide synthesis strategies and GP1, 2, 3, and 9.

[0279] [ka]

[0280] The resulting chelator was used for either fragment condensation with CXCR4-binding peptides or for prior deacetylation. HO-(s(tBu))3-TMAA

[0281] [ka]

[0282] HO-(s(tBu))3-TMAA:HPLC (10-90% B in 15 minutes):t R = 14.85 minutes. Monoisotopic mass calculation value (C 42 H 57 N3O8S): 763.39, Measured value: 764.7 [M+H]+ . HO-(Hcy(Gluc(OAc)4))3-TMAA

[0283]

change

[0284] HO-(Hcy(Gluc(OAc)4))3-TMAA:HPLC(15min.10~95%B):t R =12.97 points.モノアイソトピック mass calculation value (C 75 H 93 N3O 32 S4):1675.46, measured value:1677.2[M+H] + . HO-s(tBu)-(Hcy(Gluc(OAc)4))2-TMAA

[0285]

change

[0286] HO-s(tBu)-(Hcy(Gluc(OAc)4))2-TMAA:HPLC(15min.10~95%B):t R =13.0 points.モノアイソトピック mass calculation value (C 64 H 81 N3O 24 S3):1371.44, measured value:1372.3[M+H] + . HO-cit-Hcy(Lac(OAc)7)-cit-TMAA

[0287]

change

[0288] HO-cit-Hcy(Lac(OAc)7)-cit-TMAA:HPLC(15min.10~90%B):t R =7.97 points.モノアイソトピック mass calculation value (C 63 H 81 N7O24 S2): 1383.48, Measured value: 1141.1 [M-Trt+H] + . HO-s(tBu)-Hcy(Lac(OAc)7)-s(tBu)-TMAA

[0289] [ka]

[0290] HO-s(tBu)-Hcy(Lac(OAc)7)-s(tBu)-TMAA:HPLC(10~90%B in 15 minutes):t R = 8.47 minutes. Monoisotopic mass calculation value (C 65 H 85 N3O 24 S2): 1355.50, Measured value: 1001.8 [M-Trt, -2tBu+H] + . Deacetylation After cutting from the resin, the chelator was dissolved in MeOH (2 mL / 50 mg) and the pH was adjusted to 10-11 with KCN. After at least 4 hours at ambient temperature, the deacetylated chelator was precipitated in Et2O. HO-(Hcy(Gluc(OAc) 12 ))3-TMAA:HPLC (10-90% B in 15 minutes):t R = 12.97 minutes. Monoisotopic mass calculation value (C 75 H 93 N3O 32 S4): 1675.46, Measured value: 1677.2 [M+H] + . HO-(Hcy(Gluc))2-TMAA:HPLC (10~95%B in 15 minutes):t R = 8.70 minutes. Monoisotopic mass calculation value (C 48 H 65 N3O 16 S3): 1035.35, Measured value: 1036.3 [M+H] + . HO-cit-Hcy(Lac)-cit-TMAA:HPLC(10~95%B in 15 minutes):t R= 7.90 minutes. Monoisotopic mass calculation value (C 49 H 67 N7O 17 S2): 1089.40, Measured value: 1089.9 [M+H] + . HO-s(tBu)-Hcy(Lac)-s(tBu)-TMAA:HPLC(10~95%B in 15 minutes):t R = 11.59 minutes. Monoisotopic mass calculation value (C 51 H 71 N3O 17 S2): 1061.42, Measured value: 1061.8 [M+H] + . HO-a-(s(tBu))3-TMAA

[0291] [ka]

[0292] HO-a-(s(tBu))3-TMAA:RP-HPLC (40-100% B in 15 minutes):t R = 16.54 minutes. Monoisotopic mass calculation value (C 45 H 62 N4O9S): 834.42, Measured value: 835.2 [M+H] + . HO-dap(Boc)-(s(tBu))3-TMAA

[0293] [ka]

[0294] HO-dap(Boc)-(s(tBu))3-TMAA:RP-HPLC (40-100% B in 15 minutes):t R = 17.60 minutes. Monoisotopic mass calculation value (C 50 H 71 N5O 11 S): 949.49, Measured value: 950.6 [M+H] + . 2.3.8.2 Linker-Chelator-Construction Linker-chelator constructs were prepared using GP1, GP2, GP3, and GP9 via linear synthesis on resin. HO-Abz-ar(Pbf)-r(Pbf)-(s(tBu))3-TMAA

[0295] [ka]

[0296] HO-Abz-ar(Pbf)-r(Pbf)-(s(tBu))3-TMAA:RP-HPLC (10-90% B in 15 minutes):t R = 18.41 minutes. Monoisotopic mass calculation value (C 90 H 123 N 13 O 18 S3): 1769.83, Measured value: 1771.2 [M+H] + . HO-Abz-ar(Pbf)-f-(s(tBu))3-TMAA

[0297] [ka]

[0298] HO-Abz-ar(Pbf)-f-(s(tBu))3-TMAA:RP-HPLC (10~90%B in 15 minutes):t R = 17.44 minutes. Monoisotopic mass calculation value (C 80 H 104 N 10 O 15 S2): 1508.71, Measured value: 1509.8 [M+H] + . HO-Abz-ar(Pbf)-h(Trt)-(s(tBu))3-TMAA

[0299] [ka]

[0300] HO-Abz-ar(Pbf)-h(Trt)-(s(tBu))3-TMAA:RP-HPLC(10~90%B in 15 minutes):t R = 17.80 minutes. Monoisotopic mass calculation value (C 96 H 116 N 12 O 15 S2): 1740.81, Measured value: 1742.3 [M+H] + . (tBu)e(HO-Ahx-dap(N4(Boc)4)ue(tBu)2

[0301] [ka]

[0302] (tBu)e(HO-Ahx-dap(N4(Boc)4)ue(tBu)2:RP-HPLC (10-95% B in 15 minutes):t R = 16.57 minutes. Monoisotopic mass calculation value (C 60 H 107 N9O 20 ): 1273.76, Measured value: 1274.6 [M+H] + . (tBu)e(HO-Ahx-dap(R-DOTAGA(tBu)4-dap(SiFa-BA)ue(tBu)2

[0303] [ka]

[0304] (tBu)e(HO-Ahx-dap(R-DOTAGA(tBu)4-dap(SiFa-BA)ue(tBu)2:RP-HPLC(10~95%B in 15 minutes):t R = 15.62 minutes. Monoisotopic mass calculation value (C 85 H 146 FN 11 O 22 Si): 1720.04, Measured value: 1721.3 [M+H] + , 861.4[M+2H] 2+ . HO-Ahx-dap(R-DOTAGA(tBu)4)-SiFalin

[0305] [ka]

[0306] HO-Ahx-dap(R-DOTAGA(tBu)4)-SiFalin:RP-HPLC(10~95%B in 15 minutes):t R = 11.64 minutes. Monoisotopic mass calculation value (C 63 H 112 FN8O 12 Si + ): 1235.81, Measured value: 1235.9 [M+H] + . HO-Ahx-dap(DOTA(tBu)3)-D-HCy(lactosyl)-SiFalin

[0307] [ka]

[0308] HO-Ahx-dap(DOTA(tBu)3)-D-HCy(lactosyl)-SiFalin:RP-HPLC (10-95% B at 15 mins):t R = 9.55 minutes. Monoisotopic mass calculation value (C 72 H 127 FN9O 22 SSi + ): 1548.86, Measured value: 1550.3 [M+H] + , 775.3[M+2H] 2+ . HO-Ahx-dap(R-DOTAGA(tBu)4)-D-HCy(Trt)-Ac

[0309] [ka]

[0310] HO-Ahx-dap(R-DOTAGA(tBu)4)-D-HCy(Trt)-Ac: RP-HPLC (10 - 95% B in 15 min): t R = 12.49 min. Calculated monoisotopic mass for (C 69 H 104 N8O 14 S): 1300.74, found: 1301.5 [M + H] + . HO-Ahx-dap(DOTA(tBu)3)-Fmoc

[0311] [[ID=I5]] [Chem.]<000342I>

[0312] HO-Ahx-dap(DOTA(tBu)4)-Fmoc: RP-HPLC (10 - 95% B in 15 min): t R = 16.66 min. Calculated monoisotopic mass for (C 52 H 79 N7O 12 S): %93.%8, found: 938.7 [M - tBu + H] + . 2.3.8.3 HO-HYNIC(BOC)

[0313] [Chem.]

[0314] The synthesis of HO-HYNIC(Boc) was achieved according to the published procedure (45, 46). Briefly, hydrazino-nicotinic acid was reacted overnight with Boc2O (1.00 eq) and triethylamine (1.30 eq) in DMF. The solvent was evaporated under reduced pressure and the crude product was subjected to silica flash chromatography using EtOAc, then EtOAc + 1 vol% AcOH. After evaporation of the solvent, the desired product was obtained as a white powder. ESI-MS: Calculated monoisotopic mass for (C 11 H 15 N3O4): 253.11, found: 254.4 [M + H] + . 2.3.8.4 3-((tert-butoxycarbonyl)(2-((tert-butoxycarbonyl)amino)ethyl)amino)-2-(((tert-butoxycarbonyl)(2-((tert-butoxycarbonyl)amino)ethyl)amino)methyl)propanoic acid (=N4 chelator

[11] )

[0315] [ka]

[0316] 4.00 equivalents of tert-butyl-(2-aminoethyl)-carbamate were slowly added to 1.00 equivalent of 3-bromo-2-(bromomethyl)propanoic acid in 25 mL / mmol of THF while vigorously stirring.

[0317] [ka]

[0318] The mixture was stirred at ambient temperature for 4 hours, and then the solvent was removed under reduced pressure at room temperature. The crude product

[10] was dissolved in acetone:H2O (1:1, 25 mL / mmol), cooled to 0°C, and NEt3 (3.00 equivalents) was added. After pre-activation for 5 minutes, Boc2O (4.00 equivalents) was added. The mixture was stirred for 15 hours (0°C to room temperature), the solvent was removed under reduced pressure, and the crude product was purified by flash chromatography (35-95% MeCN in H2O, 15 minutes). R f (alkyl + 0.5% AcOH) = 0.65. MS (ESI, cation): C 28 H 52 N4O 10 Calculated monoisotopic mass: 604.37; measured value by ESI-MS: m / z = 605.0 [M+H] + . 1H-NMR (400 MHz, DMSO-d6) δ = 7.17-6.19 (m, 2H, NH), 3.28-3.17 (m, 6H, CH2), 3.10-2.95 (m, 6H, CH2), 2.94- 2.90 (m, 1H, CH), 1.38 (s, 18H, CH3), 1.36 (s, 18H, CH3). 3. Synthesized peptides The majority of peptides in this study were synthesized by fragment condensation. The CPCR4 binding motif, as well as the linker unit in most cases (see 2.3.6), were synthesized and purified separately (see 2.3.1). These fragments were condensed according to GP8, the Fmoc was deprotected by dissolution in 20% piperidine / DMF (v / v), and purified by RP-HPLC. The resulting CPCR4-linker constructs were used as starting materials for derivatization with the corresponding chelator or functional group such as the labeling moiety. The resulting peptides are listed below along with their respective analytical data.

[0319] [ka]

[0320] 3.1 Technetium-99M SPECT Tracer 3.1.1 mas3 conjugate peptide Tc-CXCR4-1

[0321] [ka]

[0322] The synthesis of Tc-CXCR4-1 was facilitated by fragment condensation according to GP8. CPCR4 (see 2.3.1) was condensed with the linker HO-Abz-ar(Pbf)-Fmoc (see 2.3.6), the resulting peptide was deprotected with Fmoc, and then further coupled with HO-(s(tBu))3-TMAA (see 2.3.8.1). The crude product was deprotected (see GP5) and subjected to semi-preparative RP-HPLC purification. Tc-CXCR4-1:RP-HPLC (10-90% B in 15 minutes):t R = 9.82 minutes. Monoisotopic mass calculation value (C 63 H 86 N 18 O 16 S): 1382.62, Measured value: 692.2 [M+2H] 2+ . Tc-CXCR4-2

[0323] [ka]

[0324] Tc-CXCR4-2 was synthesized in the same manner as Tc-CXCR4-1. CPCR4 (see 2.3.1) was condensed with the linker HO-Abz-ar(Pbf)-Fmoc (see 2.3.6), the resulting peptide was deprotected with Fmoc, and then further coupled with HO-a-(s(tBu))3-TMAA (see 2.3.8.1). The crude product was deprotected (see GP5) and subjected to semi-preparative RP-HPLC purification. Tc-CXCR4-2:RP-HPLC (15-45% B in 15 minutes):t R = 9.10 minutes. Monoisotopic mass calculation value (C 66 H 91 N 19 O 17 S): 1453.66, Measured value: 728.6 [M+2H] 2+ . Tc-CXCR4-3

[0325] [ka]

[0326] Tc-CXCR4-3 was synthesized by coupling CPCR4 (see 2.3.1) with the linker-chelator construct HO-Abz-ar(Pbf)-r(Pbf)-(s(tBu))3-TMAA (see 2.3.8.2). The crude product was deprotected (see GP5) and subjected to semi-preparative RP-HPLC purification. Tc-CXCR4-3:RP-HPLC (10-90% B in 15 minutes):t R = 9.00 minutes. Monoisotopic mass calculation value (C 69 H 98 N 22 O 17 S): 1538.72, Measured value: 770.4 [M+2H] 2+ . Tc-CXCR4-4

[0327] [ka]

[0328] Tc-CXCR4-4 was synthesized in the same manner as Tc-CXCR4-3 by coupling CPCR4 (see 2.3.1) with the linker-chelator construct HO-Abz-ar(Pbf)-f-(s(tBu))3-TMAA (see 2.3.8.2). The crude product was deprotected (see GP5) and subjected to semi-preparative RP-HPLC purification. Tc-CXCR4-4:RP-HPLC (10-90% B in 15 minutes):t R = 10.20 minutes. Monoisotopic mass calculation value (C 72 H 95 N 19 O 17 S): 1529.69, Measured value: 766.1 [M+2H] 2+ . Tc-CXCR4-5

[0329] [ka]

[0330] Tc-CXCR4-5 was synthesized in the same manner as Tc-CXCR4-3 and -4 by coupling CPCR4 (see 2.3.1) with the linker-chelator construct HO-Abz-ar(Pbf)-h(Trt)-(s(tBu))3-TMAA (see 2.3.8.2). The crude product was deprotected (see GP5) and subjected to semi-preparative RP-HPLC purification. Tc-CXCR4-5:RP-HPLC (10-90% B in 15 minutes):t R = 9.53 minutes. Monoisotopic mass calculation value (C 69 H 93 N 21 O 17 S): 1519.68, Measured value: 761.2 [M+2H] 2+ . Tc-CXCR4-6

[0331] [ka]

[0332] The synthesis of Tc-CXCR4-6 was carried out in the same manner as for Tc-CXCR4-1. CPCR4 (see 2.3.1) was condensed with the linker HO-Abz-ar(Pbf)-Fmoc (see 2.3.6), the resulting peptide was deprotected with Fmoc, and then further coupled with HO-dap(Boc)-(s(tBu))3-TMAA (see 2.3.8.1). The crude product was deprotected (see GP5) and subjected to semi-preparative RP-HPLC purification. Tc-CXCR4-6:RP-HPLC (35-65% B in 15 minutes):t R = 8.85 minutes. Monoisotopic mass calculation value (C 66 H 92 N 20 O 17 S): 1468.67, Measured value: 735.1 [M+2H] 2+ . Tc-CXCR4-7

[0333] [ka]

[0334] The synthesis was carried out by fragment condensation (GP8) of the CPCR4-linker construct CPCR4-Ambz-ar(Pbf)-his(Trt)-NH2 (see 2.3.7) and the chelator HO-(s(tBu))3-TMAA (see 2.3.8.1). The desired product was obtained by subsequent deprotection of the peptide (GP5) and purification by semi-preparative RP-HPLC. Tc-CXCR4-8

[0335] [ka]

[0336] The synthesis was carried out by fragment condensation (GP8) of the CPCR4-linker construct CPCR4-Ambz-ar(Pbf)-NH2 (see 2.3.7) and the modified chelator HO-a-(s(tBu))3-TMAA (see 2.3.8.1). The desired product was obtained by subsequent deprotection of the peptide (GP5) and purification by semi-preparative RP-HPLC. Tc-CXCR4-8:RP-HPLC (20-60% B in 15 minutes):t R = 7.26 minutes. Monoisotopic mass calculation value (C 67 H 94 N 20 O 17 S): 1482.68, Measured value: 742.4 [M+2H] 2+ . 3.1.2 Modified MAS3 Conjugate Peptides Tc-CXCR4-9

[0337] [ka]

[0338] Tc-CXCR4-9 was synthesized by fragment condensation (see GP8). The CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7) was reacted with the protected chelator HO-(Hcy(Gluc(OAc)4))3-TMAA (see 2.3.8.1). The desired crude product was obtained by deacetylation and removal of the subsequent acid-unstable protecting group as described (see GP5), and this was purified by semi-preparative RP-HPLC. Tc-CXCR4-9:RP-HPLC (5-55% B in 15 minutes):t R = 9.51 minutes. Monoisotopic mass calculation value (C 87 H 128 N 20 O 29 S4): 2044.80, Measured value: 1023.7 [M+2H] 2+ . Tc-CXCR4-10

[0339] [ka]

[0340] Tc-CXCR4-10 was synthesized in the same manner as Tc-CXCR4-9, but the difference was that the chelator HO-s(tBu)-(Hcy(Gluc))2-TMAA (see 2.3.8.1) was deacetylated and then coupled with the CPCR4-linker construct. The crude product was completely deprotected (see GP5) and purified by semi-preparative RP-HPLC. Tc-CXCR4-10:RP-HPLC (10-60% B in 15 minutes):t R = 9.00 minutes. Monoisotopic mass calculation value (C 80 H 116 N 20 O 25 S3): 1852.76, Measured value: 927.4 [M+2H] 2+ , 618.7[M+3H] 3+ . Tc-CXCR4-11

[0341] [Chemical formula]

[0342] Tc-CXCR4-11 was synthesized in the same manner as Tc-CXCR4-10 using the deacetylated chelator HO-s(tBu)-Hcy(Lac)-s(tBu)-TMAA (see 2.3.8.1). The crude product was fully deprotected (see GP5) and purified by semi-preparative RP-HPLC. Tc-CXCR4-11: RP-HPLC (10 - 60% B in 15 minutes): t R = 7.90 minutes. Calculated monoisotopic mass (C 79 H 114 N 20 O 26 S2): 1822.77, found: 920.4 [M + H2O + 2H] 2+ , 614.1 [M + H2O + 3H] 3+ . Tc-CXCR4-12

[0343] [Chemical formula]

[0344] Tc-CXCR4-12 was synthesized in the same manner as Tc-CXCR4-10 and -11 using the deacetylated chelator HO-cit-Hcy(Lac)-cit-TMAA (see 2.3.8.1). The crude product was fully deprotected (see GP5) and purified by semi-preparative RP-HPLC. Tc-CXCR4-12: RP-HPLC (10 - 60% B in 15 minutes): t R = 8.00 minutes. Calculated monoisotopic mass (C 85 H 126 N 24 O 26 S2): 1962.87, found: 982.6 [M + 2H] 2+ , 655.5 [M + 3H] 3+ . 3.1.3 Immobilized Chelator Peptides Tc-CXCR4-13

[0345] [ka]

[0346] The synthesis of Tc-CXCR4-13 was carried out according to GP8 by coupling HO-HYNIC(Boc) (see 2.3.8.3) to the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7), followed by deprotection with GP5 and purification by semi-preparative RP-HPLC. Tc-CXCR4-13:RP-HPLC (5-55% B in 15 minutes):t R = 7.88 minutes. Monoisotopic mass calculation value (C 61 H 80 N 20 O 11 ): 1268.63, Measured value: 635.6 [M+2H] 2+ . Tc-CXCR4-14

[0347] [ka]

[0348] Tc-CXCR4-14 was synthesized in the same manner as Tc-CXCR4-13. According to GP8, HO-N4(Boc)4 (see 2.3.8.4) was coupled to the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7), followed by deprotection with GP5 and purification by semi-preparative RP-HPLC. Tc-CXCR4-14:RP-HPLC (10-60% B in 15 minutes):t R = 8.03 minutes. Monoisotopic mass calculation value (C 63 H 93 N 21 O 11 ): 1319.74, Measured value: 660.7 [M+2H] 2+ , 441.0[M+3H] 3+ . Tc-CXCR4-15

[0349] [ka]

[0350] Tc-CXCR4-15 was synthesized in the same manner as Tc-CXCR4-13 and -14. According to GP8, the scaffold (tBu)e(HO-dap(N4(Boc)4))ue(tBu)2 with a chelator was coupled to the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7), then deprotected with GP5 and purified by semi-preparative RP-HPLC.

[0351] [ka]

[0352] (tBu)e(HO-dap(N4(Boc)4))ue(tBu)2:RP-HPLC (10-95% B in 15 minutes):t R = 16.75 minutes. Monoisotopic mass calculation value (C 54 H 96 N8O 19 ): 1160.68, Measured value: 1162.2 [M+H] + , 531[M-Boc+2H] 2+ . Tc-CXCR4-15:RP-HPLC (10-60% B in 15 minutes):t R = 8.03 minutes. Monoisotopic mass calculation value (C 77 H 113 N 25 O 20 ): 1707.86, Measured value: 855.5 [M+2H] 2+ , 570.6[M+3H] 3+ , 427.1[M+4H] 4+ . Tc-CXCR4-16

[0353] [ka]

[0354] Tc-CXCR4-16 was synthesized in the same manner as Tc-CXCR4-15. According to GP8, the chelator-containing Ahx-system scaffold (tBu)e(HO-Ahx-dap(N4(Boc)4))ue(tBu)2 (see 0) was coupled to the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7), then deprotected with GP5 and purified by semi-preparative RP-HPLC. Tc-CXCR4-16:RP-HPLC (10-60% B in 15 minutes):t R = 7.83 minutes. Monoisotopic mass calculation value (C 83 H 124 N 26 O 21 ): 1820.94, Measured value: 1821.5 [M+H] + , 910.8[M+2H] 2+ , 607.4[M+3H] 3+ . 3.2 DOTA Conjugate Peptides Conjugate of DOTA to scaffolds having either Ambz or Abz (see 2.3.7) was carried out according to a recently published procedure (47). Briefly, DOTA (4.00 equivalents) was pre-activated in H2O for 30 minutes with NHS (5.00 equivalents), EDCI (5.00 equivalents), and 2,4,6-collidine (6.00 equivalents). The Fmoc-deprotected peptide was dissolved in DMF (30 μL / mg) and added to the mixture, which was stirred at room temperature for 2–4 hours. The solvent was evaporated under vacuum, and the crude product was treated with TFA for 1–2 hours (see GP5). After removing TFA in a stream of N2, the product was purified by semi-preparative RP-HPLC.

[0355] Iodination of the binding motif containing tyr was achieved using purified and completely deprotected peptides as a substrate, following the general procedure GP7. CXCR4-DOTA-1

[0356] [ka]

[0357] The synthesis of CXCR4-DOTA-1 was carried out by applying general synthetic procedures GP1, GP2, and GP3 to obtain CPCR4-Ambz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7). DOTA was coupled as described above, and the peptide was deprotected according to GP5. CXCR4-DOTA-1:RP-HPLC (10-60% B in 15 minutes):t R = 7.97 minutes. Monoisotopic mass calculation value (C 72 H 103 N 21 O 17 ): 1533.78, Measured value: 768.6 [M+2H] 2+ , 512.6[M+3H] 3+ . [ nat Ga]CXCR4-DOTA-1:RP-HPLC (10-60% B at 15 mins):t R = 7.97 minutes. Monoisotopic mass calculation value (C 72 H 101 GaN 21 O 17 ): 1600.69, Measured value: 801.0 [M+2H] 2+ , 534.8 [M+3H] 3+ . [ nat Lu]CXCR4-DOTA-1:RP-HPLC (10-60% B at 15 mins):t R = 7.99 minutes. Monoisotopic mass calculation value (C 72 H 100 LuN 21 O 17 ): 1705.70, Measured value: 570.1 [M+3H] 3+ . CXCR4-DOTA-2

[0358] [ka]

[0359] CXCR4-DOTA-2 was synthesized by iodination with GP7 using completely deprotected and purified CXCR4-DOTA-1 as the substrate. The desired product was isolated by semi-preparative RP-HPLC. CXCR4-DOTA-2:RP-HPLC (10-60% B in 15 minutes):t R = 8.85 minutes. Monoisotopic mass calculation value (C 72 H 102 IN 21 O 17 ): 1659.68, Measured value: 1660.6 [M+H] + , 830.7[M+2H] 2+ , 553.9 [M+3H] 3+ . [ nat Ga]CXCR4-DOTA-2:RP-HPLC (10-60% B at 15 mins):t R = 8.73 minutes. Monoisotopic mass calculation value (C 72 H 100 Gain 21 O 17 ): 1726.59, Measured value: 863.9 [M+2H] 2+ , 576.6 [M+3H] 3+ . [ nat Lu]CXCR4-DOTA-2:RP-HPLC (10-60% B at 15 mins):t R = 8.80 minutes. Monoisotopic mass calculation value (C 72 H 99 ILuN 21 O 17 ): 1831.60, Measured value: 916.8 [M+2H] 2+ , 611.6[M+3H] 3+ . CXCR4-DOTA-3

[0360] [ka]

[0361] CXCR4-DOTA-3 was synthesized in the same way as CXCR4-DOTA-1, but the difference was the linker used. In this case, HO-Abz-ar-(Pbf)-dap(Boc)-Fmoc (see 2.3.6) was coupled to CPCR4, then Fmoc was deprotected, purified, and DOTA coupling was performed. CXCR4-DOTA-3:RP-HPLC (20-75% B in 15 minutes):t R = 5.36 minutes. Monoisotopic mass calculation value (C 71 H 101 N 21 O 17 ): 1519.77, Measured value: 1521.0 [M+H] + , 760.8[M+2H] 2+ , 507.9 [M+3H] 3+ . [ nat Ga]CXCR4-DOTA-3:RP-HPLC (20-75% B at 15 mins):t R = 6.65 minutes. Monoisotopic mass calculation value (C 71 H 99 GaN 21 O 17 ): 1586.68, Measured value: 1569.5 [M+H] + , 795.0[M+2H] 2+ , 529.6 [M+3H] 3+ . [ nat Lu]CXCR4-DOTA-3:RP-HPLC (20-75% B at 15 mins):t R = 6.62 minutes. Monoisotopic mass calculation value (C 71 H 98 LuN 21 O 17 ): 1691.69, Measured value: 1691.9 [M+H] + , 846.8 [M+2H] 2+ , 565.1[M+3H] 3+ . CXCR4-DOTA-4

[0362] [ka]

[0363] The synthesis of CXCR4-DOTA-4 was achieved by iodizing completely deprotected and purified CXCR4-DOTA-3 according to GP7. The desired product was isolated by semi-preparative RP-HPLC. CXCR4-DOTA-4:RP-HPLC (20-80% B in 15 minutes):t R = 5.72 minutes. Monoisotopic mass calculation value (C 71 H 100 IN 21 O 17 ): 1645.67, Measured value: 1647.2 [M+H] + , 824.4[M+2H] 2+ , 550.0[M+3H] 3+ . [ nat Ga]CXCR4-DOTA-4:RP-HPLC (20-80% B at 15 mins):t R = 5.52 minutes. Monoisotopic mass calculation value (C 71 H 98 Gain 21 O 17 ): 1712.58, Measured value: 857.1 [M+2H] 2+ , 572.2[M+3H] 3+ . [ nat Lu]CXCR4-DOTA-4:RP-HPLC (20-80% B at 15 mins):t R = 5.55 minutes. Monoisotopic mass calculation value (C 71 H 97 ILuN 21 O 17 ): 1817.58, Measured value: 910.2 [M+2H] 2+ , 607.1[M+3H] 3+ . CXCR4-DOTA-5

[0364] [ka]

[0365] The synthesis of CXCR4-DOTA-5 was carried out in the same manner as CXCR4-DOTA-3, the only difference being the CPCR4-linker construct CPCR4-Abz-a-cit-dap(Boc)-NH2 (see 2.3.7). After Fmoc deprotection, DOTA was coupled as described above, the resulting peptide was deprotected (GP5), and then purified by semi-preparative RP-HPLC. CXCR4-DOTA-5:RP-HPLC (10-60% B in 15 minutes):t R = 8.40 minutes. Monoisotopic mass calculation value (C 71 H 100 N 20 O 18 ): 1520.75, Measured value: 1522.0 [M+H] + , 761.6[M+2H] 2+ . [ nat Ga]CXCR4-DOTA-5:RP-HPLC (10-60% B at 15 mins):t R = 8.33 minutes. Monoisotopic mass calculation value (C 71 H 98 N 20 GaO 18 ): 1587.66, Measured value: 794.6 [M+2H] 2+ , 529.8 [M+3H] 3+ . [ nat Lu]CXCR4-DOTA-5:RP-HPLC (10-60% B in 15 minutes):t R = 8.42 minutes. Monoisotopic mass calculation value (C 71 H 97 LuN 20 O 18 ): 1692.67, Measured value: 847.2 [M+2H] 2+ , 565.1[M+3H] 3+ . Penchixafol

[0366] [ka]

[0367] Penthixafor was synthesized according to the previously published literature (48). Briefly, HO-Ambz-Fmoc was coupled to CPCR4 (GP8), and the product was deprotected with Fmoc. After purification by semi-preparative RP-HPLC, DOTA was coupled as described above (47) according to the literature. The resulting peptide was deprotected (GP5) and purified by semi-preparative RP-HPLC. Penthixafor: RP-HPLC (15-45% B in 15 minutes): t R = 14.96 minutes. Monoisotopic mass calculation value (C 60 H 80 N 14 O 14 ): 1220.60, Measured value: 1220.8 [M+H] + . [ nat [Ga] Pentixafol: RP-HPLC (10-60% B in 15 minutes): t R = 8.73 minutes. Monoisotopic mass calculation value (C 60 H 78 GaN 14 O 14 ): 1287.51, Measured value: 863.9 [M+2H] 2+ , 576.6 [M+3H] 3+ . Penchixatel

[0368] [ka]

[0369] Pentixatel was obtained by iodination of pentixaphor using a completely deprotected and purified peptide as a substrate, according to reference (39) and GP7. The resulting mixture was subjected to semi-preparative RP-HPLC. Penticosatel: RP-HPLC (15-45% B in 15 minutes): t R = 10.60 minutes. Monoisotopic mass calculation value (C 60 H 79 IN 14 O 14 ): 1346.59, Measured value: 1347.7 [M+H] + , 676.2[M+2H]2+ . [ nat Lu] Penticosatel: RP-HPLC (10-60% B in 15 minutes): t R = 9.34 minutes. Monoisotopic mass calculation value (C 60 H 79 ILuN 14 O 14 ): 1518.41, Measured value: 1519.2 [M+H] + , 760.4[M+2H] 2+ . 3.3 Fluorine-18PET Tracer / Radiation Hybrid CXCR4-SiFA-1

[0370] [ka]

[0371] CXCR4-SiFA-1 was synthesized by fragment condensation (see GP8) of CPCR4 with the SiFA-containing linker HO-Abz-ar(Pbf)-dap(SiFA-BA)-Fmoc. The desired product was obtained by subsequent Fmoc deprotection and coupling with DOTA-GA anhydride, followed by deprotection and purification by semi-preparative RP-HPLC.

[0372] [ka]

[0373] [ka]

[0374] HO-Abz-ar-(Pbf)-dap(SiFA-BA)-Fmoc:RP-HPLC (50-95% B at 15 min):t R = 17.70 minutes. Monoisotopic mass calculation value (C 62 H 77 FN8O 11 SSi): 1188.52, Measured value: 1189.3 [M+H] +. [ nat Ga]CXCR4-SiFA-1:RP-HPLC (10-90% B at 15 mins):t R = 8.98 minutes. Monoisotopic mass calculation value (C 89 H 124 FGaN 21 O 20 Si): 1922.83, Measured value: 1924.0 [M+H] + , 962.9[M+2H] 2+ . [ nat Lu]CXCR4-SiFA-1:RP-HPLC (10-90% B at 15 mins):t R = 9.77 minutes. Monoisotopic mass calculation value (C 89 H 124 FLuN 21 O 20 Si): 2028.85, Measured value: 1015.1 [M+2H] 2+ . CXCR4-SiFA-2

[0375] [ka]

[0376] CXCR4-SiFA-2 was synthesized in the same manner as CXCR4-SiFA-1. The Abz-based linker unit was extended with Fmoc-h(Trt)-OH, and then extended with Fmoc-dap(Dde)-OH. Subsequent Dde deprotection, SiFa-BA coupling, and resin cleavage yielded a linker containing SiFa. Coupling with CPCR4, Fmoc deprotection, DOTA-GA linking, and overall deprotection were carried out similarly.

[0377] [ka]

[0378] HO-Abz-ar-(Pbf)-h(Trt)-dap(SiFA-BA)-Fmoc:RP-HPLC (70-95% B at 15 min):t R= 19.04 minutes. Monoisotopic mass calculation value (C 87 H 98 FN 11 O 12 SSi): 1567.69, Measured value: 1568.9 [M+H] + . CXCR4-SiFA-2:RP-HPLC (10-90% B in 15 minutes):t R = 8.43 minutes. Monoisotopic mass calculation value (C 95 H 133 FN 24 O 21 Si): 1992.98, Measured value: 997.2 [M+2H] 2+ , 665.7[M+3H] 3+ . [ nat Ga]CXCR4-SiFA-2:RP-HPLC (10-90% B at 15 mins):t R = 5.40 minutes. Monoisotopic mass calculation value (C 95 H 131 FGaN 24 O 21 Si): 2059.89, Measured value: 1031.2 [M+2H] 2+ , 688.8[M+3H] 3+ . [ nat Lu]CXCR4-SiFA-2:RP-HPLC (10-90% B at 15 mins):t R = 5.89 minutes. Monoisotopic mass calculation value (C 95 H 130 FLuN 24 O 21 Si): 2164.90, Measured value: 1084.8 [M+2H] 2+ , 723.1[M+3H] 3+ . CXCR4-SiFA-3

[0379] [ka]

[0380] CXCR4-SiFA-3 was synthesized by condensation of the SiFA-containing fragment HO-dap(SiFA-BA)-k((R)-DOTA-GA(tBu)4)-(R)-DOTA-GA(tBu)4 with the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7, GP8). The resulting peptide was deprotected for 12 hours (GP5) and purified by semi-preparative RP-HPLC.

[0381] [ka]

[0382] HO-dap(SiFA-BA)-k((R)-DOTA-GA(tBu)4)-(R)-DOTA-GA(tBu)4:RP-HPLC(25~95%B in 15 min):t R = 12.32 minutes. Monoisotopic mass calculation value (C 94 H 165 FN 12 O 22 Si): 1861.19, Measured value: 931.2 [M+2H] 2+ . CXCR4-SiFA-3:RP-HPLC (20-60% B at 15 mins):t R = 8.77 minutes. Monoisotopic mass calculation value (C 117 H 174 FN 29 O 31 Si): 2528.27, Measured value: 844.5 [M+3H] 3+ , 633.4[M+4H] 4+ . [ nat Ga]CXCR4-SiFA-3:RP-HPLC (20-60% B at 15 mins):t R = 8.82 minutes. Monoisotopic mass calculation value (C 117 H 170 FGa2N 29 O 31 Si): 2662.09, Measured value: 1333.1 [M+2H] 2+ , 889.5[M+3H] 3+ . [nat Lu]CXCR4-SiFA-3:RP-HPLC (20-60% B at 15 mins):t R = 9.71 minutes. Monoisotopic mass calculation value (C 117 H 168 FLuN 29 O 31 Si): 2872.10, Measured value: 1438.5 [M+2H] 2+ , 958.7 [M+3H] 3+ . CXCR4-SiFA-4

[0383] [ka]

[0384] CXCR4-SiFA-4 was synthesized according to GP8 by linking the SiFA-containing moiety (tBu)e(HO-dap((R)-DOTAGA(tBu)4)-dap(SiFA-BA))ue(tBu)2 with CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7). The desired product was obtained by subsequent 12-hour deprotection (GP5) and semi-preparative RP-HPLC.

[0385] [ka]

[0386] (tBu)e(HO-dap((R)-DOTAGA(tBu)4)-dap(SiFA-BA))ue(tBu)2:RP-HPLC(10~95%B in 15 minutes):t R = 15.97 minutes. Monoisotopic mass calculation value (C 79 H 135 FN 10 O 21 Si): 1606.96, Measured value: 1609.1 [M+H] + , 805.4[M+2H] 2+ . CXCR4-SiFA-4:RP-HPLC (10-60% B in 15 minutes):t R= 11.70 minutes. Monoisotopic mass calculation value (C 106 H 152 FN 27 O 30 Si): 2330.10, Measured value: 1167.2 [M+2H] 2+ , 778.6[M+3H] 3+ , 584.3 [M+4H] 4+ . [ nat Ga]CXCR4-SiFA-4:RP-HPLC (10-60% B at 15 mins):t R = 12.32 minutes. Monoisotopic mass calculation value (C 106 H 150 FGaN 27 O 30 Si): 2397.01, Measured value: 1200.7 [M+2H] 2+ , 801.0[M+3H] 3+ . [ nat Lu]CXCR4-SiFA-4:RP-HPLC (20-60% B at 15 mins):t R = 11.72 minutes. Monoisotopic mass calculation value (C 106 H 149 FLuN 27 O 30 Si): 2502.01, Measured value: 835.7 [M+3H] 3+ . CXCR4-SiFA-5

[0387] [ka]

[0388] The synthesis of CXCR4-SiFA-5 was carried out in the same manner as for CXCR4-SiFA-4, the difference being the SiFA-containing moiety (tBu)e(HO-Ahx-dap((R)-DOTAGA(tBu)4)-dap(SiFA-BA))ue(tBu)2 (see 0) coupled with CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7). Subsequent 12 hours of deprotection (GP5) and semi-preparative RP-HPLC yielded the desired product.

[0389] [ka]

[0390] (tBu)e(HO-Ahx-dap((R)-DOTAGA(tBu)4)-dap(SiFA-BA))ue(tBu)2:RP-HPLC(10~95%B in 15 min):t R = 15.62 minutes. Monoisotopic mass calculation value (C 85 H 146 FN 11 O 22 Si): 1720.04, Measured value: 1721.3 [M+H] + , 861.6[M+2H] 2+ . CXCR4-SiFA-5:RP-HPLC (10-60% B in 15 minutes):t R = 11.70 minutes. Monoisotopic mass calculation value (C 112 H 163 FN 28 O 31 Si): 2443.18, Measured value: 1221.9 [M+2H] 2+ , 814.8[M+3H] 3+ , 611.3[M+4H] 4+ . [ nat Ga]CXCR4-SiFA-5:RP-HPLC (10-60% B at 15 mins):t R = 11.82 minutes. Monoisotopic mass calculation value (C 112 H 160 FGaN 28 O 31 Si): 2509.08, Measured value: 1255.3 [M+2H] 2+ , 837.0[M+3H] 3+ . [ nat Lu]CXCR4-SiFA-5:RP-HPLC (10-60% B at 15 mins):t R = 12.72 minutes. Monoisotopic mass calculation value (C 112 H 159 FLuN 28 O 31 Si): 2614.09, Measured value: 1307.8 [M+2H]2+ , 872.1[M+3H] 3+ . CXCR4-SiFA-6

[0391] [ka]

[0392] CXCR4-SiFA-6 was synthesized by fragment condensation of CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7) and HO-Ahx-dap((R)-DOTAGA(tBu)4)-SiFAlin (see 0) containing SiFA (GP8). SiFA-benzyl bromide was obtained during the synthesis of SiFA-BA synthon (see 2.3.4), and this was coupled overnight to the DMG N-terminus in DCM (3.00 equivalents of SiFA-benzyl bromide, 3.00 equivalents of DIPEA). The desired product was obtained by final deprotection of the peptide (GP5) and semi-preparative RP-HPLC purification.

[0393] [ka]

[0394] HO-Ahx-dap((R)-DOTAGA(tBu)4)-SiFAlin:RP-HPLC(10~95%B in 15 min):t R = 11.64 minutes. Monoisotopic mass calculation value (C 63 H 112 FN8O 13 Si + ): 1235.81, Measured value: 1235.9 [M+H] + . CXCR4-SiFA-6:RP-HPLC (10-60% B at 15 mins):t R = 11.70 minutes. Monoisotopic mass calculation value (C 102 H 153 FN 25 O 22 Si + ): 2127.14, Measured value: 1063.4 [M+2H] 2+, 709.2[M+3H] 3+ . [ nat Ga]CXCR4-SiFA-6:RP-HPLC (10-60% B at 15 mins):t R = 11.73 minutes. Monoisotopic mass calculation value (C 102 H 150 FGaN 25 O 22 Si + ): 2193.04, Measured value: 1096.7 [M+2H] 2+ , 731.3[M+3H] 3+ , 548.7 [M+4H] 4+ . [ nat Lu]CXCR4-SiFA-6:RP-HPLC (10-60% B at 15 mins):t R = 12.71 minutes. Monoisotopic mass calculation value (C 102 H 149 FLuN 25 O 22 Si + ): 2298.05, Measured value: 1149.3 [M+2H] 2+ , 766.4[M+3H] 3+ . CXCR4-SiFA-7

[0395] [ka]

[0396] CXCR4-SiFA-7 was synthesized in the same manner as CXCR4-SiFA-6 using HO-Ahx-dap(DOTA(tBu)3)-D-HCy(lactosyl)-SiFAlin (see 0) containing SiFA (GP8). Final deprotection of the peptide (GP5) and semi-preparative RP-HPLC purification yielded the desired product.

[0397] [ka]

[0398] HO-Ahx-dap(DOTA(tBu)3)-D-HCy(lactosyl)-SiFalin:RP-HPLC (10-95% B at 15 mins):t R = 9.55 minutes. Monoisotopic mass calculation value (C 72 H 127 FN9O 22 SSi + ): 1548.86, Measured value: 1550.3 [M+H] + , 775.3[M+2H] 2+ . CXCR4-SiFA-7:RP-HPLC (10-95% B in 15 minutes):t R = 7.61 minutes. Monoisotopic mass calculation value (C 102 H 153 FN 25 O 22 Si + ): 2496.25, Measured value: 838.8 [M + H2O + 3H] 3+ . [ nat Ga]CXCR4-SiFA-7:RP-HPLC (10-60% B at 15 mins):t R = 11.23 minutes. Monoisotopic mass calculation value (C 115 H 174 FGaN 26 O 31 SSi + ): 2563.16, Measured value: 861.5 [M + H2O + 3H] 3+ . [ nat Lu]CXCR4-SiFA-7:RP-HPLC (10-60% B at 15 mins):t R = 11.47 minutes. Monoisotopic mass calculation value (C 115 H 173 FLuN 26 O 31 SSi + ): 2668.16, Measured value: 896.3 [M + H2O + 3H] 3+ . 3.4 Peptides conjugated with cytotoxic substances CXCR4-MMAE-1

[0399] [ka]

[0400] The synthesis of the cysteine-containing binding scaffold HO-Abz-ar(Pbf)-c(Trt)-Fmoc (see 2.3.6) was carried out as described. Condensation of the CPCR4 scaffold (see 2.3.1) with the linker moiety, subsequent Fmoc deprotection, and deprotection of the acid-unstable protecting group were carried out using GP5 and GP8. Sulfhydryl-maleimide coupling was performed as described above (see GP10), and the resulting peptide was purified by semi-preparative RP-HPLC.

[0401] [ka]

[0402] CPCR4-Abz-arc-NH2:RP-HPLC:(10-90% B at 15 mins):t R = 9.80 minutes. Monoisotopic mass calculation value (C 55 H 74 N 16 O 10 S): 1150.55, Measured value: 1151.3 [M+H] + , 576.4[M+2H] 2+ . CXCR4-MMAE-1:RP-HPLC:(25-70% B in 15 minutes):t R = 9.31 minutes. Monoisotopic mass calculation value (C 123 H 179 N 27 O 25 S): 2466.33, Measured value: 1234.0 [M+2H] 2+ , 823.2[M+3H] 3+ . CXCR4-MMAE-2

[0403] [ka]

[0404] CXCR4-MMAE-2 was synthesized in the same manner as CXCR4-MMAE-1, except that the N-terminus was D-Hcy(Trt)-NH2 instead of c(Trt)-NH2 (see 2.3.6). The deprotected intermediate product CPCR4-Abz-ar-Hcy(SH)-NH2 was similarly coupled to VcMMAE (see GP10) and purified by semi-preparative RP-HPLC.

[0405] [ka]

[0406] CXCR4-MMAE-2:RP-HPLC:(25-70% B in 15 minutes):t R = 9.39 minutes. Monoisotopic mass calculation value (C 124 H 181 N 27 O 25 S): 2480.34, Measured value: 1241.0 [M+2H] 2+ , 827.8 [M+3H] 3+ . 125 I-CXCR4-MMAE-2:HPLC (0%B at 2 minutes, 0-35%B at 1 minute, 35-50%B at 15 minutes): R =14.19 minutes. CXCR4-MMAE-3

[0407] [ka]

[0408] CXCR4-MMAE-3 was synthesized in the same manner as CXCR4-MMAE-1 and -2. The linker HO-Abz-ar(Pbf)-h(Trt)-Fmoc (see 2.3.6) was coupled to CPCR4 (GP8), Fmoc was deprotected, and the peptide was coupled to Fmoc-D-Hcy(Trt)-OH. After complete deprotection, sulfhydryl-maleimide coupling was performed as described (see GP10), and the peptide was purified by semi-preparative RP-HPLC.

[0409] [ka]

[0410] CXCR4-MMAE-3:RP-HPLC (30-70% B in 15 minutes):t R = 5.93 minutes. Monoisotopic mass calculation value (C 130 H 188 N 30 O 26 S): 2617.40, Measured value: 1310.1 [M+2H] 2+ , 873.8[M+3H] 3+ . 125 I-CXCR4-MMAE-3:HPLC (0%B at 2 minutes, 0-35%B at 1 minute, 35-50%B at 15 minutes): R =15.18 minutes. CXCR4-MMAE-4

[0411] [ka]

[0412] CXCR4-MMAE-4 was synthesized by fragment condensation of the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7) and the Ahx-system chelator-containing moiety HO-Ahx-dap(R-DOTAGA(tBu)4)-D-HCy(Trt)-Ac (see 0). The peptide was deprotected overall, the damaging substance was coupled according to GP10, and the peptide was purified by semi-preparative RP-HPLC.

[0413] [ka]

[0414] HO-Ahx-dap((R)-DOTA-GA(tBu)4)-D-HCy(Trt)-Ac:RP-HPLC(10~95%B in 15 min):t R= 12.64 minutes. Monoisotopic mass calculation value (C 69 H 104 N8O 14 S): 1300.74, Measured value: 1060.2 [M-Trt+H] + , 651.6[M+2H] 2+ . CPCR4-Abz-ar-dap-Ahx-dap((R)-DOTA-GA)-D-HCy(VcMMAE)-Ac:RP-HPLC(10~95%B in 15 min):t R = 6.22 minutes. Monoisotopic mass calculation value (C 89 H 131 N 25 O 23 S): 1949.96, Measured value: 652.3 [M+3H]3 + . CXCR4-MMAE-4:RP-HPLC (20-70% B in 15 minutes):t R = 9.63 minutes. Monoisotopic mass calculation value (C 157 H 236 N 36 O 38 S): 3265.74, Measured value: 1634.4 [M+2H] 2+ , 1089.8 [M+3H] 3+ , 817.7[M+4H] 4+ . [ nat Lu]CXCR4-MMAE-4:RP-HPLC (20-70% B in 15 minutes):t R = 10.46 minutes. Monoisotopic mass calculation value (C 157 H 233 N 36 O 38 S): 3437.65, Measured value: 1720.1 [M+2H] 2+ , 1146.9 [M+3H] 3+ . 3.5 Optical Imaging Compounds CXCR4-OI-1

[0415] [ka]

[0416] The synthesis of CXCR4-OI-1 was based on the fragment condensation of the CPCR4-linker construct CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 (see 2.3.7) with a sulfo-Cy5-carboxylic acid (GP8). The resulting peptide was treated with TFA (GP5) and purified by semi-preparative RP-HPLC. CXCR4-OI-1:RP-HPLC (5-55% B at 15 mins):t R = 8.73 minutes. Monoisotopic mass calculation value (C 87 H 111 N 19 O 17 S2): 1757.78, Measured value: 880.5 [M+2H] 2+ , 587.3 [M+3H] 3+ . CXCR4-OI-2

[0417] [ka]

[0418] CXCR4-OI-2 was synthesized by iodination using completely deprotected and purified CXCR4-OI-1 as a substrate, according to the described method (see GP7). The mixture was subjected to semi-preparative RP-HPLC to obtain the desired product. CXCR4-OI-2:RP-HPLC (5-55% B at 15 mins):t R = 11.00 minutes. Monoisotopic mass calculation value (C 87 H 110 IN 19 O 17 S2): 1883.68, Measured value: 942.7 [M+2H] 2+ , 630.4[M+3H] 3+ . CXCR4-OI-3

[0419] [ka]

[0420] CXCR4-OI-3 was synthesized by fragment condensation of CPCR4-Abz-ar(Pbf)-dap(Boc)-NH2 and HO-Ahx-dap(DOTA(tBu)3)-Fmoc (see 0). The desired product was obtained by subsequent Fmoc deprotection and standard coupling of Cy5.5 according to GP3, GP5, and GP8. HO-Ahx-dap(DOTA(tBu)4)-Fmoc:RP-HPLC (10-95% B at 15 mins):t R = 16.66 minutes. Monoisotopic mass calculation value (C 52 H 79 N7O 12 S): 993.58, Measured value: 938.7 [M-tBu+H] + . CPCR4-Abz-ar(Pbf)-dap(Boc)-Ahx-dap(DOTA(tBu)3)-NH2:RP-HPLC(10~95%B in 15 min):t R = 9.14 minutes. Monoisotopic mass calculation value (C 110 H 166 N 24 O 24 S): 2239.22, Measured value: 1120.3 [M+2H] 2+ . CXCR4-OI-3:RP-HPLC (10-60% B in 15 minutes):t R = 8.86 minutes. Monoisotopic mass calculation value (C 112 H 154 N 26 O 26 S2): 2343.10, Measured value: 1173.4 [M+2H] 2+ , 782.7[M+3H] 3+ . [ nat Lu]CXCR4-OI-3:RP-HPLC (10~60%B in 15 minutes):t R = 8.78 minutes. Monoisotopic mass calculation value (C 112 H 151 N 26 O 26 S2): 2515.01, Measured value: 1258.4 [M+2H] 2+ , 839.3[M+3H] 3+ . 4. Radial labeling 4.1 125 I label / 125 I-FC-131 Approximately 50-150 μg of unlabeled precursor was dissolved in DMSO (20 μL), and 280 μL of Tris buffer (25 mM Tris-HCl, 0.40 mM NaCl, pH=7.5) was added. 125 After adding 5 μL of [I]NaI solution (15-20 MBq, see 1.2), the mixture was transferred to a reaction tube and coated with 150 μg of Iodogen®. After incubation at room temperature for 15 minutes, the mixture was removed from the oxidized product and subjected to RP-HPLC purification. 125 I-Fc-131

[0421] [ka]

[0422] 125 Prepare I-Fc-131 as described above, and then follow the RP-HPLC procedure: RP-HPLC (20-55% B in 15 minutes): t R =9.35 minutes It was purified according to the following method. 4.2 99M TC sign Peptides 99m TcO4 - Labeling was performed depending on the chelator used. The peptides and their respective mixtures (either freshly obtained from aqueous solutions or pre-formulated and lyophilized as a kit) were freshly eluted. 99m It was reacted with Tc-pertecnetate (see 1.2). 4.2.1 Cherators induced from mas3 The labeled mixture contained the same components whether in solution or when using the lyophilized formulation (49): Stock 1: 1.78 g of sodium phosphate dibasic dihydrate was dissolved in H2O (50.0 mL) (=Solution 1). 1.38 g of sodium phosphate monobasic monohydrate was dissolved in H2O (50.0 mL) (=Solution 2). Solution 1 (47.35 mL) and Solution 2 (2.65 mL) were mixed to obtain Stock 1. Stock 2: Stock 1 (5.00 mL) was diluted with H2O (5.00 mL) to obtain Stock 2. Stock 3: Each peptide was dissolved in DMSO, H2O, or a mixture thereof to a concentration of typically 1.00 mM. Stock 4 was prepared by dissolving 2.50 g of disodium tartrate dehydrate in Stock 1. Stock 5: 30.0 mg of ascorbic acid was mixed with 10.0 mL (10.0 mM) of aqueous HCl solution. Stock 6: 4.00 mg of tin(II) chloride dihydrate was dissolved in Stock 5 (1.0 mL). This solution was prepared fresh for each labeling experiment.

[0423] The labeled mixture or kit was prepared by mixing the stock solution according to the following instructions. Stock 1: 6.76 μL Stock 2: 10.0 μL Stock 3: 5.00 μL Stock 4: 8.00 μL Stock 6: 2.00 μL Newly eluted 99m Tc-pertecnetate (0.10–5.00 μL, 50.0–850 MBq) was added, and the mixture was heated at 95°C for 30 minutes. Quality control was performed directly from the reaction mixture by radioactive TLC and radioactive RP-HPLC.

[0424] Radioactive RP-HPLC:R t ( 99m TcO4 - )=t0, R t ( 99m Tc-colloid) = t0, R t ( 99mTc-Tartrate) = 2-3 minutes, R t ( 99m Tc-peptide) => 3 minutes.

[0425] 60RP-18F coated with silica using different mobile phases 254 Radioactive TLC in s fragments: NH4OAc / DMF(1 / 1, v / v):R f ( 99m TcO4 - )=1, R f ( 99m Tc-colloid) = 0, R f ( 99m Tc-Tartrate) = 0.5~0.8 min, R f ( 99m Tc-peptide) = 0.8~1. 2-Butanone:R f ( 99m TcO4 - )=1, R f ( 99m Tc-colloid) = 0, R f ( 99m Tc-Tartrate) = 0 minutes, R f ( 99m Tc-peptide) = 0. NaCl (25vol% in H2O):R f ( 99m TcO4 - )=0, R f ( 99m Tc-colloid) = 1, R f ( 99m Tc-Tartrate) = 0.5~0.8 min, R f ( 99m Tc-peptide) = 0. 4.2.2 HYNIC as a chelator The labeled mixture contained the same components whether in solution or when using the lyophilized formulation (50). Stock 1: Ethylenediaminediacetic acid (EDDA) was dissolved in an aqueous NaOH solution (0.10 M) to a concentration of 10.0 g / L. Stock 2: Disodium tartrate dihydrate was dissolved in NaH2PO4 buffer (40.0 g / L) to a concentration of 40.0 g / L. Stock 3: Tin(II) chloride dihydrate was dissolved in an aqueous sodium ascorbate solution (3.00 g / L in 0.01 M HCl) to a concentration of 1.50 g / L. This mixture was freshly prepared for each labeling experiment. Stock 4: Each peptide was dissolved in DMSO, H2O, or a mixture thereof to a concentration of typically 1.00 mM.

[0426] The labeled mixture or kit was prepared by mixing the stock solution according to the following instructions. Stock 1: 50.0 μL Stock 2: 50.0 μL Stock 3: 5.33 μL Stock 4: 5.00 μL Newly eluted 99m Tc-pertecnetate (0.10–5.00 μL, 50.0–850 MBq) was added, and the mixture was heated at 95°C for 20 minutes. Quality control was performed directly from the reaction mixture by radioactive TLC and radioactive RP-HPLC as described above (see 4.2.1). 4.2.3 N4 as a chelator The labeled mixture contained the same components whether in solution or when using a lyophilized formulation. Stock 1: Na2HPO4 was dissolved in H2O to a concentration of 0.05M (pH=11.5). Stock 2: Disodium citrate sesquihydrate was dissolved in H2O to a concentration of 0.10 M. Stock 3: Tin(II) chloride dihydrate was dissolved in an aqueous sodium ascorbate solution (3.00 g / L in 0.01 M HCl) to a concentration of 1.00 g / L. This mixture was freshly prepared for each labeling experiment. Stock 4: Each peptide was dissolved in DMSO, H2O, or a mixture thereof to a concentration of typically 1.00 mM.

[0427] The labeled mixture or kit was prepared by mixing the stock solution according to the following instructions. Stock 1: 25.0 μL Stock 2: 3.00 μL Stock 3: 5.00 μL Stock 4: 7.50 μL Newly eluted 99m Tc-pertecnetate (0.10–5.00 μL, 50.0–850 MBq) was added, and the mixture was heated at 90°C for 10 minutes. Quality control was performed directly from the reaction mixture by radioactive TLC and radioactive RP-HPLC as described above (see 4.2.1). 4.3 177 LU indicator Peptides containing DOTA or DOTA-GA 177 For Lu labeling, 0.5–2 nmol of each peptide (directly from stock, DMSO, H2O, or a mixture thereof) was mixed with 10 μL of sodium acetate aqueous buffer (1.00 M, pH=5.50). Typically, the desired radioactivity level was between 5–80 MBq. 177 Lu]LuCl3 (0.04 M in HCl) was added, and this mixture was diluted with HCl (0.04 M) to a total volume of 100 μL. After 30 minutes at 95°C, 10 μL (0.10 M) of sodium ascorbate was added to prevent radiolysis, and the reaction was controlled by radioactive RP-HPLC and radioactive TLC. Silica-coated 60RP-18F 254 s and mobile phase NH4OAc / DMF(1 / 1;v / v): R f ( 177 LuCl3)=0, R f ( 177 Lu-colloid) = 0, R f ( 177 Lu-peptide) = 1. Cellulose ITLC-SG paper and mobile phase trisodium citrate (0.10M): R f ( 177 LuCl3)=1, R f ( 177 Lu-colloid) = 0, R f ( 177 Lu-peptide) = 0. 4.4 18 F sign Labeling of peptides containing SiFA was achieved according to a forthcoming document (51). In short, a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate Light) was conditioned with H2O (10 mL), and then... 18 An aqueous solution of F-fluoride was passed through the cartridge. The cartridge was purged with 10 mL of air, dried with 10 mL of ACN, and then purged again with 20 mL of air to remove any trace amounts of water. 18 100 μmol of F-fluoride [K + ⊂2.2.2]OH - The sample was eluted in 500 μL of ACN, and the pH was adjusted by adding 25 μL of oxalic acid (1.00 M in ACN).

[0428] This mixture was used in one or more labeling experiments. The desired amount of radioactivity (typically 30-500 MBq) was mixed with 10-25 μmol of each SiFA-containing peptide (directly from stock in DMSO), and this mixture was incubated at room temperature for 5 minutes. The reaction mixture was then diluted with 9 mL of HEPES buffer (0.10 M, pH=3). Unreacted material was removed by passing this mixture through a Sep-PakC18 light cartridge. 18 The product was isolated from F-fluoride. After purging the cartridge with H2O (10 mL), the peptide was eluted with 500 μL of EtOH / PBS mixture (1 / 1; v / v). Radiochemical purity was determined using radioactive RP-HPLC and radioactive TLC. Silica-coated 60RP-18F 254 s and mobile phase ACN / PBS (1 / 1; v / v; +10 vol% NaOAc (2.00 M in H2O); +1 vol% TFA): R f ( 18 F-Fluoride) = 0, R f ( 18 F-peptide) = 0.8~1. 4.5 68 GA sign Peptides containing DOTA and peptides containing DOTA-GA 68Ga labeling was implemented according to reference (34). A Scintomics automated GallElutch system was used. + I used a system. In short, it's made by IThemba LABS. 68 Ge / 68 The Ga generator was eluted with HCl aqueous solution (1.00 M), and the fraction (typically 1.25 mL, 500-700 MBq) was transferred to a reaction vial (ALLTECH, 5 mL) pre-filled with 2-5 nmol of each peptide. The reaction mixture was heated at 95°C for 5 minutes, and then passed through a Sep-Pak C8 light cartridge pre-conditioned with H2O (10 mL). The product was eluted with EtOH / H2O (1 / 1; v / v) 2 mL, the cartridge was purged with PBS (1 mL) and H2O (1 mL), and then the EtOH was removed under vacuum. Radiochemical purity was assessed by radioactive TLC. Silica-coated 60RP-18F 254 s and mobile phase NH4OAc / DMF(1 / 1;v / v): R f ( 68 GaCl3)=0, R f ( 68 Ga-colloid) = 0, R f ( 68 Ga-peptide) = 1. Cellulose ITLC-SG paper and mobile phase trisodium citrate (0.10M): R f ( 68 GaCl3)=1, R f ( 68 Ga-colloid) = 0, R f ( 68 Ga-peptide) = 0. 4.6 67 GA sign Peptides containing DOTA 67 Labeling with Ga was performed in the same manner as described in reference (52). In short, [ 67 Ga[G]citrate was immobilized on a SEP-Pak silica light cartridge, washed with H2O (10 mL), and eluted with the desired volume of HCl (0.1 M). Then, the obtained 67The GaCl3 solution fraction was diluted with HEPES to a total volume of 200 μL and added to the peptide (5 nmol, H2O). This mixture was then heated at 95°C for 30 minutes, diluted with PBS to a volume of at least 3 mL, and passed through a SEP Pak C8 light cartridge to remove excess. 67 GaCl3 was removed. The labeled peptide was eluted with 0.5 mL of EtOH / PBS (1 / 1; v / v) mixture. Radiochemical yield and purity were assessed by radioactive TLC. Silica-coated 60RP-18F 254 s and mobile phase NH4OAc / DMF(1 / 1;v / v): R f ( 67 GaCl3)=0, R f ( 67 Ga-colloid) = 0, R f ( 67 Ga-peptide) = 1. Cellulose ITLC-SG paper and mobile phase trisodium citrate (0.10M): R f ( 67 GaCl3)=1, R f ( 67 Ga-colloid) = 0, R f ( 67 Ga-peptide) = 0. 5. In vitro experiments 5.1 IC 50 decision CXCR4-positive Jurkat T lymphocytes were grown in Gibco's RPMI1640GlutaMAX medium supplemented with 10 vol% FBS and maintained at 37°C in a 5% CO2 atmosphere.

[0429] In in vitro experiments, cells were counted using a hemocytometer with trypan blue as a contrast agent. The cell suspension was centrifuged, and the pellet was resuspended in HBSS (+1 wt% BSA) to a concentration of 2 million cells / mL. Standard ligands were placed in 8x3 polystyrene tubes. 125 25 μL of I-Fc-131 (see 4.1, 1.00 nM in HBSS) and 25 μL of ligand for investigation at their respective concentrations (10-4 ~10 -10 M was added (n=3 for each concentration). 200 μL of cell suspension (400,000 cells / well) was added, and 10 -5 ~10 -11 The final peptide concentration range for M was obtained. The tubes were cooled at 8°C for 2 hours to prevent internalization. The experiment was stopped by removing the supernatant. HBSS (200 μL) was added to the cells, the suspension was centrifuged, and the supernatant was pooled with each of the initial fractions. This step was repeated, and then the tubes containing the supernatant and the cell pellets were measured for radioactivity in a gamma counter. 5.2 invIC 50 decision Reverse IC 50 (invIC 50 Regarding the determination of the value, a normal IC 50 The protocol for determining the values ​​was restarted with minor modifications. The peptide under investigation was radiolabeled, and a stock solution was prepared in HBSS (2.00 nM). A concentration gradient of the standard ligand Fc-131 was prepared in HBSS (10 -4 ~10 -10 The solution was prepared in M). Then, 25 μL of the radioactive peptide solution, 25 μL of each Fc-131 solution, and 200 μL of the cell suspension (400,000 cells) were placed in a cell tube. The experiment was carried out as described above. 5.3 Decision on Internalization Chem_1 cells expressing CXCR4 were grown in DMEM-F12 medium supplemented with 10 vol% FBS, 1 vol% NEA, 1 vol% PenStrep, and 1 vol% HEPES (1.00 M). The cells were maintained at 37°C in a 5% CO2 atmosphere.

[0430] In the in vitro experiment, the culture medium was removed and the cells were harvested by incubation with trypsin / EDTA (0.05% / 0.02% w / v) at 37°C for 30 minutes. The cells were counted and seeded into 24-well plates 24 ± 2 hours before the experiment (100,000 cells / well).

[0431] The culture medium was removed, and the cells were incubated in 200 μL of DMEM-F12 (+5 wt% BSA) at 37°C for 15 minutes. To block the receptor, each well (n=3 at each time point) was treated with either 25 μL of DMEM-F12 (+5 wt% BSA) or 25 μL of AMD3100 stock (1 mM in H2O). The experiment was performed as a dual tracer method, so a standard ligand was used. 125 A radioactive tracer solution containing I-Fc-131 and the radiolabeled peptide under investigation was prepared at a concentration of 2.00 nM each. 25 μL of this stock was added to the wells ( 125 The cells were incubated at 37°C for the respective times (final concentration of Fc-131: 0.20 nM; final concentration of the peptide under investigation: 0.20 nM).

[0432] The experiment was stopped by placing the well plate on ice and removing the supernatant. The cells were washed with non-radioactive HBSS (250 μL), and both fractions, each containing a certain amount of unbound radioactive ligand, were combined for each well.

[0433] 250 μL of cold acid washing agent (0.02 M NaOAc in aqueous acetic acid solution, pH=5) was added, and the cells were incubated on ice for 15 minutes. The supernatant was removed, the cells were washed with cold HBSS, and the respective fractions were combined to obtain a certain amount of surface-bound radioligand.

[0434] Next, the cells were incubated in 300 μL of NaOH (1.00 M in H2O) at room temperature for at least 30 minutes, after which the supernatant was removed. The wells were washed with another 300 μL of NaOH, and the fractions containing a certain amount of internalized radioligand were combined.

[0435] Three different fractions were measured for radioactivity in a gamma counter, and the radioactivity of the radionuclide used to label the peptide under investigation was measured first. After an appropriate time interval according to the half-life of the first radionuclide, 125 To investigate the radioactivity of I, the same fraction was measured again. The data was corrected for nonspecific internalization and compared with a standard ligand. 125Specific internalization of I-Fc-131 was referenced. 5.4 Determination of Octanol / PBS Partition Coefficient The ligand under investigation (typically 0.50–3.00 MBq depending on the radioisotope) was diluted in PBS (pH=7.4) to a total volume of 1.00 mL and mixed with 1.00 mL of n-octanol in a low-adsorption Eppendorf tube (n=8). The tube was vortexed at maximum speed for 3 minutes to ensure equilibrium, and then centrifuged at 15.000 × g for 5 minutes using a Biofuge 15 (Heraeus Holding GmbH, Osterode, Germany). 100 μL aliquots of each fraction were measured in a gamma counter, and logD was obtained. 7.4 We calculated it as follows:

[0436]

number

[0437] 6. In vivo experiments 6.1 Mouse models and tumor models All animal experiments were conducted in accordance with German general animal welfare regulations and organizational guidelines for animal husbandry and use. To establish tumor xenotransplantation, Jurkat cells (2-3 × 10⁶) were used. 7 The cells were suspended in a mixture of Gibco's RPMI1640 medium and BD Biosciences (Heidelberg, Germany) Matrigel (1 / 1;v / v) and subcutaneously inoculated into the right shoulder of 6-10 week old CB17-SCID mice procured from either Charles River GmbH (Sulzfeld, Germany) or the company's own mouse breeding facility. Mice were used when the tumors had grown to a diameter of 5-8 mm (4-10 weeks after inoculation). 6.2 μSPECT / μPET / CT Imaging Imaging experiments were conducted using a VECTor manufactured by MILabs BV. (Utrecht, Netherlands). 4The study was performed using small animal SPECT / PET / OI / CT. The resulting data was analyzed using the accompanying PMOD (version 4.0) software. Mice were anesthetized with isoflurane and injected with radiolabeled compounds via the tail vein. After distribution to each distance, the mice were euthanized, and blood samples for subsequent in vivo distribution studies were collected by puncture, followed by image acquisition. Static images were acquired with a 45-minute imaging time, using an HE-GP-RM collimator and stepped multi-planar bed movement for SPECT isotopes, and an HE-UHR-M collimator and stepped helical bed movement for PET isotopes. All images were reconstructed using MILabs-Rec software (version 10.02) and the pixel-based Similarity-Regulated Ordered Subsets Expectation Maximization (SROSEM) algorithm with window-based confusion correction (below 20% and above 20% of the photoelectric peak, respectively). (Voxel size CT: 80 μm, Voxel size SPECT / PET: 0.8 mm, 1.6 mm (FWHM) Gaussian blurring post-processing filter, calibration coefficient (unit kBq / mL) and attenuation correction included, absorption correction not included) 6.3 In vivo distribution study Approximately 0.5 to 20 MBq (0.02 to 0.20 nmol), 125 I sign, 177 Lu label, 99m Tc label, 18 F-mark or 68 Ga-labeled ligands were injected into the tail veins of Jurkat tumor-bearing CB-17SCID mice. After allowing time for distribution within the organism, the animals were disposed of. Selected organs were removed, weighed, and measured in a gamma counter. II. Results 1. Technetium-99M SPECT tracer 1.1 mas3 conjugate peptide 1.1.1 Chemical structure The chemical structures of the mas3 conjugate compounds Tc-CXCR4-1~-8 are shown below.

[0438] [ka]

[0439] [Table 1]

[0440] 1.1.2 In-vitro data

[0441] [Table 2]

[0442] 1.1.3 In vivo distribution test

[0443] [Table 3]

[0444] [Table 4]

[0445] [Table 5]

[0446] [Table 6]

[0447] 1.1.4 Mouse imaging test Figure 1 shows the following in female Jurkat tumor-bearing mice: 99m The images show the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Tc-CXCR4-6 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. 1.1.5 Internalization Test

[0448] [Table 7]

[0449] 1.2 Modified MAS3 Conjugate Peptides 1.2.1 Chemical structure The chemical structures of the modified mas3 conjugate compound Tc-CXCR4-9~-12 are shown below.

[0450] [ka]

[0451] [Table 8]

[0452] 1.2.2 In vitro data

[0453] [Table 9]

[0454] 1.2.3 In vivo distribution study

[0455] [Table 10]

[0456] [Table 11]

[0457] 1.2.4 Mouse imaging test Figure 2 shows the following in female Jurkat tumor-bearing mice: 99mThis image shows the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Tc-CXCR4-9 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0458] Figure 3 shows the results in female Jurkat tumor-bearing mice. 99m This image shows the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Tc-CXCR4-11 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0459] Figure 4 shows the results in female Jurkat tumor-bearing mice. 99m The images show the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Tc-CXCR4-12 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. 1.2.5 Internalization Test

[0460] [Table 12]

[0461] [Table 13]

[0462] 1.3 Immobilized chelator peptides 1.3.1 Chemical structure The chemical structures of Tc-CXCR4-13~-16, peptides with immobilized chelators containing HYNIC and N4, are shown below.

[0463] [ka]

[0464] [Table 14]

[0465] 1.3.2 In vitro data

[0466] [Table 15]

[0467] 1.3.3 In vivo distribution test

[0468] [Table 16]

[0469] [Table 17]

[0470] 1.3.4 Mouse imaging test Figure 5 shows the following in female Jurkat tumor-bearing mice: 99m The images show the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Tc-CXCR4-13 injection. No competing substances, 1-10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0471] Figure 6 shows the results in female Jurkat tumor-bearing mice. 99m The images show the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Tc-CXCR4-14 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0472] Figure 7 shows the results in female Jurkat tumor-bearing mice. 99mThe images show the MIP (Maximum Intensity) of both CT and SPECT scans 2 hours after Tc-CXCR4-14 injection. No competing substances, 1–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. 1.3.5 Internalization Test

[0473] [Table 18]

[0474] [Table 19]

[0475] 1.3.6 In vivo testing [ 99m Because favorable preclinical data were obtained for Tc]CXCR4-Tc-14, this ligand was selected for the first proof-of-concept trial in patients with multiple myeloma. 99m The radioactive synthesis of Tc]CXCR4-Tc-14 was performed manually as described in Chapter 4.2.3 above. 430~604 MBq [ 99m Images were acquired 5 minutes to 21 hours after injection of Tc]CXCR4-Tc-14. Detailed scanning procedures are described below.

[0476] Figure 8 shows maximum projection (MIP) images obtained from SPECT and PET imaging of a female patient with multiple myeloma. A)[ 18 F] PET MIP (1 hour after injection, 189 MBq) 18 F)FDG), B)[ 99m SPECT MIP of Tc]CXCR4-Tc-14 (3 hours after injection, 604 MBq) 99m Tc]CXCR4-Tc-14). Straight arrows indicate tumor lesions, and dotted arrows indicate [ in the heart 18 Physiological uptake of F]FDG, and in the spleen [ 99m This shows the physiological uptake of Tc]CXCR4-Tc-14.

[0477] [ 99m The in vivo distribution of Tc]CXCR4-Tc-14 is established using PET tracers. 68 Its biodistribution is similar to that of [Ga]pentixafor (Lapa C, Schreder M, Schirbel A et al., 68GaPentixafor-PET / CT for imaging of chemokine receptor CXCR4 expression in multiple myeloma-Comparison to 18FFDG and laboratory values. Theranostics. 2017;7:205~212. doi:10.7150 / thno.16576). In healthy organs, significant uptake is observed in the kidneys. The relatively high uptake in the spleen reflects known CXCR4 expression and ligand uptake toward CXCR4 in humans. Significant uptake is also observed in the liver and bone marrow, both of which express CXCR4 physiologically but express it even more in pathological conditions (Philipp-Abbrederis K, Herrmann K, Knop S et al., In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma. EMBO Mol Med. 2015;7:477~487.doi:10.15252 / emmm.201404698; Vag T, Gerngross C, Herhaus P et al., First Experience with Chemokine Receptor CXCR4-Targeted PET imaging of Patients with Solid Cancers. J Nucl Med. 2016;57:741~746.doi:10.2967 / jnumed.115.161034). The low uptake in the blood pool and the rapid clearance of tracers from non-target tissues indicate the significant targeting potential of the tracers, as confirmed in preclinical trials (InvIC). 50 =10.2nM, internalized ( 125Combined with I-FC-131 (in percentages) = 742%) [ 99m Appropriate hydrophilicity of Tc]CXCR4-Tc-14 (logD 7.4 The result is almost certainly -1.75. High tracer uptake in tumor lesions and low accumulation in background tissues allows for high-contrast visualization of multiple metastases (Figure 8, B). SPECT / CT images of the axial section are [ 18 Consistent with PET / CT scans using [F]FDG, the height of lesion uptake and in osteolytic lesions within the pelvis [ 99m The useful resolution of Tc]CXCR4-Tc-14 further demonstrates the tracer's potential (Figure 9). 18 Among F]FDG-positive lesions, those targeting CXCR4 [ 99m No cases were found to be negative in SPECT scans using [99mTc]CXCR4-Tc-14. Figure 9A) shows axial SPECT / CT images 3 hours after injection of 604 MBq of [99mTc]CXCR4-Tc-14 in a patient with multiple myeloma, and Figure 9B) shows the same patient with 189 MBq of [ 18 The image shows an axial PET / CT image taken one hour after injection of F]FDG, with straight arrows indicating the bladder and dashed arrows indicating osteolytic lesions.

[0478] Contrary to the findings obtained in preclinical studies in tumor-bearing mice, no substantial increase in ligand uptake was detected in the human liver and lungs, confirming that the increased uptake in the mouse liver and lungs was due to the ligand's mCXCR4 affinity.

[0479] Physiological uptake in the stomach and thyroid gland is low, comparable to other normal organs, and this indicates that, 99mThe organisms showed no contamination with [99m]Tc-pertechnetate, or in vivo disappearance of radioactive metals (Franken PR, Guglielmi J, Vanhove C et al., Distribution and dynamics of (99m)Tc-pertechnetate uptake in the thyroid and other organs assessed by single-photon emission computed tomography in living mice. Thyroid. 2010;20:519~526. doi:10.1089 / thy.2009.0213).

[0480] Figure 10 shows [ in the same patient 99m This shows the in vivo distribution of Tc]CXCR4-Tc-14 between 5 minutes and 21 hours after injection. 99m The specific doses of Tc]CXCR4-Tc-14 in various target tissues and organs were determined.

[0481] Figure 10A) shows the results of 604 MBq in female patients with multiple myeloma. 99m Figure 10B) shows MIP images obtained from SPECT imaging at 5, 60, 120, 5, and 21 minutes after injection of Tc]CXCR4-Tc-14, and the specific dose [μGy / MBq] calculated for selected organs and tissues of the same patient.

[0482] [ 99mThe time-resolved in vivo distribution of Tc]CXCR4-Tc-14 reaffirms rapid background clearance, as well as uptake in the lesion and spleen and bone marrow, even at early time points such as 5 minutes after injection. At later time points, no delay in biliary clearance was detected, indicating tracer clearance via the kidney. However, uptake in the kidney and bladder was found to be low throughout the observation period, suggesting long-term ligand retention in CXCR4-expressing tissues (Figure 10 10, B). This appears to be a direct result of the high target affinity and marked internalization into CXCR4-expressing cancer cells determined in preclinical studies.

[0483] The highest specific dose is determined for the spleen (47 μGy / MBq), followed by the liver (14 μGy / MBq), osteogenic cells (13 μGy / MBq), red bone marrow (11 μGy / MBq), and kidney (10 μGy / MBq) (Figure 10 10, B). The whole-body effective dose is the 604 MBq injected [ 99m For [Tc]CXCR4-Tc-14, the calculated dose was 6.3 μSv / MBq, which represents a delivered whole-body dose of 3.8 mSv. The specific dose delivered to organs and the whole-body effective dose have been found to be similar to those of other technetium-99m labeled ligands such as [99mTc]PSMA I&S targeting PSMA or [99mTc]CXCR4-L targeting CXCR4 (P. Vallejo-Armenta et al., Contrast Media and Molecular Imaging, 2020, citation ID 2525037, https: / / doi.org / 10.1155 / 2020 / 2525037; S. Urban et al., J. Nucl. Med. 2021, 62(8), 1075~1081). Compared to dosimetry data obtained for [68Ga]pentixafor in patients with multiple myeloma, [99mTc]CXCR4-Tc-14 shows a higher whole-body absorbed dose but a lower specific organ dose (K. Herrmann et al., J. Nucl. Med. 2015, 56(3), 410-416).

[0484] This proof-of-concept trial is conducted in a clinical setting. 99m This provides sufficient grounds for further evaluation of Tc]CXCR4-Tc-14. Clinical SPECT / CT Imaging In patients [ 99m The clinical evaluation of Tc]CXCR4-Tc-14 was conducted under compassionate use, in compliance with Article 132b of the German Medicinal Products Act (AMG), and in accordance with the responsible authorities (the government of Oberbayern). The initial proof-of-concept trial was conducted at the Universitatsklinikum Augsburg (Augsburg, Germany).

[0485] All subjects were examined using a Discovery MN CT670Pro (GE Healthcare, Solingen, Germany) equipped with an Optima540CT. Whole-body SPECT / CT scans were acquired at 30 cm / min at 5 and 60 minutes after tracer injection, at 120 and 300 minutes at 12 cm / min, and at 21 hours at 5 cm / min. Using double-head technology, 60 subsets of scans were acquired in a 128x128 matrix (zoom 1). SPECT scans at 60 and 180 minutes after tracer injection were acquired with an exposure time of 8 seconds per subset, and scans at 21 hours were acquired with an exposure time of 16 seconds per subset. Ejection data were smoothed by applying a Butterworth filter (0.48) and iteratively reconstructed using a subsetted expectation maximization algorithm (2 iterations, 10 subsets). 430~604 MBq [ 99m Images were acquired 5 minutes to 21 hours after injection of Tc]CXCR4-Tc-14.

[0486] Specific and effective doses were analyzed in selected organs. For dose calculation, target voxels were defined around areas of high uptake in whole-body images. Time-activity curves for each organ were automatically determined, and tracer residence times were calculated using specific SPECT camera calibrations and standard radioactivity values. Residence times were used as input data for the Olinda / EXM software. The output of these calculations was compared against the International Commission on Radiological Protection (ICD) dosimetry guidelines. Clinical PET / CT Imaging In patients [ 18 The clinical evaluation of F]FDG was conducted in accordance with the responsible authorities (the government of Oberbayern). The clinical trial was conducted at the Universitatsklinikum Augsburg (Augsburg, Germany).

[0487] All subjects were tested using the Biograph mCT-S40 (Siemens Healthineers, Erlangen, Germany). 18 Whole-body PET / CT scans were acquired 1 hour after injection of [F]FDG at a scan rate of 2 minutes per bed position. Emission data were iteratively reconstructed. Low-dose CT was performed for attenuation correction and anatomical correlation. 189 MBq [ 18 Images were acquired one hour after injection of [F]FDG. 2. Lutetium-177 / Gallium-68 Theranostic 2.1 Chemical structure The chemical structures of the DOTA-containing peptides CXCR4-DOTA-1 to -5 are shown below.

[0488] [ka]

[0489] [Table 20]

[0490] 2.2 In vitro data

[0491] [Table 21]

[0492] 2.3 In vivo distribution study

[0493] [Table 22]

[0494] [Table 23]

[0495] [Table 24]

[0496] [Table 25]

[0497] [Table 26]

[0498] [Table 27]

[0499] [Table 28]

[0500] 2.4 Mouse imaging test Figure 11 shows the results in female Jurkat tumor-bearing mice. 177The images show the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Lu-CXCR4-DOTA-1 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0501] Figure 12 shows the results in female Jurkat tumor-bearing mice. 177 The images show the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Lu-CXCR4-DOTA-2 injection. No competing substances, 2-10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0502] Figure 13 shows the results in female Jurkat tumor-bearing mice. 177 This image shows the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Lu-CXCR4-DOTA-3 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0503] Figure 14 shows the results in female Jurkat tumor-bearing mice. 177 This image shows the MIP (Maximum Intensity) of both CT and SPECT scans one hour after Lu-CXCR4-DOTA-4 injection. With and without competing substance (left) and with competing substance (right, 100 nmol AMD3100), 2–10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver.

[0504] Figure 15 shows the results in female Jurkat tumor-bearing mice. 177 The images show the MIP (Maximum Intake) of both CT and SPECT scans 6 hours after Lu-CXCR4-DOTA-4 injection. No competing substances, 2-10% iD / mL. White arrows indicate the target organ: solid line = tumor, dotted line = kidney, dashed line = liver. 2.5 Internalization Test

[0505] [Table 29-1]

[0506] [Table 29-2]

[0507] 3. Fluorine-18 PET tracer / radiation hybrid 3.1 Chemical structure The chemical structures of the SiFA-containing peptide and radiative hybrid are shown below.

[0508] [ka]

[0509] [Table 30]

[0510] 3.2 In vitro data

[0511] [Table 31]

[0512] 3.3 In vivo distribution study

[0513] [Table 32]

[0514] Later, two additional in vivo distribution studies were conducted to reinvestigate this issue by simultaneously injecting 1 and 2 nanomoles of non-radioactive material, respectively. The aim of this approach was partial and complete blockade of the mouse CXCR4 receptor by the non-radioactive material.

[0515] Figure 16 shows the [ ] in female CB-17SCID mice carrying Jurkat tumors. 18 F, natThis shows the in vivo distribution profile of Ga]CXCR4-SiFA-07 1 hour after injection. Different amounts of radioligand, i.e., 36 pmol, 1,000 pmol, and 2,000 pmol, were applied. Data are expressed as %iD / g values, and for the 36 pmol experiment, the values ​​are the mean ± SD of 5 animals. One animal was used for each of the other two experiments.

[0516] To save the animals' lives, each additional test was conducted using only one mouse. Therefore, the data obtained from these experiments are not typical. However, certain trends can be inferred. Co-injection of 1,000 pmol of non-radioactive material resulted in an increase in the levels of circulating radioactive ligands. Blood radioactivity levels increased by 310%, including in the pancreas (196%) and muscles (226%), as well as in CXCR4 - This leads to increased uptake in the tissue. This expansion of blood circulation is CXCR4 + This was particularly understated in the organs of the liver (121%) and spleen (114%), suggesting initial receptor saturation. With higher ligand circulation, nearly twice the tumor uptake (180%) was observed compared to injection without supplementation.

[0517] When 2,000 pmol of non-radioactive material is simultaneously injected, significant receptor saturation is reached. Compared to the initial in vivo distribution with only 36 pmol of ligand, the most notable change in organ uptake is observed in CXCR4. + Uptake is observed in organs such as the spleen (61%) and liver (70%), with even more pronounced changes seen in tumors (40%). These datasets show that uptake in tumors is CXCR4 + This supports the above idea that it depends on the availability of mCXCR4 receptors in organs. 3.4 Internalization Test

[0518] [Table 33]

[0519] [Table 34]

[0520] [Table 35]

[0521] 4. Peptides conjugated with cytotoxic substances 4.1 Chemical structure The chemical structures of the compounds CXCR4-MMAE-1 to -4, to which cytotoxic substances are bound, are shown below.

[0522] [ka]

[0523] [Table 36]

[0524] 4.2 In vitro data

[0525] [Table 37]

[0526] 4.3 In vivo distribution study

[0527] [Table 38]

[0528] 4.4 Internalization Test

[0529] [Table 39]

[0530] Cytotoxic effect The evaluation of cytotoxic efficacy was conducted in collaboration with the group of Professor Ulrich Keller, M.D., who currently leads the biological investigation of Myc-related cancers at the Klinikum Rechts der Isar (TUM) in Munich. This group received the compound CXCR4-MMAE-02 for the analysis of its in vitro cytotoxic capacity.

[0531] Two cell lines were selected for each experiment: the B-cell lymphoma cell line U2932 and the lymphoblastoid-like Raji cell line that more highly expresses CXCR4. Both cell lines were incubated with PDCs for 24, 48, 72, and 96 hours at various concentrations of 0 (blank), 10, 20, 40, and 100 nM. Cell viability was then assessed for dead cells using flow cytometry experiments, with propidium iodide as a marker (Figure 17).

[0532] Figure 17 shows the results of flow cytometry analysis of cell viability using Raji cells (left) and U2932 cells (right). Cells were incubated with CXCR4-MMAE-02 at 0 (blank), 10, 20, 40, and 100 nM for 0, 24, 48, 72, and 96 hours, then stained with propidium iodide and subjected to flow cytometry analysis. Cell viability is defined as the number of surviving cells relative to the total number of cells.

[0533] Experiments with both cell lines show that cell viability decreased when incubated with higher concentrations of PDC. Furthermore, a time-dependent effect may be observed, as cell viability decreased overall with longer incubation times. In other words, cell death induced by incubation with CXCR4-MMAE-02 is consistent with the amount of PDC used and the incubation time. Deviant data points may be due to small sample size and therefore experimental failure. Figure 18 shows the results from Figure 18, visualizing the amount of cancer cells killed by incubation with PDC.

[0534] Figure 18 shows the results of flow cytometry analysis of cell viability using Raji cells and U2932 cells. Cells were incubated with CXCR4-MMAE-02 at concentrations of 0 (blank), 10, 20, 40, and 100 nM for 96 hours, then stained with propidium iodide, and flow cytometry analysis of dead cells was performed.

[0535] At concentrations of 10, 20, and 40 nM, a significantly higher proportion of dead Raji cells can be detected compared to U2932 cells. This finding is consistent with higher CXCR4 expression in Raji cells and possibly more efficient PDC uptake compared to U2932 cells. In this case as well, the deviation of the 100 nM data point is likely due to the small sample size.

[0536] Cell cycle profiling experiments of U2932 cells were performed after incubation with various concentrations of CXCR4-MMAE-02 for 72 hours. For this purpose, cells were fixed, permeabilized, and treated with propidium iodide. Next, the number of cells in the G0 / G1, S, and G2 / M phases was measured relative to the amount of PDC used.

[0537] Figure 19 shows the results of cell cycle profiling experiments using U2932 cells. Cells were incubated with 0 (blank), 10, 20, 40, and 100 nM CXCR4-MMAE-02 for 72 hours, then fixed, permeabilized, and stained with propidium iodide. The percentage of cells in the G0 / G1, S, or G2 / M phases was determined.

[0538] Figure 19 shows the results, indicating that increasing the amount of PDC and incubating it leads to a greater number of cells remaining in the G2 / M phase (mitotic / cell division phase). This data suggests that MMAE is released into the cell, inhibiting microtubule polymerization and leading to cell arrest before replication. This induced cell cycle arrest then likely leads to the activation of checkpoint sentinels, such as kinases that manipulate apoptosis in cancer cells. 5. Optical imaging compounds 5.1 Chemical structure The chemical structures of the optical imaging compounds CXCR4-OI-1~-3 are shown below.

[0539] [ka]

[0540] [Table 40]

[0541] 5.2 In vitro data

[0542] [Table 41]

[0543] 5.3 In vivo distribution study

[0544] [Table 42]

[0545] [Table 43]

[0546] CXCR4 +In a separate in vivo distribution study, the benefit of partial organ blockade for tumor uptake was investigated using a radioactive ligand at low molar activity. To save animal lives, only one mouse was used in this study. Therefore, the resulting in vivo distribution is not typical. However, certain trends can be inferred. Figure 20 summarizes the experimental results.

[0547] Figure 20 shows the [ ] in female CB-17SCID mice carrying Jurkat tumors. 177 This shows the in vivo distribution profile of Lu]CXCR4-OI-03 1 hour after injection. Different amounts of radioactive ligand, namely 59 pmol and 1,000 pmol, were applied. Data are expressed as %iD / g values, with the 59 pmol experiment being the mean ± SD of 5 animals, and the other experiment using 1 animal.

[0548] Injecting 1,000 pmol of non-radioactive peptides triggers CXCR4 + A decrease in uptake is observed in the organs of the lungs (-23%), liver (-10%), and spleen (-23%). Therefore, tumor uptake increases 1.6 times, which indicates that a larger amount of the peptide in circulation accumulates within the tumor. Thus, if tumor uptake is low, it is because CXCR4 + This appears to be due to ligand capture within the organ, and not a result of a lack of targeting potential. 5.4 Internalization Test

[0549] [Table 44]

Images