CXCR4-Ligands for Diagnostic and Therapeutic Use and Precursors thereof

The present invention relates to compounds that are capable of binding to the seven transmembrane G-protein coupled chemokine receptor subtype (CXCR4) with high affinity and are thus considered CXCR4 ligands. They preferably act as agonists, or may also act as antagonists, inverse or partial agonists. The compounds are suitable for use in diagnostic and therapeutic applications.

The binding of stromal cell-derived factor 1 (SDF-1) (now referred to as C-X-C motif chemokine 12 (CXCL12)) to CXCR4 (1) activates the downstream protein kinase B (AKT)/mitogen- activated protein kinases (MARK) signaling pathway, which leads to the alteration of gene expression, actin polymerization, cell skeleton rearrangement and cell migration. The physiological functions of the CXCL12/CXCR4 axis include embryogenesis, immune response, hematopoiesis, brain development and neo-angiogenesis (2-6). Besides its fundamental involvement in physiological processes, elevated CXCR4 expression is associated with diverse malignancies. It mediates HIV-1 entry into T-cells as a co-receptor where it was first identified (3). CXCR4 is involved in B-cell trafficking and tissue localization in chronic leukemia patients (7) as well as the regulation of organ specific metastasis in different breast cancer models (8). Hence, CXCR4 overexpression is known in more than 20 human tumor types, including hematopoietic malignancies, brain neoplasm, gastrointestinal cancer and other cancer types (2, 8-10). Increasing evidence indicates that the CXCL12/CXCR4 axis functions as a critical communication bridge between tumor cells and stromal cells to create a permissive microenvironment (11). Cytokine CXCL12 and its receptor CXCR4 therefore represent a promising actionable target for therapeutic strategies, since the aberrant expression of CXCR4 strongly promotes proliferation, migration and invasion of different cancer types (12).

Several peptidic and non-peptidic CXCR4 antagonists that target the CXCL12/CXCR4 axis have been developed, due to their potential use for medicinal applications. The most established example is the bicyclam AMD3100 (Plerixafor®, Tetrazacyclotetradec-1- ylmethyl)phenyl]methyl}-1,4,8,11-tetrazacyclo-tetradecan) that has been approved by the FDA for the treatment of non-Hodgkin's lymphoma and multiple myeloma. Further peptidic CXCR4 antagonists have been developed, e.g. T140 and its derivatives which are side-chain cyclized peptides that contain one or two cyclization sites (13-15). A less cytotoxic and biologically stable derivative of T140 is TN 14003 (16). Introduction of a 4-fluorobenzoyl group constituted a novel pharmacophore for T140-based CXCR4 antagonists, TF14016, with subnanomolar binding affinity (16). This peptide CXCR4 antagonist was further employed in ¹⁸ F-or ⁶⁸ Ga based positron emission tomography (PET) imaging of CXCR4 expression in vivo (17-19). T140-based CXCR4 antagonists are already used for the prevention and/or therapy of cancers and chronic rheumatoid arthritis (US 8410059 B2, US 8765683 B2).

LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-D-Arg-2-Nal-Gly-D-Glu]-Lys(iPr)-NH ₂ ), a potent CXCR4 antagonist was demonstrated to exhibit good antitumor activities in solid tumor and breast cancer metastatic models and is currently in phase II clinical studies (NCT01391130 and NCT1439568) (20).

Moreover, three cyclic pentapeptides (peptide R, I and S) that are based on the N-terminal sequence of CXCL12 significantly inhibit subcutaneous growth of renal cancer cells. They also effect lung metastases and primary tumor growth (21). In addition, lactam-cyclized heptapeptides were reported to be potent CXCR4 antagonists useful in the treatment of cancers, rheumatoid arthritis, pulmonary fibrosis, and HIV infection (WO 2008/150689 A1).

T140-derived, cyclic pentapeptides based on cyc/o(D-Tyr ¹ -Arg ² -Arg ³ -Nal ⁴ -Gly ⁵ ) (from now on referred to as Fc-131) are used for therapy of cancer and anti-inflammation (22). SAR studies including alanine scanning, N-methyl amino acid scanning, optimization of amino acid residues and design of retro-inverso sequence peptides all failed to improve the binding affinity or anti- HIV activity compared to Fc-131 (23-25) and demonstrated the highly optimized binding scaffold of Fc-131. However, the introduction of amidine type dipeptide equivalents resulted in new lead structures of cyclic pentapeptides addressing CXCR4 (WO 2012/118124 A1). In addition, N-methylation of the peptide bonds of Fc-131 significantly affected its activity, resulting in the cyclic pentapeptide-based CXCR4 antagonist Fc-122 that shows a significant enhancement of CXCR4 antagonistic activity (26-28).

Within the scope of the development of molecular imaging probes for CXCR4, the N- methylation approach was employed to enhance binding affinity, while all side chains of Fc- 131 were tested for their feasibility of exchange. The substitution of Arg ² with D-Omithine and subsequent methylation of the N-terminus yielded CPCR4 (cyclo(D-Tyr ¹ -D-[NMe]Orn ² -Arg ³ - Nal ⁴ -Gly ⁵ )), which exhibits good binding affinity towards CXCR4. This lead structure served as an anchor point for further modifications (29), (WO 2007/096662). Dimeric derivatives of CPCR4 have been described, although the application for in vivo diagnostics was prevented by elevated accumulation of the CXCR4 ligands in the liver (WO 2009/027706) (29, 30).

Recently, a minimalistic approach was employed in which the Technetium chelator hydrazino-nicotinic acid was attached directly at the D-Orn ² side chain, resulting in a CXCR4 SPECT imaging agent (31). This compound is currently under examination in a first proof of concept study in men (32).

Nevertheless, further structural modifications, e.g. the introduction of an aromatic spacer attached to the side chain of [NMe]Orn ² of cyc/o(D-Tyr ¹ -D-[NMe]Orn ² -Arg ³ -Nal ⁴ -Gly ⁵ ) facilitated the introduction of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a labeling moiety (WO 2011/131735) for medicinal applications (33-35).

In the majority of the cases, additional structural changes on the detectable label (36) resulted in severe losses of binding affinity. An additional modification (introduction of (3-iodo)tyrosine) in the cyclic pentapeptide scaffold of cyclo(D-Tyr ¹ -D-[NMe](DOTA)-Om ² Arg ³ -Nal ⁴ -Gly ⁵ ) (pentixafor) was described, which significantly increased the binding affinity towards hCXCR4. Consequently, the resulting ¹⁷⁷ Lu- and ⁹⁰ Y-pentixather was utilized in therapeutic approaches of CXCR4 associated malignancies (WO 2015/185162) (37).

Building on the success of these clinically applied peptides, further optimization was undertaken to enhance affinity and versatility of the peptide scope. Replacement of the short aromatic spacer with a tailormade peptidic linker unit ultimately resulted in a CXCR4 binding motif with enhanced affinity and significantly increased internalization rates due to newly induced semi-agonistic properties of the peptide scaffold (38). These modifications allowed the introduction of a variety of functional modalities such as mas ₃ -based technetium chelators or AmBF ₃ as a ¹⁸ F-labeling unit without greater impairment of the affinity of the resulting peptides (WO 2020/053255).

The diagnostic and therapeutic potential of CXCR4 ligands has been shown in many different cases e.g. for the treatment of HIV infection and cancer, or for the visualization of CXCR4 expression in patients. Especially, the cyclic pentapeptides are optimized for the perfect interaction of the compounds in the binding cavity of CXCR4 and small modifications, e.g. the introduction of labeling moieties or residues to influence the pharmacokinetics of the CXCR4 ligands, result in considerably high affinity losses (36) and therefore have to be structurally optimized for each desired application. Consequently, the design and development of novel CXCR4 targeting compounds requires elaborated structure-activity-relationship studies (SAR studies).

For this reason, there is a need for a universally applicable ligand design which ensures high affinity towards the human CXCR4 receptor while allowing the linkage of a broad variety of functional groups with diagnostic or therapeutic utility. The present invention provides such novel ligand compounds and their uses in medical and scientific applications. The compounds of the invention are capable of binding the human CXCR4 receptor with high affinity and the murine receptor with moderate affinity and, hence, are suitable as CXCR4 ligands. These ligands may function as agonists, inverse agonists, partial agonists or antagonists. The structure of the linker in the ligand compounds provided by the invention surprisingly results in high flexibility of the compounds towards the attachment of various functional moieties while preserving or even enhancing CXCR4 affinity. Moreover, based on favorable in vitro characteristics like higher affinity and boosted internalization, higher and more consistent tumor uptake is often reached. The compounds of the invention are thus particularly suitable for medical applications such as preclinical and clinical imaging, and therapeutic applications, such as endoradiotherapy.

Before the background described above, the present invention provides a compound of formula (I) or a salt thereof, which is a C-X-C chemokine receptor type 4 ligand compound, or briefly a CXCR4 receptor ligand compound: wherein: a is 0 or 1 , preferably 0; b is 0 or 1 ; c is 0 or 1, and d is 0 or 1 , with the proviso that at least one of c and d is 1 ; e is an integer of 1 to 4, preferably 2 to 4; R ^CP is a cyclopeptide group of formula (II): wherein, in formula (II)

R ^B1 is H or I, preferably H;

R ^B2 is an alkanediyl chain; and wherein the dashed line marks a bond which attaches the group R ^CP to the remainder of the compound of formula (I);

R ^L1 is H or alkyl;

R ^L2 is substituted alkyl, which substituted alkyl is substituted with at least one group selected from -NH ₂ and -NH-C(=X)-NH ₂ with X being selected from NH and O;

_RL3 is -CH ₂ -NH ₂ or -CH ₂ -(1H-imidazol-4-yl);

R ^L4 is -NH ₂ ;

X ¹ is a coupling group; R ^S is a divalent spacer group; and

R ^A is a functional group comprising a moiety with diagnostic or therapeutic utility. In the CXCR4 receptor ligand compounds of the present invention, the binding motif R ^CP and the functional group R ^A are linked by a linker of the following structure.

This linker forming part of the compounds of formula (I) is characterized by the presence of a substituent with a small size and a functional group that typically carries a positive charge under physiological conditions, provided as R ^L3 , R ^L4 or as R ^L3 and R ^L4 . In the context of the invention, it has been found that this linker structure allows, amongst other beneficial properties, a high CXCR4 affinity to be retained for the compounds of the present invention with no or only little detrimental influence of the functional group R ^A . Thus, relying on the combination of the binding motif and the linker contained in the compounds in accordance with the invention, CXCR4 receptor ligands with a broad variety of functional groups can be provided without concerns about a significant loss of the affinity provided by the binding motif.

In accordance with further related aspects, the invention provides a therapeutic or a diagnostic composition comprising a CXCR4 receptor ligand compound of formula (I) or a salt thereof.

As noted above, salts, typically pharmaceutically acceptable salts, of the compounds of formula (I) are encompassed by the present invention. Thus, unless indicated to the contrary, any reference to a compound in accordance with the invention herein encompasses both the compound of formula (I) and the preferred embodiments of this formula disclosed herein, and the salts thereof. Moreover, any racemates, enantiomers, or diastereomers of the compounds of formula (I) are encompassed, unless a specific stereochemistry of the compound under consideration is indicated in a specific context.

As illustrated by the reference to the compounds of formula (I) as CXCR4 receptor ligand compounds, the compounds are capable of acting as ligand compounds which bind to the CXCR4 receptor. To that extent, the compounds of formula (I) and their salts comprise a cyclopeptide group R ^CP , which may also be referred to herein as the “binding motif’ of the CXCR4 receptor ligand compounds of formula (I), since it ensures a binding interaction between the compounds in accordance with the invention or their salts and the CXCR4 receptor and thus serves as an affinity anchor for the compounds towards the CXCR4 receptor.

A binding motif of a CXCR4 receptor ligand compound is preferably capable of specifically binding to the CXCR4 receptor. In this connection, specifically binding preferably means that the binding motif of the CXCR4 receptor ligand compound does not, or essentially does not, bind to other proteins than the CXCR4 receptor, in particular does not bind, or essentially does not bind, to other members of the CXC chemokine receptor family. The term “essentially does not bind” preferably means that the binding affinity of the binding motif, as determined e.g. by an IC ₅₀ value, to CXCR4 is at least 100, preferably at least 1000 and most preferably at least 10000 times stronger than the binding affinity to other proteins, in particular other members of the CXC chemokine receptor family.

The cyclopeptide group R ^CP as a binding motif in formula (I) is a group of the formula (II): wherein

R ^B1 is H or I, preferably H; and

R ^B2 is an alkanediyl chain.

As will be understood by the skilled reader, the bond marked by the dashed line in formula (II) does not carry a methyl group at its end opposite to the nitrogen atom, but represents a bond which attaches the group R ^CP to the remainder of the compound of formula (I), i.e. in this case to the point of attachment of R ^CP in formula (I). In other words, the bond marked by the dashed line in formula (II) represents a covalent bond which is present in the compounds in accordance with the invention between the nitrogen atom of the -NH- group indicated in formula (II) and the carbon atom of the carbonyl group shown at the left side of formula (I) to which R ^CP is attached. Thus, an amide bond is provided using the -NH- group of R ^CP shown in formula (II), and the carbonyl group shown in formula (I).

In the group of formula (II), -R ^B2 - is an alkanediyl chain, preferably a C2-C6 alkanediyl chain, more preferably a C2-C3 alkanediyl chain, and most preferably -CH ₂ -CH ₂ -CH ₂ -. Thus, R ^CP is preferably a group of formula (Ila): wherein R ^B1 is defined as for formula (II), including any preferred embodiments of this variable as further defined herein, and wherein the dashed line marks a bond which attaches the group R ^CP to the remainder of the compound of formula (I).

It is still further preferred that R ^CP is a group of formula (IIb):

wherein R ^B1 is defined as for formula (II), including any preferred embodiments of this variable as further defined herein, and wherein the dashed line marks a bond which attaches the group R ^CP to the remainder of the compound of formula (I).

The variable a in formula (I) is either 0 or 1 , such that the group -CH ₂ - attached to the phenylene ring in the linker structure of formula (I) is an optional group which can be present or absent. As will be understood, if it is absent, i.e. if a is 0, the group -CH ₂ - is replaced by a direct bond between the atoms adjacent to the group in formula (I).

The group R ^L1 in formula (I) is H or C1-C6 alkyl, more preferably H or C1-3 alkyl, still more preferably H or methyl, and most preferably methyl.

R ^L2 in formula (I) is substituted alkyl, which substituted alkyl is substituted with at least one group selected from -NH ₂ and -NH-C(=X)-NH ₂ . X is selected from NH and O. The alkyl moiety of the substituted alkyl is preferably C1-C6 alkyl, more preferably C1-C4 alkyl, and still more preferably C2-C4 alkyl. The alkyl moiety is preferably a linear alkyl moiety. The alkyl moiety carries at least one, preferably exactly one, substituent which is selected from -NH ₂ and the group -NH-C(=X)-NH ₂ . X is preferably NH, in which case the group -NH-C(=X)-NH ₂ is a guanidino group. In line with the above, R ^L2 is preferably a group selected from -(CH ₂ ) _A -NH ₂ and -(CH ₂ )A-NH-C(=NH)-NH ₂ , wherein A is an integer of 1 to 6, preferably 1 to 4, and more preferably 2 to 4. Most preferably, R ¹² is -(CH ₂ )3-NH-C(=NH)-NH ₂ .

R ^L3 in formula (I) and its preferred embodiments as defined herein is -CH ₂ -NH ₂ or -CH ₂ -(1H- imidazol-4-yl) and is preferably -CH ₂ -NH ₂ . The group -CH ₂ -(1H-imidazol-4-yl) can be illustrated by the following formula, wherein the dashed line marks a bond which attaches the group to the remainder of the compound of formula (I):

R ^L4 in formula (I) and its preferred embodiments as defined herein is -NH ₂ .

The variable e in formula (I) and its preferred embodiments as defined herein is an integer of 1 to 4, preferably 2 to 4, and is more preferably 2.

X ¹ in formula (I) is a coupling group. As will be understood by the skilled reader, the coupling group X ¹ is a functional group which allows R ^S or R ^A to be coupled to the remainder of the compound of formula (I) via a covalent bond which is formed between the group X ¹ and R ^S or between the group X ¹ and R ^A . The coupling group may consist of one or more atoms. A preferred coupling group X ¹ is selected from -NH-, -C(O)-, -O-, and -S-. Typically, the coupling group X ¹ is covalently linked to a further, complementary coupling group comprised in R ^S or R ^A , so that the two coupling groups combine to form a binding unit, such as an amide bond -C(O)-NH-, an ester bond -C(O)-O-, or a thiosuccinimidyl group which can be illustrated by the following formula, wherein each dashed line marks a bond which attaches the group to an adjacent atom or group within the compound of formula (I). For example, a coupling group X ¹ = -NH- may form an amide bond -NH-C(O)- with a complementary group -C(O)- comprised in R ^S or R ^A ; a coupling group X ¹ = -C(O)- may form an amide bond -C(O)-NH- with a complementary group -NH- comprised in R ^S or R ^A , or may form an ester bond -C(O)-0- with a complementary group -O- comprised in R ^S or R ^A ; a coupling group X ¹ = -O- may form an ester bond -O-C(O)- with a complementary group -C(O)- comprised in R ^S or R ^A , or may form an ether bond -O- with a carbon atom comprised in R ^S or R ^A ; a coupling group X ¹ = -S- may form a thioester bond -S-C(O)- with a complementary group -C(O)- comprised in R ^S or R ^A , or may form a thioether bond -S- with a carbon atom comprised in R ^S or R ^A . In accordance with a preferred embodiment, X ¹ is the sulfur atom -S- and forms a covalent bond with a complementary succinimidyl group comprised in R ^S or R ^A . It will be understood that the latter combination can be conveniently achieved by allowing a compound with a thiol group to react with a compound containing a maleimidyl group.

The variable c in formula (I) is either 0 or 1 , such that the group carrying the substituent R ^L3 contained within the brackets [...] carrying the index c can be present or absent. As will be understood, if it is absent, i.e. if c is 0, the group is replaced by a direct bond. Preferably, c is 1. Likewise, the variable d in formula (I) is either 0 or 1 , such that the group carrying the substituent R ^L4 contained within the brackets [...] carrying the index d can be present or absent. As will be understood, if it is absent, i.e. if d is 0, the group is replaced by a direct bond. However, in the compounds in accordance with the invention, at least one of c and d must be 1.

Thus, it is preferred that the compound of formula (I) has the formula (la), (lb) or (Ic): wherein the variables R ^CP , a, R ^L1 , R ^L2 , R ^L3 , R ^S , b, R ^L4 , e, X ¹ and R ^A are defined as for formula (I), including any preferred embodiments of these variables as further defined herein.

Among these preferred formulae for the compound of formula (I), further preference is given to formula (la):

It is particularly preferred that the compound of formula (I) is a compound of formula (laa): wherein the variables R ^CP , a, R ^L1 , R ^L2 , R ^L3 , R ^S , b and R ^A are defined as for formula (I), including any preferred embodiments of these variables as further defined herein. R ^S in formula (I) and its preferred embodiments as defined herein is a divalent spacer group. The variable b is 0 or 1 , such that the spacer group R ^S is an optional group which can be present or absent. As will be understood, if it is absent, i.e. if e is 0, the group R ^S is replaced by a direct bond between the atoms adjacent to R ^S in formula (I). The group R ^S preferably comprises a linear chain of 3 to 10, preferably of 4 to 6 carbon atoms. This linear chain of carbon atoms extends between the group NH and the group R ^A (if b is 1 and d is 0) or between the group X ¹ and the group R ^A (if b is 1 and d is 1). In addition to the linear chain of 3 to 10, preferably of 4 to 6 carbon atoms, R ^S may comprise one or two, preferably two, coupling groups which allow the chain of carbon atoms to be attached to the group NH and the group R ^A or the group X ¹ and the group R ^A , respectively. Typically, the spacer group R ^S is unbranched and comprises no charged group.

More preferably, the group R ^S comprises a linear alkanediyl chain having 3 to 10, preferably having 4 to 6 carbon atoms. This alkanediyl chain extends between the group NH and the group R ^A (if b is 1 and d is 0) or the group X ¹ and the group R ^A (if b is 1 and d is 1 ). In addition to the linear alkanediyl chain having 3 to 10, preferably having 4 to 6 carbon atoms, R ^6L may comprise one or two, preferably two, coupling groups which allow the alkanediyl chain to be attached to the group NH and the group R ^A or the group X ¹ and the group R ^A , respectively.

Thus, it is preferred that the group R ^S is a group of the formula

-X ² -(CH ₂ ) _B -X ³ - wherein

X ² is a coupling group attached to the group NH in formula (I) if d is 0 or to the group X ¹ in formula (I) if d is 1;

B is an integer of 3 to 10, preferably 4 to 6; and

X ³ is a coupling group attached to R ^A .

For the coupling groups X ² and X ³ , similar considerations apply as for the coupling group X ¹ discussed above. Thus, the coupling group X ² is a functional group which allows R ^S to be coupled either to the NH group via a covalent bond which is formed between the group X ² and NH, or to the group X ¹ via a covalent bond which is formed between the group X ² and X ¹ . The coupling group X ² may consist of one or more atoms. Preferred coupling groups are selected from -NH-, -C(O)-, -O-, and -S-. If X ² is linked to NH, it is preferably -C(O)-, such that X ² and X ¹ form an amide bond -C(O)-NH-. If X ² is linked to X ¹ , the coupling group X ² and X ¹ are typically complementary coupling groups which combine to form a binding unit, such as an amide bond -C(O)-NH-, an ester bond -C(O)-O-, or a thiosuccinimidyl group

A particularly preferred group R ^S is a group of the formula -C(O)-(CH ₂ )B-NH-, wherein B is as defined above, and wherein the bond at the N-terminus is attached to R ^A .

In line with the above, it will be understood that a preferred combination of variables for the compound of formula (I) is the one wherein R ^B2 in R ^CP is -CH ₂ -CH ₂ -CH ₂ -, R ^L1 is methyl, R ^L2 is -(CH ₂ )3-NH-C(=NH)-NH ₂ , c is 1, and the remaining variables are defined as for formula (I) and (II), including any preferred embodiments of these variables as further defined herein. A still more preferred combination of variables for the compound of formula (I) is the one wherein R ^B2 in R ^CP is -CH ₂ -CH ₂ -CH ₂ -, R ^L1 is methyl, R ^L2 is -(CH ₂ ) ₃ -NH-C(=NH)-NH ₂ , c is 1 , d is 0, b is 0 or 1 , R ^S , if present, is -C(O)-(CH ₂ )B-NH-, wherein B is as defined above and wherein the bond at the N-terminus is attached to R ^A , and the remaining variables are defined as for formula (I) and (II), including any preferred embodiments of these variables as further defined herein.

Thus, a particularly preferred compound of formula (I) has the following formula (lab) or the following formula (lac)

wherein R ^B1 , a, R ^S and R ^A are defined as for formulae (I) and (II), respectively, including any preferred embodiments of these variables as further defined herein.

R ^A is a functional group comprising a moiety with diagnostic or therapeutic utility, e.g. a labeling group. As will be understood by the skilled person, a moiety with diagnostic utility is a group which facilitates the detection of the ligand compound in accordance with the invention after administration to a patient or after it has been brought into contact in vitro or ex vivo with a physiological sample, or a precursor of such a group. An example of such a precursor is a group which can carry a radioactive element that can be detected, but wherein such an element is not yet contained, e.g. a SiFA moiety or a chelating moiety. Preferably, a moiety with diagnostic utility is a group or a precursor thereof which allows the compound in accordance with the invention to be detected and located in the body of a patient after its administration to the patient. Due to its affinity to CXCR4, the compound of the invention comprising a moiety with diagnostic utility may function in particular as a tracer for CXCR4. A moiety with therapeutic utility is a group which allows the compound of the invention to treat or prevent a disease or disorder after its administration to a patient, in particular a disease or disorder which can be treated or prevented by blocking of the CXCR4 receptor or which is associated with an increased or aberrant expression of CXCR4, or a precursor of such a group. An example of such a precursor is a group which can carry a radioactive element that has a therapeutic effect, but wherein such an element is not yet contained, e.g. a chelating moiety.

The group R ^A comprises a moiety with diagnostic or therapeutic utility. Preferably, it comprises one or two of these moieties. A combination of two moieties can be useful, e.g., to provide a compound of the invention which combines diagnostic and therapeutic utility.

Preferably, R ^A in the compound formula (I) and its preferred embodiments defined herein comprises, or consist of, at least one of the following moieties with diagnostic or therapeutic utility: (i) a chelating moiety; (ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, preferably a chelated radioactive or non-radioactive cation;

(iii) a silicon-fluoride acceptor (SiFA) moiety which comprises a silicon atom and a fluorine atom, wherein the fluorine atom is linked via a covalent bond directly to the silicon atom, and which SiFA moiety can be labeled with ¹⁸ F by isotopic exchange of ¹⁹ F by ¹⁸ F or which is labeled by ¹⁸ F;

(iv) a cytotoxic moiety; and

(v) a fluorescent moiety.

More preferably, R ^A comprises, or consists of, one of the moieties (i) to (v), or a combination of one moiety selected from the chelating moiety (i) and the chelate (ii), and one SiFA moiety (iii).

The chelating moiety of (i) and (ii) is suitable to form a chelate with a radioactive or non- radioactive cation or anion, preferably a radioactive cation. Suitable chelating agents providing chelating moieties for diverse cations and anions are well known in the art and can be used in the context of the present invention. Metal- or cation-chelating agents, e.g. macrocyclic or acyclic compounds, which are suitable to provide a chelating moiety, are available from a number of manufacturers. It will be understood that numerous chelating agents can be used in an off-the-shelf manner by a skilled person without further ado. It will further be understood that the suitability of the chelating moiety to form a chelate with a given anion or cation requires the chelating moiety to be able to provide a chelate ligand in a chelate complex comprising the anion or cation under consideration, but does not require the chelating moiety to form the only ligand of the anion or cation in the chelate complex. Thus, a chelate in accordance with option (ii) above may comprise a chelated cation or anion, the chelating moiety (i) as a chelating ligand, and an additional ligand coordinated with the chelated cation or anion.

For example, a chelating moiety, may comprise at least one of a macrocyclic ring structure with 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 an acyclic, open chain chelating structure with 8 to 20 main chain atoms of which 2 or more, preferably 3 or more are heteroatoms selected from oxygen atoms, sulfur atoms and nitrogen atoms. Thus, preferred chelating agents which can be used to provide a chelating moiety of (i) or (ii) above are selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1 ,4,8,11-tetraazacyclotetradec- 1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl- (hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandi amide (DFO), 4,11- bis(carboxymethyl)-1,4,8,11-tetraazabicycle[6.6.2]hexadecan (DO2A), 1,4,7,10- tetraazacyclododecan-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'-dipyridoxylethylendiamine-N.N'-diacetate-5,5'-bis(phosp hat) (DROP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N.N'-tetraacetic acid (EDTA), ethyleneglykol-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-tetraazacyclodecan-4,7,10-triacet ate (HP-DOA3), 1,4,7-triazacyclononan-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3- carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1 ,4,7- triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraaza- bicyclo[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- tetraazacyclotridecan-N,N ^, ,N",N'''-tetraacetic acid (TRITA), and triethylenetetra- aminehexaacetic acid (TTHA), N,N' -bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H ₂ macropa), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyri din-2-ylmethyl)- carbamoyl]-ethyl} heptanedioic acid bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-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- tetraazacyclododecan-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-tetraazacyclododecane tetraacetic acid, mercaptoacetyl- triserine (mas ₃ ), hydrazinonicotinic acid (HYNIC) and 3-(2-aminoethylamino)-2-[(2- aminoethylamino)methyl]propanoic acid (N4 chelator, 6-carboxy-1,4,8,11-tetraazaundecane). As a further preferred example, reference can be made to a modified mercaptoacetylserine chelating agent (modified mas ₃ ), wherein one or more of the serine residues are replaced by another amino acid containing a hydrophilic side chain.

Among these preferred chelating agents which can be used to provide a chelating moiety of (i) or (ii) above, particularly preferred is a chelating agent selected from mas ₃ , modified mas ₃ , HYNIC, N4 chelator, DOTA and DOTAGA.

As will be understood by the skilled reader, the preferred and particularly preferred chelating agents listed above can conveniently provide a chelating moiety in a compound in accordance with the invention by using a functional group contained in the chelating agent to provide a binding unit which attaches the chelating moiety to the remainder of the compound. As examples of such a binding unit, reference can be made to an amide bond (-C(O)-NH-) or an ester bond (-C(O)-O-) which can be provided e.g. using a carboxyl group or an amino group which may be contained as a functional group in the chelating agent. chelating moiety provided by the compound of formula (I) or its salt, one or more additional ligands which are coordinated to the chelated cation and which are not part of the compound of formula (I) or its salt, such as an oxo-ligand in a chelate including a ^99m Tc(V)-oxo core.

Particularly preferred as a group R ^A is a group comprising a chelating moiety which can form a chelate with a cation selected from a cation of ^99m Tc, ¹⁷⁷ Lu, ⁶⁷ Ga and ⁶⁸ Ga. Likewise, preferred as a group R ^A is a group comprising a chelate with a chelated cation selected from a cation of 9 ^9m Tc, ¹⁷⁷ Lu, ⁶⁷ Ga and ⁶⁸ Ga.

As noted above, the structure of the linker forming part of the compound of the invention allows a broad variety of functional groups R ^A to be used in these compounds while retaining a high CXCR4 affinity. In the case of a functional group R ^A comprising a chelating moiety as referred to in options (i) and (ii) above, the positive influence of the linker is particularly pronounced for compounds of formula (I) and their salts wherein R ^A is a group other than a group consisting of a DOTA or DOTAGA residue, or other than a group consisting of a residue of a chelator for M ³⁺ (i.e. for three-valent metal cations) in general.

A silicon-fluoride acceptor (SiFA) moiety in line with option (iii) above preferably comprises a group of formula (S-1): wherein

R ^S1 and R ^S2 are independently a linear or branched C3 to C10 alkyl group, preferably R ^S1 and R ^S2 are independently selected from isopropyl and tert-butyl, and more preferably R ^S1 and R ^S2 are both tert-butyl, and wherein the bond marked with the dashed line attaches the group to the remainder of the compound of formula (I). Preferably, the group of formula (S-1) is attached as a substituent to a phenyl ring.

More preferably, the SiFA moiety is a group selected from a group of formula (S-2) and a group of formula (S-3). wherein n is 1 , 2, or 3 and is preferably 1 , R ^S1 and R ^S2 are independently a linear or branched C3 to C10 alkyl group, preferably R ^S1 and R ^S2 are independently selected from isopropyl and tert- butyl, and more preferably R ^S1 and R ^S2 are both tert-butyl, and wherein the bond marked by the dashed line attaches the group to the remainder of the compound of formula (I). Suitable counterions for the positively charged quarternary nitrogen atom indicated in formula (S-3), which carries two methyl substituents, include anions as they are discussed herein with regard to salts formed of the compound of formula (I), and may include, e.g., trifluoro acetate or acetate anions.

As will be understood by the skilled reader, the bond at the carbonyl group marked by the dashed line in formulae (S-2) and (S-3) does not carry a methyl group at its end opposite to the carbonyl group, but represents a bond which attaches the SiFA moiety to the remainder of the compound of formula (I). In other words, the bond marked by the dashed line represents a covalent bond which is present between the carbon atom of the carbonyl group shown in formulae (S-2) and (S-3) and an atom or group adjacent to the group (S-2) or (S-3) in the compounds in accordance with the invention. For example, an amide bond (-C(O)-NH-) or an ester bond (-C(O)-O-), preferably an amide bond, is provided using the carbonyl group shown in formulae (S-2) and (S-3) and a group -NH- or -O- adjacent to these groups. For example, such a group -NH- or -O- may be comprised by a linker forming part of R ^A .

As will be further understood, the fluorine atom contained in formulae (S-1) to (S-3) may be a ¹⁸ F atom or a ¹⁹ F atom which can be exchanged to provide ¹⁸ F by isotopic exchange of ¹⁹ F by ¹⁸ F.

A cytotoxic moiety as option (iv) discussed above may be provided, for example, by a residue of a cytotoxic compound, e.g. an auristatin analogue, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or PF-06380101. The residue may be provided using a functional group contained in the cytotoxic compound to form a binding unit which attaches the cytotoxic moiety to the remainder of the compound in accordance with the invention. Optionally, a metabolically cleavable linker may further be provided as a linker attaching the cytotoxic moiety to the remainder of the compound in accordance with the invention.

A fluorescent moiety as option (v) discussed above may be provided, for example, by a residue of a fluorescent dye. Such fluorescent dyes are known in the art, and include, e.g., Cy5- and Cy7-based cyanine dyes. The residue may be provided using a functional group contained in the fluorescent dye to provide a binding unit which attaches the cytotoxic moiety to the remainder of the compound in accordance with the invention.

In addition to a moiety with diagnostic or therapeutic utility as discussed above, R ^A may comprise one or more further moieties intended for a different purpose. For example, R ^A may comprise a divalent or higher valent linker which allows one or more moieties with diagnostic or therapeutic utility to be attached the remainder of the compound. As another example, reference may be made to a moiety which is comprised by R ^A in order to adjust the hydrophilic/hydrophobic characteristics of the compound in accordance with the invention, e.g. a moiety which carries one or more polar groups.

As will be understood by the skilled person, R ^A comprises a coupling group which allows R ^A to be covalently attached to a group -NH- (if, in the compound of formula (I), d is 0 and b is 0), to a group -X ¹ - (if, in the compound of formula (I), d is 1 and b is 0), or to the terminus of -R ^S - (if, in the compound of formula (I), b is 1). Suitable coupling groups can be selected and provided relying on well-established principles of synthetic chemistry. For example, a coupling group can be contained in a linker optionally comprised by R ^A , or can be a part of the moiety with diagnostic or therapeutic utility comprised by R ^A . For example, if, in the compound of formula (I), d is 0 and b is 0, it is preferred that R ^A comprises a coupling group -C(O)- for attachment to -NH- to provide an amide bond. Such a coupling group can be conveniently derived e.g. from a carboxyl group. Likewise, if, in the compound of formula (I), b is 1 , and R ^S is a group of formula -C(O)-(CH ₂ )B-NH-, it is preferred that R ^A comprises a coupling group -C(O)- for attachment to -NH- to provide an amide bond. As another example, if, in the compound of formula (I), d is 1, b is 0, and X ¹ is -S-, R ^A may comprise a succinimidyl group as a coupling group for attachment to -S- to yield a thiosuccinimidyl group. Such a coupling group can be conveniently derived e.g. from a maleimidyl group.

In line with the above, particularly preferred embodiments of R ^A can be exemplified as follows:

- R ^A is a chelating moiety provided by a mercaptoacetyl triserine (mas ₃ ) chelating agent attached via an amide bond to the remainder of the compound of formula (I);

- R ^A is a chelate comprising a chelating moiety provided by a mercaptoacetyl triserine (mas ₃ ) chelating agent attached via an amide bond to the remainder of the compound of formula (I) and a chelated cation, such as a ^99m Tc cation; - R ^A is a chelating moiety provided by a modified mercaptoacetyl triserine (mas ₃ ) chelating agent attached via an amide bond to the remainder of the compound of formula (I), wherein one or more of the serine residues are replaced by another amino acid residue carrying a hydrophilic side chain, such as citrulline, or by an amino acid residue with a glycosylated side chain;

- R ^A is a chelate comprising a chelating moiety provided by a modified mercaptoacetyl triserine (mas ₃ ) chelating agent attached via an amide bond to the remainder of the compound of formula (I), wherein one or more of the serine residues are replaced by another amino acid residue carrying a hydrophilic side chain, such as citrulline, or by an amino acid residue with a glycosylated side chain, and a chelated cation, such as a ^99m Tc cation;

- R ^A is a chelating moiety provided by a hydrazinonicotinic acid (HYNIC) chelating agent attached via an amide bond to the remainder of the compound of formula (I);

- R ^A is a chelate comprising a chelating moiety provided by a hydrazinonicotinic acid (HYNIC) chelating agent attached via an amide bond to the remainder of the compound of formula (I) and a chelated cation, such as a ^99m Tc cation.

R ^A is a chelating moiety provided by a 3-(2-aminoethylamino)-2-[(2- aminoethylamino)methyl]propanoic acid (N4) chelating agent attached via an amide bond to the remainder of the compound of formula (I) or R ^A comprises a chelating moiety provided by an N4 chelating agent attached via an amide bond to the remainder of the compound of formula (I):

- R ^A is a chelate comprising a chelating moiety provided by a 3-(2-aminoethylamino)-2-[(2- aminoethylamino)methyl]propanoic acid (N4) chelating agent attached via an amide bond to the remainder of the compound of formula (I) and a chelated cation, such as a ^99m Tc cation, or R ^A comprises a chelate comprising a chelating moiety provided by an N4 chelating agent attached via an amide bond to the remainder of the compound of formula (I) and a chelated cation, such as a ^99m Tc cation;

- R ^A comprises a SiFA moiety and a chelating moiety, such as a chelating moiety provided by a DOTA or a DOTAGA chelating agent attached via an amide bond to the remainder of the compound of formula (I);

- R ^A comprises a SiFA moiety and a chelate which comprises a chelating moiety, such as a chelating moiety provided by a DOTA or a DOTAGA chelating agent, and a chelated cation, such as a ¹⁷⁷ Lu cation a ⁶⁸ Ga cation or a ⁶⁹ Ga cation;

- R ^A comprises a cytotoxic moiety provided by MMAE; or

- R ^A comprises a fluorescent moiety provided by the fluorescent dye Cy5.5. The presence of R ^S (i.e. the selection of b as 1 in formula (I)) may lead to an additional benefit with a view to a high CXCR4 affinity of the compounds of the present invention in particular if the group R ^A has a high molecular weight, if it contains a bulky group in the proximity to its point of attachment to the remainder of the compound of formula (I), or if the group R ^A comprises negatively charged functional groups (without taking into account functional groups wherein a negative charge is neutralized by the formation of a chelate complex). Thus, as an orientation, it is expected that the presence of the optional spacer group R ^S additionally improves the affinity for compounds containing a group R ^A which fulfills two of the following three requirements: i) the molecular weight of R ^A is more than 300 g/mol; ii) R ^A has a charge of < -1 if the compound of formula (I) is kept in a solution at neutral pH (not taking into account charged groups in chelating agents which are neutralized by the formation of a chelate complex); iii) R ^A comprises a phenyl ring or a larger aromatic group in the proximity to the point of attachment of R ^A to the remainder of the compound of formula (I), e.g. less than 7 covalent bonds (C-C bonds, C-O-bonds, or C-N bonds) away from the atom to which R ^A is attached.

As noted above, the ligand compounds in accordance with the invention encompass the compounds of formula (I) and their salts, preferably pharmaceutically acceptable salts. Such salts may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, 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, e.g. by neutralization with a base. Other charged groups which may be present in the compounds in accordance with the invention include groups which are continuously charged, such as quaternary ammonium cations substituted by four organyl groups or charged chelate complexes.

As exemplary anions which may be present in salt forms of the compounds of the invention if the salt form comprises a positively charged form of the compound of formula (I), mention may be made, for example, of an anion selected from chloride, bromide, iodide, sulfate, nitrate, phosphate (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate, hydrogencarbonate or perchlorate; acetate, trifluoroacetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, undecanoate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, nicotinate, benzoate, salicylate or ascorbate; sulfonates such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate (tosylate), 2-naphthalenesulfonate, 3-phenylsulfonate, or camphorsulfonate. Since trifluoroacetic acid is frequently used during the synthesis of peptides, trifluoroacetate salts are typical salts which are provided if a compound comprising a peptide structure is formed. Such trifluoroacetate salts may be converted to acetate salts during their workup. As exemplary cations which may be present in salt forms of the compounds of the invention if the salt form comprises a negatively charged form of the compound of formula (I), mention may be made, for example, of a cation selected from alkali metal cations, such as lithium, sodium or potassium, alkaline earth metal cations, such as calcium or magnesium; and ammonium (including ammonium ions substituted by organic groups).

The ligand compound in accordance with the invention is preferably capable of binding to human CXCR4 with an affinity reflected by an IC ₅₀ value of 100 nM or less, more preferably 10 nM or less, and still more preferably 5 nM or less.

As exemplary compounds in accordance with the invention, the following are mentioned.

A compound of the following formula, a compound wherein the mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these:

a compound of the following formula, a compound wherein the mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the modified mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these:

a compound of the following formula, a compound wherein the modified mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the modified mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the modified mas ₃ chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the HYNIC chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the N4 chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the N4 chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the N4 chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTAGA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein any of the DOTAGA chelating moieties shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTAGA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTAGA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTAGA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, or a salt thereof: a compound of the following formula, or a salt thereof:

a compound of the following formula, or a salt thereof: a compound of the following formula, a compound wherein the DOTAGA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these: a compound of the following formula, or a salt thereof: a compound of the following formula, or a salt thereof: and a compound of the following formula, a compound wherein the DOTA chelating moiety shown in the formula forms a chelate with a chelated radioactive or non-radioactive cation, or a salt of any of these:

In a further aspect, the present invention provides a pharmaceutical composition (also referred to as a therapeutic composition) comprising or consisting of one or more types, preferably one type, of the ligand compound in accordance with the invention, i.e. a compound of formula (I) including any preferred embodiments thereof as discussed herein, or a salt thereof. In a related aspect, the ligand compound in accordance with the invention is provided for use in therapy or for use as a medicament. Thus, the ligand compound of the invention can be used in a therapeutic method, which method may comprise administering the ligand compound to a subject. The subject may be a human or an animal and is preferably a human. It is to be understood that in accordance with these medical aspects of the invention the functional group R ^A generally comprises a moiety with therapeutic utility, e.g. a group which carries a radioactive element that has a therapeutic effect, or a cytotoxic moiety.

The therapy or therapeutic method referred to above aims at the treatment or prevention of a disease or disorder of the human or animal body, generally a disease or disorder that can be treated or prevented by blocking the CXCR4 receptor or which is associated with increased or aberrant expression of the CXCR4 receptor, such as cancer, a cardiovascular disorder or an inflammatory disorder. Thus, in terms of a therapeutic application, the compound of the invention is preferably provided for use in the treatment or prevention of cancer, an inflammatory disorder or a cardiovascular disorder, such as atherosclerosis, myocardial infarction, or stroke.

Due to the versatility provided by the invention in terms of the choice of the group R ^A in formula (I), the compounds of the invention can be conveniently adapted to various therapeutic approaches. For example, a compound in accordance with the invention comprising a chelating moiety capable of forming a chelate with a radioactive component, e.g. a radioactive metal cation, or a compound comprising such a chelate, can be used for radiotherapy, in particular targeted radioligand therapy (RLT). For radiotherapy, the chelated radioactive cation is preferably a gamma or beta emitter since they may emit a radiation dose in the target area that weakens or destroys particular targeted cells. Examples of gamma or beta emitters are ¹⁷⁷ Lu, ⁸⁹ Zr and ¹⁸⁶ Re. As another example, a compound in accordance with the invention comprising a cytotoxic moiety may be provided for use in a therapy involving the chemotherapeutic destruction of cells, e.g. cancer cells.

In another aspect, the present invention provides a diagnostic composition comprising or consisting of one or more types, preferably one type, of the ligand compound in accordance with the invention, i.e. a compound of formula (I) including any preferred embodiments thereof as discussed herein, or a salt thereof. In a related aspect, the ligand compound in accordance with the invention is provided for use in a method of diagnosis in vivo of a disease or disorder. Thus, the ligand compound in accordance with the invention can be used in a method of diagnosis, which method may comprise 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 thereby detecting or monitoring the disease to be diagnosed. The subject may be a human or an animal and is preferably human. Alternatively, a method of diagnosis may also comprise adding the ligand compound to a sample, e.g. a physiological sample obtained from a subject in vitro or ex vivo and detecting the ligand compound in the sample. It is to be understood that in accordance with these diagnostic aspects of the invention the functional group R ^A generally comprises a moiety with diagnostic utility, such as a group carrying a detectable radioactive element, or a fluorescent moiety.

The method of diagnosis referred to above aims at the identification of a disease or disorder of the human or animal body, generally a disease or disorder that can be treated or prevented by blocking the CXCR4 receptor or which is associated with increased expression of the CXCR4 receptor, such as cancer, a cardiovascular disorder or an inflammatory disorder. Thus, in terms of a diagnostic application, the compounds of the invention are preferably provided for use in a method of diagnosis in vivo of cancer, a cardiovascular disorder or an inflammatory disorder, e.g. a disorder such as atherosclerosis, myocardial infarction, or stroke.

Due to the versatility provided by the invention in terms of the choice of the group R ^A in formula (I), the compounds of the invention can be conveniently adapted to various diagnostic approaches. For example, a compound in accordance with the invention comprising a chelating moiety capable of forming a chelate with a radioactive component, e.g. a radioactive metal cation, a compound comprising such a chelate, or a compound comprising an ¹⁸ F atom, can be used for nuclear diagnostic imaging. For instance, if the compound in accordance with the invention comprises a positron emitter, such as a chelated ⁶⁴ Cu cation, a chelated ⁶⁸ Ga cation, or a ¹⁸ F atom bound in a SiFA moiety, the compound can be used for diagnosis via positron emission tomography (PET) imaging. Other compounds in accordance with the invention may be used for diagnosis via single photon emission computerised tomography (SPECT) imaging, e.g. a compound comprising a chelated ^99m Tc cation. As further examples, a compound in accordance with the present invention comprising a SiFA moiety can be used for diagnosis via ¹⁹ F MRI, or a compound in accordance with the invention comprising a fluorescent moiety can be used in a diagnostic method involving optical imaging.

It will be understood that suitability for a therapeutic and a diagnostic application is not mutually exclusive, i.e. a compound in accordance with the invention may be suitable for both applications and, thus, a functional group R ^A may comprise a moiety with both diagnostic and therapeutic utility, or a moiety with diagnostic utility and a moiety with therapeutic utility as separate moieties. For example, the compounds in accordance with the invention may comprise a radioactive species active in radiotherapy and diagnostic imaging. Moreover, the compounds of the invention encompass radiohybrid compounds which comprise both a SiFA moiety and a chelating moiety suitable to form a chelate with a therapeutically active radioactive cation. Such a radiohybrid compound may be used for diagnostic purposes if the SiFA moiety carries a ¹⁸ F atom, and the chelating moiety forms a chelate with a cold (non- radioactive) cation, and it may be used for therapeutic purposes if the SiFA moiety carries a ¹⁹ F atom and the chelating moiety forms a chelate with a corresponding hot (radioactive) cation. Advantageously, if the radioactive and the non-radioactive cation are different isotopes of the same chemical species, the pharmacokinetic properties of the diagnostic and the therapeutic variant of the radiohybrid compound remain the same. Exemplary radiohybrid ligand compounds for use in diagnosis and therapy are a compound combining an ¹⁸ F atom/a ^nat Ga cation and compound combining a ¹⁹ F atom/a ⁶⁸ Ga cation, a compound combining an ¹⁸ F atom/a ^nat Y cation and compound combining a ¹⁹ F atom/a ⁹⁰ Y cation, or a compound combining an ¹⁸ F atom/a ^nat Lu cation and compound combining a ¹⁹ F atom/a ¹⁷⁷ Lu cation.

The 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 solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be accomplished in different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery. The compositions may be administered directly to the target site. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present e.g. in amounts between 0,1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

The following items summarize aspects of the invention. It will be understood that these items are closely related to the above parts of the description, and that the information provided in these items may supplement the above parts of the description and vice versa.

1. A compound of formula (I) or a salt thereof: wherein: a is 0 or 1 , preferably 0; b is 0 or 1 ; c is 0 or 1 , and d is 0 or 1 , with the proviso that at least one of c and d is 1 ; e is an integer of 1 to 4, preferably 2 to 4;

R ^CP is a cyclopeptide group of formula (II): wherein, in formula (II)

R ^B1 is H or I, preferably H;

R ^B2 is an alkanediyl chain; and wherein the dashed line marks a bond which attaches the group R ^CP to the remainder of the compound of formula (I);

R ^L1 is H or alkyl;

R ^L2 is substituted alkyl, which substituted alkyl is substituted with at least one group selected from -NH ₂ and -NH-C(=X)-NH ₂ with X being selected from NH and O;

R ^L3 is -CH ₂ -NH ₂ or -CH ₂ -(1H-imidazol-4-yl);

R ^L4 is -NH ₂ ;

X ¹ is a coupling group; R ^S is a divalent spacer group; and

R ^A is a functional group comprising a moiety with diagnostic or therapeutic utility.

2. The compound or salt of item 1 , wherein R ^CP in formula (I) is a group of formula (Ila):

wherein R ^B1 is defined as in item 1 , and wherein the dashed line marks a bond which attaches the group R ^CP to the remainder of the compound of formula (I).

3. The compound or salt of item 1 or 2, wherein the group R ^L1 in formula (I) is H or C1-C6 alkyl, more preferably H or C1-3 alkyl.

4. The compound or salt of item 3, wherein the group R ^L1 in formula (I) is methyl.

5. The compound or salt of any of items 1 to 4, wherein R ^L2 in formula (I) is C1-C6 alkyl, more preferably C1-C4 alkyl, and still more preferably C2-C4 alkyl, carrying one substituent which is selected from -NH ₂ and the group -NH-C(=X)-NH ₂ , wherein X is NH or O.

6. The compound or salt of item 5, wherein R ^L2 in formula (I) is a group selected from -(CH ₂ )A-NH ₂ and -(CH ₂ )A-NH-C(=NH)-NH ₂ , wherein A is an integer of 1 to 6, preferably 1 to 4, and more preferably 2 to 4.

7. The compound or salt of item 6, wherein R ^L2 in formula (I) is -(CH ₂ )3-NH-C(=NH)-NH ₂ .

8. The compound or salt of any of items 1 to 7, wherein R ^L3 in formula (I) is -CH ₂ -NH ₂ . 9. The compound or salt of any of items 1 to 8, wherein e in formula (I) is 2.

10. The compound or salt of any of items 1 to 9, wherein X ¹ in formula (I) is -S'

11. The compound or salt of any of items 1 to 10, wherein c in formula (I) is 1 and d in formula (I) is 0, or wherein c in formula (I) is 0 and d in formula (I) is 1.

12. The compound or salt of any of items 1 to 10, wherein c in formula (I) is 1 and d in formula (I) is 0.

13. The compound or salt of any of items 1 to 12 wherein b in formula (I) is 0.

14. The compound or salt of any of items 1 to 12, wherein R ^S in formula (I) is -C(O)-(CH ₂ )B-

NH wherein B is an integer of 3 to 10, preferably 4 to 6, and wherein the bond at the N- terminus is attached to R ^A .

15. The compound or salt of any of items 1 to 14, wherein R ^A in formula (I) comprises one or two moieties with diagnostic or therapeutic utility.

16. The compound of or salt any of items 1 to 15, wherein the moiety with diagnostic or therapeutic utility comprised by R ^A in formula (I) is selected from:

(i) a chelating moiety;

(ii) a chelate formed by a chelating moiety (i) with a chelated radioactive or non-radioactive cation or anion, preferably a chelated radioactive or non-radioactive cation;

(iii) a silicon-fluoride acceptor (SiFA) moiety which comprises a silicon atom and a fluorine atom, wherein the fluorine atom is linked via a covalent bond directly to the silicon atom, and which SiFA moiety can be labeled with ¹⁸ F by isotopic exchange of ¹⁹ F by ¹⁸ F or which is labeled by ¹⁸ F;

(iv) a cytotoxic moiety; and

(v) a fluorescent moiety.

17. The compound or salt of item 16, wherein R ^A in formula (I) comprises one of the moieties (i) to (v), or comprises a combination of one moiety selected from the chelating moiety (i) and the chelate (ii), and one SiFA moiety (iii). 18. The compound or salt of item 16 or 17, wherein the chelating moiety referred to in (i)

19. The compound or salt of any of items 16 to 18, wherein the chelating moiety referred to in (i) and (ii) is provided by a chelating agent selected from bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5- [acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydro xy)amino]-4- oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11- tetraazabicycle[6.6.2]hexadecan (DO2A), 1,4,7,10-tetraazacyclododecan-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'-dipyridoxylethylendiamine-N.N'-diacetate- 5,5'-bis(phosphat) (DROP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine- N,N’-tetraacetic acid (EDTA), ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (MEDIA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan- 4,7,10-triacetate (HP-DOA3), 1,4,7-triazacyclononan-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacycl ononane

(NODAGA), 1,4,7-triazacyclononanetriacetic 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 -tetra- acetic acid (TETA), terpyridine-bis(methyleneamine) tetraacetic acid (TMT), 1,4,7,10- tetraazacyclotridecan-N,N ^, ,N",N'" -tetraacetic acid (TRITA), and triethylenetetra- aminehexaacetic acid (TTHA), N,N'-bis[(6-carboxy-2-pyridyl)methyl]-4,13-diaza-18-crown-6 (H ₂ macropa), 4-amino-4-{2-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyri din-2-ylmethyl)- carbamoylj-ethyl) heptanedioic acid bis-[(3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-pyridin-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- tetraazacyclododecan-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-tetraazacyclododecane tetraacetic acid, mercaptoacetyl- triserine (mas ₃ ), hydrazinonicotinic acid (HYNIC) 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, wherein one or more of the serine residues are replaced by another amino acid containing a hydrophilic side chain.

20. The compound or salt of any of items 16 to 19, wherein R ^A comprises a chelating moiety which can form a chelate with a cation selected from a cation of ^99m Tc, ¹⁷⁷ Lu and ⁶⁸ Ga, or wherein R ^A comprises a chelate with a chelated cation selected from a cation of ^99m Tc, ¹⁷⁷ Lu and ⁶⁸ Ga.

21. The compound or salt of any of items 16 to 20, wherein the SiFA moiety (iii) comprises a group of formula (S-1): wherein

R ^S1 and R ^S2 are independently a linear or branched C3 to C10 alkyl group, preferably R ^S1 and R ^S2 are independently selected from isopropyl and tert-butyl, and more preferably R ^S1 and R ^S2 are both tert-butyl, and wherein the dashed bond attaches the group to the remainder of the compound of formula (I).

22. The compound or salt of item 21, wherein the SiFA moiety (iii) is selected from a group of formula (S-2) and a group of formula (S-3). wherein n is 1 , 2, or 3 and is preferably 1 , R ^S1 and R ^S2 are independently a linear or branched C3 to C10 alkyl group, preferably R ^S1 and R ^S2 are independently selected from isopropyl and tert- butyl, and more preferably R ^S1 and R ^S2 are both tert-butyl, and wherein the bond marked by the dashed line attaches the group to the remainder of the compound of formula (I).

23. The compound or salt of any of items 16 to 22, wherein the cytotoxic moiety (iv) is provided by a residue of an auristatin analogue, preferably selected from monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF), or by a residue of PF-06380101.

24. The compound or salt of any of items 16 to 23, wherein the fluorescent moiety (v) is provided by a residue of a fluorescent dye, preferably a Cy5- or Cy7-based cyanine dye.

25. A pharmaceutical composition comprising or consisting of a compound or salt of any of items 1 to 24.

26. The compound or salt of any of items 1 to 24 for use as a medicament.

27. The compound or salt of any of items 1 to 24 for use in the treatment or prevention of a disease or disorder that can be treated or prevented by blocking the CXCR4 receptor, or of a disease or disorder that is associated with an increased or aberrant expression of the CXCR4 receptor.

28. The compound or salt of any of items 1 to 24 and 27 for use in the treatment or prevention of cancer, a cardiovascular disorder or an inflammatory disorder.

29. A diagnostic composition comprising or consisting of a compound or salt of any of items 1 to 24. 30. The compound or salt of any of items 1 to 24 for use in a method of diagnosis in vivo of a disease or disorder.

31. The compound or salt of any of items 1 to 24 for use in a method of diagnosis in vivo of a disease or disorder that can be treated or prevented by blocking the CXCR4 receptor or of a disease or disorder that is associated with an increased or aberrant expression of the CXCR4 receptor.

32. The compound or salt of any of items 1 to 24 and 31 for use in a method of diagnosis in vivo of cancer, a cardiovascular disorder or an inflammatory disorder.

In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

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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

 Examples

I. Materials and Methods

1. GENERAL INFORMATION

1.1 REAGENTS AND SOLVENTS

Purchased reagents were used without further purification. The 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). The 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).

Chematech (Dijon, France) delivered the used DOTA-GA chelators and DOTA derivatives was provided by Macrocyclics (Plano, USA). The Cy5-carboxylic acid was obtained from Lumiprobe (Hunt Valley, USA). Cytotoxic MMAE derivatives were purchased from Creative Biolabs (Shirley, USA).

Biochemicals, such as DMEM (Ham’s F-12, with stable Gin) and RPM1 1640 (w. Gin) medium, fetal bovine serum (FBS superior), phosphate-buffered saline (PBS Dulbecco, w/o Ca ²⁺ , Mg ²⁺ ), trypsine/EDTA (0.05%/0.02% in PBS, w/o Ca ²⁺ , Mg ²⁺ ) and Hank’s buffered salt solution (HBSS, with 0.35 g/L NaHCO ₃ and Ca ²⁺ , Mg ² ) were obtained from Biochrom GmbH (Berlin, Germany) or Sigma-Aldrich (Munich, Germany).

Water for RP-HPLC solvents was obtained from the in-house Millipore system from Thermo Fischer Scientific Inc. (Waltham MA, USA). Tracepure water for labeling experiments was received from Merck Millipore (Darmstadt, Germany).

1.2 RADIOACTIVE ISOTOPES

Labeling with ¹²⁵ l was carried out with a [ ¹²⁵ l]Nal solution in NaOH (40 mM, 74 TBq/mmol) from Hartmann Analytik GmbH (Braunschweig, Deutschland). [ ^99m Tc]-Pertechnetate was obtained by elution of a Drytech™ Technetium Generator from GE Healthcare (Munich, Germany) with physiological NaCI solution (0.9%; v/v). The generator was provided by the Klinikum Rechts der Isar (Technical University Munich, Munich, Germany).

A solution of [ ¹⁷⁷ Lu]LuCI ₃ (HCI (0.04 M); SA > 3 TBq/mg, 740 MBq/mL) was provided by ITM GmbH (Garching, Germany) and used directly for labeling experiments.

[ ¹⁸ F]-Fluoride in target water was provided by the Klinikum Rechts der Isar (Technical University Munich, Munich, Germany).

[ ⁶⁸ Ga]GaCI ₃ for radiosynthesis was obtained by elution of a ⁶⁸ Ge/ ⁶⁸ Ga-generator from iThemba LABS (Cape Town, South Afrika) with aqueous HCI (1.00 M). Synthesis was carried out on an automated GallElut ⁺ sytem from Scintomics GmbH (Furstenfeldbruck, Germany).

[ ⁶⁷ Ga]Ga-citrate was delivered by Mallinckrodt Pharmaceuticals (Dublin, Ireland) and converted to [ ⁶⁷ ]GaCI ₃ prior to radiosynthesis.

1.3 INSTRUMENTS AND ANALYTICS

Solid-phase peptide synthesis (SPPS) was carried out by manual operation using an Intelli- Mixer syringe shaker from Neolab (Heidelberg, Germany).

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% H ₂ O. Analytical and semi-preparative reversed-phase high pressure chromatography (RP-HPLC) runs were performed using Shimadzu gradient systems from Shimadzu Deutschland GmbH (Neufahrn, Germany), each equipped with a SPD-20A UV/Vis detector (A = 220 nm, 254 nm). A Multokrom 100 C18 (125 x 4.6 mm, 5 μm particle size) column provided by CS GmbH (Langerwehe, Germany) was used for analytical RP-HPLC runs at a flow rate of 1 mL/min. Both specific gradients and the corresponding retention times t _R are cited in the text. Semi-preparative HPLC purification was performed with a Multokrom 100 RP 18 (250 x 10 mm, 5 μm particle size) column from CS GmbH (Langerwehe, Germany) at a constant flow rate of 5 mL/min.

Analytical and semi-preparative radio-RP-HPLC was performed using a Multokrom 100 C18 (5 μm, 125 x 4.0 mm) column from CS GmbH (Langerwehe, Germany). Radioactivity was detected by connection of the outlet of the UV-photometer to a Nal(TI) well-type scintillation counter from EG&G Ortec (Munich, Germany). Radioactive probes such as mouse organs or cell-test vials were measured on a WIZARD ² ® 2480 automatic y-Counter from Perkin Elmer (Waltham MA, USA).

Radio-TLC measurements were conducted on a Scan-RAM™ from LabLogic Sstems Ltd. (Broomhill, UK). Chromatograms thereof were analyzed using the Laura™ software from LabLogic Sstems Ltd. (Broomhill, UK).

RP-HPLC chromatograms were evaluated using the LabSolution software from Shimadzu Corp. (Kyoto, Japan)

Mass spectra for characterization of organic substances were acquired on an expression ^L CMS quadrupole mass spectrometer from Advion Ltd. (Harlow, UK). NMR spectra were recorded on a Bruker AVHD-300 or AVHD-400 spectrometers from Bruker Corporation (Billerica, USA) at 300 K. pH values were measured with a SevenEasy pH-meter from Mettler Toledo (Gießen, Germany).

Purification via flash-chromatography was carried out on an Isolera™ Prime System from Biotage (Uppsala, Sweden), running a Biotage 09474 Rev. E Bio pump. A Biotage™ SNAP KP-C ₁₈ cartridge (12 g, 93 A pore diameter, 382 m ² /g surface) was used applying a linear gradient of solvent B (ACN, 0.1 vol% TEA, 2 vol% H ₂ O) in solvent A (H ₂ O, 0.1 vol% TEA).

Lyophilization of peptides was carried out using an Alpha 1-2 LDplus lyophilization instrument from Christ (Osterode am Harz, Germany), employing a RZ-2 vacuum pump from Vacubrand GmbH (Wertheim, Germany). IC ₅₀ values were calculated using GraphPad Prism 6 from GraphPad Software Inc. (San Diego, USA).

2. SYNTHESIS

2.1 SOLID-PHASE PEPTIDE SYNTHESIS FOLLOWING THE FMOC-STRATEGY

GP1: 2-CTC-resin loading

Loading of the 2-CTC resin with a Fmoc-protected amino acid (AA) was carried out by stirring a suspension of the 2-CTC-resin (1.6 mmol/g) and Fmoc-AA-OH (1.5 eq.) in DMF with DIPEA (3.0 eq.) at room temperature for 2-5 h. Remaining tritylchloride was capped by the addition of methanol (5 mL/g resin) and incubation for 15 min. Subsequently the resin was filtered off and washed with DMF (5 x 5 mL/g resin) and methanol (3 x 5 mL/g resin) and dried in vacuo. Final loading I of the resin with the Fmoc-AA-OH was determined by the following equation:

GP2: On-resin peptide coupling

The respective side-chain protected Fmoc-AA-OH (1.5 eq.) was dissolved in DMF (8 mL/g resin) and pre-activated by adding TBTU (1.5 eq.), HOBt (1.5 eq.) and DIPEA (3 eq.). For amino acids with low reactivity or peptide fragment condensation, HATU (1.5 eq.), HOAt (1.5 eq.) instead of TBTU and HOBt were used. After activation for 15 minutes, the solution was added to resin-bound free amine peptide 2-CTC-AA-NH ₂ and shaken for 2h at room temperature. For dap(Boc)-OH, dap(Dde)-OH and cys(Trt)-OH as well as fragments bearing these amino acids on their C-terminus, preactivation was shortened to 2-5 min and 2,4,6- Collidine was used as base. For peptide fragments, prolonged reaction times were often needed up to 48 h at r.t.. Subsequently, the resin was washed with DMF (6 x 5 mL/g resin) and after Fmoc-deprotection, the next amino acid was coupled analogously.

GP3: On-resin Fmoc-deprotection

The resin-bound Fmoc-peptide was treated with 20% piperidine in DMF (v/v, 8 mL/g resin) for 5 min and subsequently for 15 min. Afterwards, the resin was washed thoroughly with DMF (8 x 5 mL/g resin).

GP4: On-resin Dde-deprotection:

The Dde-protected peptide (1.0 eq.) was treated with a solution of 2% hydrazine monohydrate in DMF (v/v, 5 mL/g resin) and shaken for 15 min. In the case of present Fmoc-groups, Dde- deprotection was performed by adding a solution of imidazole (0.46 g), hydroxylamine hydrochloride (0.63 g) in NMP (2.5 mL) and DMF (0.5 mL) for 3 h at r.t.. After deprotection the resin was washed with DMF (6 x 5 mL/g resin). GP5: tBu/Boc/Pbf/Trt deprotection

Removal of tBu/Boc/Pbf-protecting groups was carried out by dissolving the crude product in TFA and stirring for 90 min at r.t.. For removal of Trt protecting groups, TIPS was added to the mixture. After removing TFA under a stream of nitrogen, the residue was dissolved in a mixture of f-butanol and water. After lyophilization the crude deprotected peptide was obtained.

GP6: N-Acetylation

Acetylation of an amine functionality was achieved by reacting the respective peptide with a mixture of DIPEA (5.00 eq.) and Ac ₂ O (5.00 eq.) in DMF for 2 h.

GP7: Iodination of CPCR4

Iodination of the CPCR4 tyrosine was carried out according to the published procedure (39). In short, the fully deprotected and purified peptide was dissolved in ACN/H ₂ O (1/1 (v/v), 1.00 mM) and 0.30 to 0.50 eq. of NIS were added. After 5 min at r.t, the reaction mixture was subjected to HPLC purification.

GP8: Condensation of fragments in solution

Connection of the CPCR4 binding motif with functional fragments was carried out in DMF employing small molar amounts of the synthesized fragment (1.10 - 1.30 eq.) and HOAt/HATU as coupling reagents. If the activated amino acid was dap, 2,4,6-Collidine was used as base, in every other case DIPEA.

GP9: Peptide cleavage off the resin

Preservation of acid labile protecting groups

The resin-bound peptide was treated with a mixture of DCM/HFIP (4/1 (v/v), 8 mL/g resin) and shaken for 60 min. The solution containing the fully protected peptide was filtered off and the resin was treated with another portion of the cleavage solution for 60 min. Both fractions were combined, and the solvents removed under reduced pressure. After lyophilization, the crude fully protected peptide was obtained. Cleavage of acid-labile protecting groups

The fully protected resin-bound peptide was treated with a mixture of TFA/TIPS/H ₂ O (95/2.5/2.5 (v/v/v)) and shaken for 30 min. The solution was filtered off and the resin was treated in the same way for another 30 min. Both filtrates were combined and concentrated under a stream of nitrogen. After dissolving the residue in a mixture of t-butanol and water and subsequent lyophilization, the crude peptide was obtained.

GP10: Sulfhydryl-Maleimido coupling

Cysteine- or homocysteine-bearing peptides were coupled with maleimide functionalities by following procedure. The fully unprotected HS-peptide was dissolved in DMF (1.00 mg/mL) and added to the Maleimide bearing substance in DMF (1.10 mg/mL). DIPEA (0.50 μL/mg peptide) was added and the reaction mixture was allowed to stir at r.t. for 2 h. Completion of the reaction was confirmed by RP-HPLC and purification carried out by semi-preparative RP- HPLC.

2.2 ^NAT GA/ ^NAT LU COMPLEXATION

For the complexation of DOTA- and DOTA-GA-bearing peptides with ^nat Ga and ^nat Lu, the fully deprotected and purified peptides were used. Peptides were dissolved in DMSO, DMSO/H ₂ O mixtures or H ₂ O, ideally to a concentration of 1 mM. The required amounts of peptide (30 - 500 nmol) were given into an Eppendorf tube and ^nat GaNO ₃ or ^nat LuCl ₃ (3.00 eq. each) in H ₂ O were added. The vial was heated to 95°C for 30 min and the quantitative conversion checked by RP-HPLC and ESI-MS. The reaction mixture was used without further purification, if no educt was traceable.

2.3 SYNTHESIS OF BUILDING BLOCKS

2.3.1 SYNTHESIS OF CYCLO(D-TYR-D-[NME]ORN-ARG-2-NAL-GLY) (= CPCR4)

The synthesis of the CXCR4 binding motif CPCR4 was realized in analogy to a previously described procedure (40). In short, 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 Fmoc deprotected and newly protected by reaction with O-NBS-CI (4.00 eq.) and 2,4,6 - Collidine (10.0 eq.) in NMP for 15 min. Methylation of the N-terminus was achieved by either employing Mitsunobu conditions (Ph ₃ P (5.00 eq.), MeOH (10.0 eq.), DIAD (5.00 eq.) in THF, 10 min) or Dimethylsulfate (Me ₂ SO ₄ (10.0 eq.), DBU (3.00 eq.) in NMP, 2x2 min) (41). Deprotection of the methylated terminus was achieved by incubation of the peptide with DBU (5.00 eq.) for 5 min before addition of mercaptoethanol (10.00 eq.). After 30 min, the resin was washed thoroughly. The following coupling of Fmoc- D-Tyr(tBu)-OH was achieved by using HOAt and HATU as coupling reagents. After final Fmoc deprotection, the peptide was cleaved off the resin under retention of acid-labile protecting groups (GP9). Cyclization was carried out using DPPA (3.00 eq.) and NaHCO ₃ (5.00 eq.) in a 1mM solution of peptide in DMF. After completion of the reaction, monitored by RP-HPLC, the product solution was concentrated under reduced pressure. The resulting crude product was fully deprotected by treatment with TFA before precipitation in Et ₂ O. Purification of the peptide by flash-chromatography yielded an off-white solid.

CPCR4: RP-HPLC (10 - 95% B in 15 min): t _R = 6.49 min. Calculated monoisotopic mass (C36H47N9O6): 701.36, found: 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-DIOXOPE NTAN- 2-YL)UREIDO)-5-OXOPENTANOIC ACID (= D-(TBU)E(OH)U-D-E(TBU) ₂ ) [3]

Di-tert-butyl-(1H-imidazole-1-carbonyl)-D-glutamate [1]

Synthesis was carried out according to the published procedure (42) with minor variation. In short, D-glu(OtBu)-OtBu (1.00 eq.) was dissolved in DCM and treated with TEA (2.50 eq.) and DMAP (0.04 eq.) on ice. GDI (1.10 eq.) was added and the mixture was stirred over night without further cooling. The reaction was stopped by addition of NaHCO ₃ , sat. and the organic layer washed with H ₂ O and brine twice each. The solvent was evaporated and the crude product, [1] was obtained as a colorless oil (91% yield). The product was used in subsequent reactions without further purification. Di-tert-butyl-(1H-imidazole-1-carbonyl)-D-glutamate [1]: RP-HPLC: (10 - 90% B in 15 min): 1R = 14.50 min. Calculated monoisotopic mass (C ₁₇ H ₂ 7N ₃ O ₅ ): 353.2, found: 376.3 [M+Na] ⁺ .

5-Benzyl-1-(tert-butyl)-(((R)-1.5-di-tert-butoxv-1.5-diox opentan-2-yl)carbamoyl)-d- qlutamate [2]

The educt [1] (1.00 eq.) was dissolved in DCE and cooled on ice before addition of TEA (2.00 eq.) and D-glu(OBn)-OtBu (1.00 eq.). The mixture was heated to 40°C over night. The solvent was then concentrated in vacuo and the crude product underwent silica flash- chromatography employing EtOAc/n-hexane/TEA (500/500/0.8 (v/v/v)). After removal of the solvents 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-diox opentan-2-yl)carbamoyl)-d-glutamate [2]: RP-HPLC: (10 - 90% B in 20 min): t _R = 17.43 min. Calculated monoisotopic mass (C ₃₀ H ₄₆ N ₂ O ₉ ): 578.3, found: 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]

The benzyl-protected educt [2] (1.00 eq.) was dissolved in EtOH and palladium (10% on activated charcoal, 0.10 eq.) was added. A H ₂ -atmosphere was maintained over night at room temperature to facilitate the deprotection reaction. 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 yield the desired product [3] as a colorless oil that solidifies (82% yield). e(tBu)ue(tBu) ₂ [3]: RP-HPLC (10 - 90% B in 15 min): t _R = 12.00 min. Calculated monoisotopic mass (C ₂₃ H ₄₉ N ₂ O ₉ ): 488.3, found: 489.4 [M+H] ⁺ , 516.4 [M+Na] ⁺ .

2.3.3 FMOC-D-HCY(GLYCOSYL/LACTOSYL)-OH

For the incorporation of a sugar moiety onto Fmoc-D-Hcy(Trt)-OH, the respective per- acetylated precursor (β-D-Glucose pentaacetate/β-D-Lactose octaacetate) was used. The sugar (1.00 eq.) and protected amino acid (1.20 eq.) were added in Argon stream to a round bottom flask and dissolved in DCM _abs . TIPS (1.30 eq.) was added as a scavenger and SnCl ₄ (2.40 eq., 1.00 M in DCM) was given drop-wise to the reaction mixture. After initial yellow coloring, the mixture became colorless and a precipitate formed after stirring over night at r.t.. The mixture was diluted with DCM and acidified with HCI (1.00 M). The organic layer was extracted with HCI and H ₂ O twice each and dried over Na ₂ SO ₄ . The solvent was evaporated in vacuo and the crude product purified via flash chromatography.

The synthesis of the silicon fluoride acceptor moiety [8] was achieved leaning on the published procedure (43) with some modifications. Reactions were carried out in dried flasks under argon atmosphere. The desired product was obtained after 5 reaction steps starting with 4- bromobenzyl alcohol.

((4-Bromobenzyl)oxv)( tert-butyl)dimethylsilane [4]

4-bromobenzyl alcohol (1.00 eq.) was dissolved in DMFabs. (15 mL/g educt) and imidazole (1.20 eq.) and TBDMSCI (1.20 eq.) were added under vigorous stirring. The reaction mixture was stirred at r.t. over night, poured into cold H ₂ O and the aqueous phase extracted 5 times with Et ₂ O. The organic phases were combined, washed twice with sat. NaHCO ₃ and brine, dried over MgSO ₄ and concentrated in vacuo. The crude product was purified via silica flash chromatography employing 5% EtOAc in petrol ether (v/v). After removal of the solvents under reduced pressure, the protected alcohol [4] was obtained as colorless oil (95% yield).

((4-Bromobenzyl)oxy)(tert-butyl)dimethylsilane [4]: RP-HPLC: (50 - 100% B in 15 min): t _R = 15.0 min. ¹ H-NMR (400 MHz, CDCI ₃ , 300 K): δ = 0.10 (6H, s, SiMe ₂ tBu), 0.95 (9H, s, SiMe ₂ tBu), 4.69 (2H, s, CH ₂ OSi), 7.21 (2H, d), 7.46 (2H, d) ppm.

Di-tert-butyl(4-((teft-butyldimethylsilyloxv)methyl)Dheny l)fluorosilane [5]

[4] (1.00 eq.) was dissolved in THFabs. (10 mL/g educt) and cooled to -78°C before addition of tBuLi (2.40 eq.) in pentane (c = 1.70 mol/L). After stirring for 30 min at -78°C, the reaction mixture was added dropwise to a solution of di-tert-butyldifluorosilane (1.20 eq.) in THFabs. (10 mL/g) at -78°C. The solution was stirred over night and allowed to warm to r.t. before addition of brine. The crude product was extracted 3 times with Et ₂ O, the combined organic phases dried over MgSO ₄ and the solvents evaporated in vacuo to afford a yellowish oil (95% yield). The crude product [5] was used without further purification.

Di-tert-butyl(4-((tert-butyldimethylsilyloxy)methyl)pheny l)fluorosilane [5]: RP-HPLC: (50 - 100% B in 20 min): t _R = 19.0 min. 4-(Di-tert-butylfluorosilanyl)benzyl alcohol [6]

Deprotection of [5] was achieved by suspension in MeOH (25 mL/g educt) and using catalytic amounts of concentrated HCI (0.25 mL/g). After stirring the mixture over night at r.t., the solvent was removed under reduced pressure. The remainder was dissolved in Et ₂ O (20 mL/g educt) and washed with sat. NaHCO ₃ solution. The aqueous layer was then three times extracted with Et ₂ O, the combined organic phases dried over MgSO ₄ , and the solvent evaporated in vacuo to afford a yellowish oil (98% yield). The crude product [6] was used without further purification.

4-(Di-tert-butylfluorosilanyl)benzyl alcohol [6]: RP-HPLC: (50 - 100% B in 15 min): t _R = 8.2 min.

4-(Di-tert-butylfluorosilyl)benzaldehvde [7]

Oxidation to the aldehyde was done employing Corey-Suggs conditions. The educt [6] (1.00 eq.) was dissolved in DCM _abs . (15 mL/g educt) and added dropwise to an ice-cooled suspension of PCC

(2.50 eq.) in DCM _abs . (20 mL/g PCC). After stirring for 30 min at 0°C and subsequently 2.5 h at r.t., Et ₂ O was added and the supernatant decanted from the solid. The black remainder was washed with Et ₂ O and the combined organic phases filtered through a pad of silica gel (10 cm/g product). The solvent was evaporated in vacuo and [7] obtained as a yellowish oil (96% yield).

4-(Di-tert-butylfluorosilyl)benzaldehyde [7]: RP-HPLC: (50 - 100% B in 15 min): t _R = 10.5 min.

4-(Di-te/t-butylfluorosilyl)benzoic acid SiFA-BA) [81

The aldehyde [7] (1.00 eq.) was dissolved in tBuOH (23 mL/g educt), DCM (2.5 mL/g educt) and NaH ₂ PO ₄ xH ₂ O (1.25M, pH = 4.0-4.5, 15 mL/g educt) and KMnO _4aq . (1 M, 23 mL/g educt) was added. After stirring for 25 min, the mixture was cooled to 5°C. KMnO4 (1.00 eq.) was added and the reaction quenched shortly afterwards by addition of sat. NaHCO ₃ .The mixture was dried over MgSO ₄ and the solvent evaporated under reduced pressure. The crude product [8] was purified by recrystallization from Et ₂ O/n-hexane (1/3, v/v) and afforded a colorless solid (60% yield).

4-(Di-tert-butylfluorosilyl)benzoic acid (= SiFA-BA [8]): RP-HPLC: (50 - 100% B in 15 min): t _R = 8.5 min. Calculated monoisotopic mass (C ₁₅ H ₂₃ FO ₂ SO: 282.4; found: m/z = 281.1 [M-H]", 235.1 [M-COOH]-. 2.3.5 (4-Bromomethylphenyl)-di-tert-butyl-fluorosilane (= SiFA-Br [9])

The Synthesis of [9] was performed according to the literature (44). In short, the SiFA benzylalcohol [6] was dissolved in DCM and the solution cooled to 0°C. CBr ₄ (1.10 eq.) was added before PPh ₃ was added over a period of 30 min in small portions. The mixture was stirred for 2 h at r.t., the solvent removed in vacuo and the remainder washed with n-hexane. The precipitate was filtered off and the liquid concentrated in vacuo. Adjacent silica flash chromatography employing n-pentane as mobile phase, afforded the desired product [9] as a colorless oil (32 - 39% yield).

(4-Bromomethylphenyl)-di-tert-butyl-fluorosilane (= SiFA-Br [9]): RP-HPLC: (50 - 100% B in 15 min): t _R = 15.10 min. Calculated monoisotopic mass (C ₁₅ H ₂₄ BrFSi): 330.08; found: m/z = 331.3 [M-H]-.

¹ H NMR (300 MHz, CDCl ₃ , 293 K) 6 7.63 - 7.54 (m, 2H, 2x-CH-), 7.40 (d, J = 8.0 Hz, 2H, 2x- CH-), 4.50 (s, 2H, -CH ₂ -), 1.06 (d, J = 1.2 Hz, 18H, 2xtBu).

¹³ C NMR (75 MHz, CDCI3, 293 K) 5 139.50 (-CH-), 135.00 (-CH-), 134.95 (-CH-), 134.84 (-CH- ), 134.66 (-CH-), 128.72 (-CH-), 33.88 (-CH ₂ -), 27.88 (6xtBu C), 20.92 (tert. C), 20.75 (tert. C).

2.3.6 SYNTHESIS OF LINKER STRUCTURES

2.3.6.1 ABZ- AND AMBZ-BASED LINKERS

The entirety of -Ambz- and -Abz- based linkers were synthesized alike (see GP1, GP2,GP3) with the difference being the coupling of Fmoc-D-Ala-OH. For -Abz- based linkers, HOAt and HATU were employed together with a prolonged reaction time of 4 h considering the lower reactivity of the amine functionality of -Abz- compared to -Ambz-. After insertion of the second or third amino acid, the respective linker was cleaved off the solid support under retention of the protecting groups (see GP9). For acetylated linker units, an additional Fmoc deprotection and subsequent acetylation as described (see GP6) was carried out on the solid support.

HO-Abz-a-r(Pbf)-Fmoc

HO-Abz-a-r(Pbf)-Fmoc: RP-HPLC (10 - 90% B in 15 min): t _R = 15.33 min. Calculated monoisotopic mass (C ₄₄ H ₅₀ N ₆ O ₉ S): 838.34, found: 839.1 [M+H] ⁺ .

HO-Abz-a-r(Pbf)-dap(Boc)-Fmoc

HO-Abz-a-r(Pbf)-dap(Boc)-Fmoc: RP-HPLC (50 - 95% B in 15 min): t _R = 13.52 min. Calculated monoisotopic mass (C ₅₂ H ₆₄ N ₈ O ₁₂ S): 1024.44, found: 1025.2 [M+H] ⁺ . The synthesis of Ahx-based peptide linkers was achieved via standard SPPS according to

GP1,

GP2. GP5 and GP4. In short, Ahx was immobilized on the 2-CTC resin, Fmoc deprotected and coupled with Fmoc-D-dap(Dde)-OH. The side chain was deprotected using imidazole and hydroxylamine before the desired chelator, namely DOTA(tBu) ₃ , R-DOTAGA(tBu) ₄ or N4(Boc) ₄ was coupled. Adjacent Fmoc-deprotection opened the possibility of further derivatization. Ahx- based linker structures were further derivatized, in detail see 2.3.8.2.

2.3.7 CPCR4-LINKER CONJUNCTIONS

CPCR4-linker conjunctions were synthesized by fragment condensation between CPCR4 and the respective Fmoc-protected linker unit under exertion of GPS. Reaction mixtures were concentrated in vacuo before Fmoc-deprotection by dissolution in 20 vol% piperidine in DMF. Adjacent semi-preparative RP-HPLC yielded the desired products.

CPCR4-Abz-a-r(Pbfi-dap(Boc)-NH ₂

The synthesis of CPCR4-Abz-a-r(Pbf)- dap(Boc)-NH ₂ was facilitated by fragment condensation according to GP8. CPCR4 (see 2.3.1) and the linker HO-Abz-a-r(Pbf)- dap(Boc)-Fmoc (see 2.3.6) were condensed and the resulting peptide Fmoc deprotected. The crude product was purified by semi- preparative RP-HPLC.

CPCR4-Abz-a-r(Pbf)-dap(Boc)-NH ₂ : RP-HPLC (10 - 95% B in 15 min): 1R = 8.83 min.

Calculated monoisotopic mass (C ₇₃ H ₉₉ N ₁₇ O ₁₅ S): 1485.72, found: 744.6[M+2H] ²⁺ . via semi-preparative RP-HPLC afforded the desired product.

CPCR4-Ambz-a-r(Pbf)-NH ₂ : RP-HPLC (10 - 90% B in 15 min): t _R = 7.94 min. Calculated monoisotopic mass (C ₈₆ H ₈₇ N ₁₅ O ₁₂ S): 1313.64, found: 658.2[M+2H] ²⁺ .

(10 - 90% B in 15 min): t _R = 13.43 min. Calculated monoisotopic mass (C ₉₁ H ₁₀₈ N ₁₈ O ₁₃ S): 1692.81, found: 848.3[M+2H] ²⁺ .

2.3.8 SYNTHESIS OF CHELATORS

2.3.8.1 MODIFIED MAS ₃ -DERIVED CHELATORS

Several mas ₃ .derived chelators were synthesized all according to standard Fmoc peptide synthesis strategy and GP1,2,3,9 .

Scheme 8: Synthesis of modified mas ₃ -derived chelators according to Fmoc peptide synthesis strategy. The resulting chelators were either used in fragment condensation with the CXCR4-binding peptides or deacetylated beforehand.

Deacetylation

After cleavage off the resin, the chelators were dissolved in MeOH (2 mL/50 mg) and the pH was adjusted to 10-11 with KCN. After at least 4 h at ambient temperature, de-acetylated chelators were precipitated in Et ₂ O.

HO-(Hcy(Gluc(OAc) ₁₂ )) ₃ -TMAA: HPLC (10 - 90% B in 15 min): t _R = 12.97 min. Calculated monoisotopic mass (C ₇₅ H ₉₃ N ₃ O ₃₂ S ₄ ): 1675.46, found: 1677.2[M+H] ⁺ .

HO-(Hcy(Gluc)) ₂ -TMAA: HPLC (10 - 95% B in 15 min): t _R = 8.70 min. Calculated monoisotopic mass (C ₄₈ H ₆₅ N ₃ O ₁₆ S ₃ ): 1035.35, found: 1036.3[M+H] ⁺ .

HO-cit-Hcy(Lac)-cit-TMAA: HPLC (10 - 95% B in 15 min): t _R = 7.90 min. Calculated monoisotopic mass (C ₄₉ H ₆₇ N ₇ O ₁₇ S ₂ ): 1089.40, found: 1089.9[M+H] ⁺ .

HO-s(tBu)-Hcy(Lac)-s(tBu)-TMAA: HPLC (10 - 95% B in 15 min): t _R = 11.59 min. Calculated monoisotopic mass (C ₅₁ H ₇₁ N ₃ O ₁₇ S ₂ ): 10 61.42, found: 1061.8 [M+H] ⁺ . 

The synthesis of HO-HYNIC(Boc) was realized following the published procedure (45,46). In short, hydrazino-nicotinic acid was reacted with Boc ₂ O (1.00 eq.) and Triethylamine (1.30 eq.) in DMF over night. The solvent was evaporated under reduced pressure and the crude product was subjected to silica flash-chromatography employing EtOAc followed by EtOAc + 1vol% AcOH. The desired product was obtained after removal of the solvent as a white powder. ESI-MS: Calculated monoisotopic mass (C ₁₁ H ₁₅ N ₃ O ₄ ): 253.11, found: 254.4 [M+H] ⁺ .

R _f (EtOAc+0.5%AcOH) = 0.65.

MS (ESI, positive): calculated monoisotopic mass for C ₂₈ H ₅₂ N ₄ O ₁₀ : 604.37; found by ESI-MS: m/z = 605.0 [M+H] ⁺ .

¹ H-NMR (400 MHz, DMSO-d ₆ ) 6 = 7.17-6.19 (m, 2H, NH), 3.28-3.17 (m, 6H, CH ₂ ), 3.10-2.95 (m, 6H, CH ₂ ), 2.94- 2.90 (m, 1H, CH), 1.38 (s, 18H, CH ₃ ), 1.36 (s, 18H, CH ₃ ). 3. SYNTHESIZED PEPTIDES

The vast majority of peptides within this study were synthesized via fragment condensation. The CPCR4 binding motif was separately synthesized and purified (see 2.3.1), as were the linker units in the most cases (see 2.3.6). These fragments were condensed according to GP8, Fmoc deprotected by dissolution in 20% piperidine/DMF (v/v) and purified via RP-HPLC. The resulting CPCR4-linker constructs were used as starting material for derivatization with the corresponding chelators or functional groups such as labeling moieties. The obtained peptides are listed in the following with their respective analytical data.

3.2 DOTA CONJUGATED PEPTIDES

Conjugation of DOTA to the respective Ambz- or Abz- bearing scaffold (see 2.3.7) was carried out according to a recently published procedure (47). In short, DOTA (4.00 eq.) was pre- activated with NHS (5.00 eq.), EDCI (5.00 eq.) and 2,4,6-Collidine (6.00 eq.) for 30 min in H ₂ O. The Fmoc deprotected peptide, dissolved in DMF (30 μL/mg) was given to the mixture and stirred for 2-4 h at r.t.. The solvent was evaporated in vacuo and the crude product treated with TFA for 1-2 h (see GP5). After removal of TFA in N ₂ stream, the product was purified via semi preparative RP-HPLC.

Iodination of the tyr-bearing binding motif was achieved according to the general procedure GP7, based on the purified and fully deprotected peptide.

CXCR4-OI-3: RP-HPLC (10 - 60% B in 15 min): t _R = 8.86 min. Calculated monoisotopic mass (C ₁₁₂ H ₁₅₄ N ₂₆ O ₂₆ S ₂ ): 2343.10, found: 1173.4 [M+2H] ²⁺ , 782.7 [M+3H] ³⁺ .

[ ^nat Lu]CXCR4-OI-3: RP-HPLC (10 - 60% B in 15 min): t _R = 8.78 min. Calculated monoisotopic mass (C ₁₁₂ H ₁₅₁ N ₂₆ O ₂₆ S ₂ ): 2515.01 , found: 1258.4 [M+2H] ²⁺ , 839.3 [M+3H] ³⁺ .

4. RADIOLABELING

4.1 ¹²⁵ I-LABELING/ ¹²⁵ I-FC-131

Approximately 50 - 150 pg of unlabeled precursor were dissolved in 20 μL DMSO and 280 μL TRIS buffer (25 mM TRIS-HCI, 0.40 mM NaCI, pH = 7.5) were added. After addition of 5 μL [ ¹²⁵ l]Nal solution (15 - 20 MBq, see 1.2), the mixture was transferred into a reaction tube, coated with 150 pg lodogen®. After incubation for 15 min at r.t., the mixture was removed from the oxidant and subjected to RP-HPLC purification.

4.2 ^99M TC-LABELING

Labeling of peptides with ^99m TcO ₄ - was carried out dependent on the chelator used. The peptides and respective admixture, either freshly taken from aqueous solutions or used as pre- formulated and freeze-dried kits, were reacted with freshly eluted ^99m Tc-Pertechnetate (see 1.2). 4.2.1 MAS ₃ -DERIVED CHELATORS

Labeling mixtures, either in solution or with a freeze-dried formulation contained the same ingredients (49):

Stock 1: 1.78 g Sodium-Phosphate-dibasic dihydrate were dissolved in 50.0 mL H ₂ O (= solution 1). 1.38 g Sodium-Phosphate-Monobasic Monohydrate were dissolved in 50.0 mL H ₂ O (= solution 2). 47.35 mL of solution 1 and 2.65 mL of solution 2 were mixed to yield stock 1.

Stock 2: 5.00 mL of stock 1 were diluted with 5.00 mL of H ₂ O to yield stock 2.

Stock 3: The respective peptide was dissolved in DMSO or H ₂ O or a mixture thereof to a concentration of usually 1.00 mM.

Stock 4: 2.50 g Disodiumtartrate dehydrate were dissolved in stock 1 to yield stock 4.

Stock 5: 30.0 mg ascorbic acid were mixed with 10.0 mL aqueous HCI (10.0 mM).

Stock 6: 4.00 mg Tin(ll)chloride dihydrate were dissolved in 1.0 mL stock 5. This solution was freshly prepared for every labeling experiment.

A labeling mixture or kit was prepared by mixing the stock solutions according to the following indication:

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

Freshly eluted ^99m Tc-Pertechnetate (0.10 - 5.00 μL, 50.0 - 850 MBq) was added and the mixture heated for 30 min at 95°C. Quality control was performed by radio-TLC and radio-RP- HPLC directly from the reaction mixture. radio-RP-HPLC: R _t ( ^99m TcO ₄ ) = to, Rt ( ^99m Tc-colloid) = to, Rt ( ^99m Tc-tartrate) = 2 - 3 min, R _t ( ^99m Tc-peptide) = > 3min. radio-TLC on Silica-coated 60 RP-18 F ₂₅₄ s strips employing different mobile phases: NH ₄ OAc/DMF (1/1 , v/v): R _f ( ^99m TcO ₄ -) = 1 , R _f ( ^99m Tc-colloid) = 0, R _f ( ^99m Tc-tartrate) = 0.5 - 0.8 min, Rf ( ^99m Tc-peptide) = 0.8 - 1.

2-Butanone: Rf ( ^99m TcO ₄ - ) = 1 , Rf ( ^99m Tc-colloid) = 0, Rf ( ^99m Tc-tartrate) = 0 min, Rf ( ^99m Tc- peptide) = 0.

NaCI (25 vol% in H ₂ O): Rf ( ^99m TcO ₄ -) = 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 CHELATOR

Labeling mixtures, either in solution or with a freeze-dried formulation contained the same ingredients (50):

Stock 1: Ethylendiaminediacetic acid (EDDA) was dissolved in aqueous NaOH (0.10 M) to a concentration of 10.0 g/L.

Stock 2: Disodiumtartrate dihydrate was dissolved in NaH ₂ PO4 buffer (40.0 g/L) to a concentration of 40.0 g/L.

Stock 3: Tin(ll)chloride dihydrate was dissolved in aqueous sodium ascorbate (3.00 g/L in 0.01 M HCI) to a concentration of 1.50 g/L. This mixture was freshly prepared for every labeling experiment.

Stock 4: The respective peptide was dissolved in DMSO or H ₂ O or a mixture thereof to a concentration of usually 1 .00 mM.

A labeling mixture or kit was prepared by mixing the stock solutions according to the following indication:

Stock 1 : 50.0 μL

Stock 2: 50.0 μL

Stock 3: 5.33 μL

Stock 4: 5.00 μL

Freshly eluted ^99m Tc-Pertechnetate (0.10 - 5.00 μL, 50.0 - 850 MBq) was added and the mixture heated for 20 min at 95°C. Quality control was performed by radio-TLC and radio-RP- HPLC directly from the reaction mixture as stated above (see 4.2.1). 4.2.3 N4 AS CHELATOR

Labeling mixtures, either in solution or with a freeze-dried formulation contained the same ingredients:

Stock 1: N ₃₂ HPO ₄ was dissolved in H ₂ O to yield a concentration of 0.05 M (pH = 11.5).

Stock 2: Disodiumcitrate sesquihydrate was dissolved in H ₂ O to a concentration of 0.10 M.

Stock 3: Tin(ll)chloride dihydrate was dissolved in aqueous sodium ascorbate (3.00 g/L in 0.01 M HCI) to a concentration of 1.00 g/L. This mixture was freshly prepared for every labeling experiment.

Stock 4: The respective peptide was dissolved in DMSO or H ₂ O or a mixture thereof to a concentration of usually 1.00 mM.

A labeling mixture or kit was prepared by mixing the stock solutions according to the following indication:

Stock 1: 25.0 μL

Stock 2: 3.00 μL

Stock 3: 5.00 μL

Stock 4: 7.50 μL

Freshly eluted ^99m Tc-Pertechnetate (0.10 - 5.00 μL, 50.0 - 850 MBq) was added and the mixture heated for 10 min at 90°C. Quality control was performed by radio-TLC and radio-RP- HPLC directly from the reaction mixture as stated above (see 4.2.1 ).

4.3 ¹⁷⁷ LU-LABELING

For ¹⁷⁷ Lu-labeling of DOTA- or DOTA-GA-bearing peptides, 0.5 - 2 nmol of the respective peptide (directly from stock, DMSO or H ₂ O or mixtures thereof) were mixed with 10 μL of an aqueous sodium acetate buffer (1.00 M, pH = 5.50). The desired activity [ ¹⁷⁷ Lu]LuCl ₃ (0.04 M in HCI), usually between 5 and 80 MBq, was added and the mixture diluted with HCI (0.04 M) to a total volume of 100 μL. After 30 min at 95°C, 10 μL sodium ascorbate (0.10 M) was added to prevent radiolysis and reaction control via radio-RP-HPLC and radio-TLC was performed.

Silica-coated 60 RP-18 F ₂₅₄ s with mobile phase NH4OAC/DMF (1/1 ; v/v):

Rf ( ¹⁷⁷ LuCl ₃ ) = 0, Rf ( ¹⁷⁷ Lu-colloid) = 0, R _f ( ¹⁷⁷ Lu-peptide) = 1. Cellulose ITLC-SG paper with mobile phase trisodium citrate (0.10 M):

Rf ( ¹⁷⁷ LuCl ₃ ) = 1 , Rf ( ¹⁷⁷ Lu-colloid) = 0, Rf ( ¹⁷⁷ Lu-peptide) = 0.

4.4 ¹⁸ F-LABELING

Labeling of SiFA-bearing peptides was achieved according to recently published literature (51). Briefly, a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light) was conditioned with 10 mL H ₂ O prior to passage of aqueous ¹⁸ F-fluoride. The cartridge was purged with 10 mL air, dried with 10 mL ACN, followed by purging with another 20 mL air to remove traces of water. ¹⁸ F-fluoride was eluted with 100 μmol [K ⁺ ⊂2.2.2]OH- (in 500 μL ACN) and the pH adjusted by addition of 25 μL oxalic acid (1.00 M in ACN).

This mixture was used for either one or several labeling experiments. The desired activity (usually 30 - 500 MBq) was mixed with 10 - 25 μmol of the respective SiFA-bearing peptide (directly from stock in DMSO) and the mixture allowed to incubate at r.t. for 5 min. The reaction mixture was then diluted with 9 mL HEPES buffer (0.10 M, pH = 3). The product was isolated from unreacted ¹⁸ F-fluoride by passage of the mixture through a Sep-Pak C18 light cartridge. After purging the cartridge with 10 mL H ₂ O, the peptide was eluted with 500 μL of a EtOH/PBS mixture (1/1 ; v/v). Radiochemical purity was determined using radio-RP-HPLC and radio-TLC.

Silica-coated 60 RP-18 F ₂₅₄ s with mobile phase ACN/PBS (1/1 ; v/v; + 10 vol% NaOAc (2.00 M in H ₂ O); + 1 vol% TFA):

Rf ( ¹⁸ F-fluoride) = 0, R _f ( ¹⁸ F-peptide) = 0.8 - 1.

4.5 ⁶⁸ GA-LABELING

⁶⁸ Ga-labeling of DOTA and DOTA-GA-bearing peptides was achieved in accordance to the literature (34). An automated GallElut ⁺ sytem from Scintomics was used. Briefly, the ⁶⁸ Ge/ ⁶⁸ Ga- generator from IThemba LABS was eluted with aqueous HCI (1.00 M) and a fraction (usually 1.25 mL, 500 - 700 MBq) was transferred into the reaction vial (ALLTECH, 5 mL), loaded beforehand with 2 - 5 nmol of the respective peptide. The reaction mixture was heated 5 min at 95°C before passage through a Sep-Pak C8 light cartridge, pre-conditioned with 10 mL H ₂ O. The product was eluted with 2 mL EtOH/H ₂ O (1/1 ; v/v), the cartridge purged with 1 mL PBS and 1 mL H ₂ O before removal of EtOH in vacuo. Radiochemical purity assessed by radio-TLC.

Silica-coated 60 RP-18 F ₂₅₄ s with mobile phase NH ₄ OAC/DMF (1/1; v/v): R _f ( ⁶⁸ GaCI ₃ ) = 0, R _f ( ⁶⁸ Ga-colloid) = 0, R _f ( ⁶⁸ Ga-peptide) = 1.

Cellulose ITLC-SG paper with mobile phase trisodium citrate (0.10 M):

R _f ( ⁶⁸ GaCI ₃ ) = 1, R _f ( ⁶⁸ Ga-colloid) = 0, R _f ( ⁶⁸ Ga-peptide) = 0.

4.6 ⁶⁷ GA-LABELING

Labeling of DOTA-bearing peptides with ⁶⁷ Ga was carried out in analogy to a method, described in literature (52). In short, [ ⁶⁷ Ga]Ga-citrate was immobilized on a SEP-Pak Silica light cartridge, washed with H ₂ O (10 mL) and eluted with the desired volume of HCI (0.1 M). A fraction of the resulting ⁶⁷ GaCI ₃ solution was then diluted with HEPES to a total volume of 200 μL and given onto the peptide (5 nmol, H ₂ O). The mixture was then heated to 95°C for 30 min, diluted with PBS to a volume of at least 3 mL and passed through a SEP Pak C8 light cartridge to remove excess ⁶⁷ GaCI ₃ . The labelled peptide was eluted with 0.5 mL of a EtOH/PBS (1/1 ; v/v) mixture. Radiochemical yields and purities were assessed via radio-TLC.

Silica-coated 60 RP-18 F254S with mobile phase NH ₄ OAC/DMF (1/1 ; v/v):

R _f ( ⁶⁷ GaCI ₃ ) = 0, R _f ( ⁶⁷ Ga-colloid) = 0, R _f ( ⁶⁷ Ga-peptide) = 1.

Cellulose ITLC-SG paper with mobile phase trisodium citrate (0.10 M):

Rf ( ⁶⁷ GaCI ₃ ) = 1 , Rf ( ⁶⁷ Ga-colloid) = 0, Rf ( ⁶⁷ Ga-peptide) = 0.

5. IN VITRO EXPERIMENTS

5.1 DETERMINATION OF IC ₅₀

CXCR4-positive Jurkat T lymphocyte cells were grown in Gibco’s RPMI 1640 GlutaMAX medium supplemented with 10 vol% FBS and maintained in a 5% CO ₂ atmosphere at 37°C.

For in vitro experiments, cells were counted in a hemocytometer using trypan blue as contrast agent. The cell suspension was centrifuged, and the pellet resuspended in HBSS (+1 wt% BSA) to a concentration of 2 mio cells/mL. 8x3 polystyrene tubes were loaded with 25 μL of the standard ligand ¹²⁵ l-Fc-131 (see 4.1 , 1.00 nM in HBSS) and 25 μL of the ligand to investigate in the respective concentration (10 ^-4 - 10- ¹⁰ M, n = 3 for each concentration). 200 μL of cell suspension (400.000 cells per well) were added to obtain a final peptide concentration range of 10 ^-5 - 10 ^-11 M. The tubes were cooled for 2 h at 8°C to prevent internalization. The experiment was stopped by removal of the supernatant. 200 μL HBSS were added onto the cells, the suspension centrifuged, and the supernatant pooled with the respective first fractions. This step was repeated once more before the tubes containing the supernatants and the ones containing the cell pellets were subjected to activity measurement in the γ-counter.

5.2 DETERMINATION OF INVICSO

For the determination of inverse IC ₅₀ (invIC ₅₀ ) values the protocol for the determination of regular IC ₅₀ values was resumed with minor changes. The peptide under investigation was radioactively labeled 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 M). A cell tube was then containing 25 μL of the radioactive peptide solution, 25 μL of the respective Fc-131 solution and 200 μL cell suspension (400.000 cells). The experiment was conducted as described above.

5.3 DETERMINATION OF INTERNALIZATION

CXCR4-expressing Chem_1 cells were grown in DMEM-F12 medium supplemented with 10 vol% FBS, 1 vol% NEA, 1 vol% PenStrep and 1 vol% HEPES (1.00 M). Cells were maintained in a 5% CO2 atmosphere at 37°C.

For in vitro experiments, cells were harvested by removal of the medium and incubation of the cells with trypsine/EDTA (0.05%/0.02%; w/v) for 30 in at 37°C. The cells were counted and seeded in 24-well plates (100.000 cell per well) 24 ± 2 h prior to the experiment.

The medium was removed, and the cells incubated in 200 μL DMEM-F12 (+5 wt% BSA) for 15 min at 37°C. Each well (n = 3 for every time point) was either treated with 25 μL DMEM- F12 (+5 wt% BSA) or 25 μL of an AMD3100 stock (1 mM in H ₂ O) for blockage of the receptors. As the experiment was carried out as a dual-tracer approach, a radio-tracer solution was prepared, containing the standard ligand ¹²⁵ l-Fc-131 and the radioactively labeled peptide under investigation each in a concentration of 2.00 nM. 25 μL of this stock was added to the wells (final ¹²⁵ Fc-131 concentration: 0.20 nM; final peptide concentration under investigation: 0.20 nM) and the cells incubated at 37°C for the respective period.

The experiment was stopped by placing the well-plate on ice and removing the supernatant. The cells were washed with 250 μL cold HBSS and the both fractions for each well combined, comprising the amount of unbound radioligand. 250 μL of cold acid wash (0.02 M NaOAc in aqueous acetic acid, pH = 5) were added and the cells incubated for 15 min on ice. The supernatant was removed, the cells washed with cold HBSS and the respective fractions combined to obtain the amount of surface-bound radioligand.

The cells were then incubated with 300 μL NaOH (1.00 M in H ₂ O) for at least 30 at r.t. before the supernatant was removed. The well was washed with another 300 μL NaOH and the fractions combined that contain the amount of internalized radioligand.

The three different fractions were subjected to activity measurement in the y-counter, measuring the activity of the radionuclide used for the labeling of the peptide under investigation first. The same fractions were measured again for the activity of ¹²⁵ l after an adequate time, in dependence on the half-life of the first radionuclide. Data was corrected for non-specific internalization and referred to the specific internalization of the standard ligand ¹²⁵ l-Fc-131.

5.4 DETERMINATION OF THE OCTANOL/PBS PARTITION COEFFICIENT

The ligand under investigation (usually 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 n-octanol in a low-bind Eppendorf tube (n = 8). The tubes were vortexed at maximum speed for 3 min to ensure equilibrium before centrifugation at 15.000xg for 5 min on a Biofuge 15 from Heraeus Holding GmbH (Osterode, Germany). An aliquot of 100 μL of each fraction was measured in the y-counter and the logD _7.4 calculated as follows:

6. IN VIVO EXPERIMENTS

6.1 MOUSE MODEL AND TUMOR MODEL

All animal experiments were conducted in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals. To establish tumor xenografts, Jurkat cells (2 - 3x10 ⁷ cells) were suspended in a mixture of Gibco’s RPMI 1640 medium and Matrigel (1/1 ; v/v) from BD Biosciences (Heidelberg, Germany), and inoculated subcutaneously onto the right shoulder of 6-10 weeks old CB17-SCID mice from either Charles River GmbH (Sulzfeld, Germany) or the in-house mouse breeding facility. Mice were used when 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 on a VECTor ⁴ small-animal SPECT/PET/OI/CT from MILabs BV. (Utrecht, The Netherlands). The resulting data were analyzed with the associated PMOD (version 4.0) software. Mice were anaesthetized with /soflurane and the radioactively labeled compounds were injected via the tail vein. Mice were euthanized after the respective distance and blood samples for later biodistribution studies were taken by cardiac puncture before image acquisition. Static images were acquired with 45 min acquisition time using the HE-GP-RM collimator and a step-wise multi-planar bed movement for SPECT isotopes and the HE-UHR-M collimator and a step-wise spiral bed movement for PET isotopes. All images were reconstructed using the MILabs-Rec software (version 10.02) and a pixel-based Similarity-Regulated Ordered Subsets Expectation Maximization (SROSEM) algorithm with a window-based scatter correction (20% below and 20% above the photopeak, respectively). (Voxel size CT: 80 μm, voxel size SPECT/PET: 0.8 mm, 1.6 mm (FWHM) Gaussian blurring post processing filter, with calibration factor in kBq/mL and decay correction, no attenuation correction)

6.3 BIODISTRIBUTION STUDIES

Approximately 0.5 - 20 MBq (0.02 - 0.20 nmol) of the ¹²⁵ l-, ¹⁷⁷ Lu-, ^99m Tc-, ¹⁸ F- or ⁶⁸ Ga-labeled ligand were injected into the tail vein of Jurkat tumor-bearing CB-17 SCID mice and the animals sacrificed after the respective biodistribution time. Selected organs were removed, weighted and measured in a y-counter.

II. Results

1. TECHNETIUM-99M SPECT TRACER

1.1 MAS ₃ -CON JUGATED PEPTIDES

1.1.1 Chemical Structures

Chemical Structures of mas ₃ -conjugated compounds Tc-CXCR4-1 to -8 are illustrated in the following. 1.1.4 Mouse Imaging Studies

Figure 1 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-6 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

1.1.5 Internalization Studies

1.2 MODIFIED MAS ₃ -CONJUGATED PEPTIDES

1.2.1 Chemical Structures

Chemical Structures of modified mas ₃ -conjugated compounds Tc-CXCR4-9 to -12 are illustrated in the following.

1.2.4 Mouse Imaging Studies

Figure 2 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-9 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 3 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-11 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 4 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-12 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

1.2.5 Internalization Studies 1.3 FIXED-CHELATOR-BASED PEPTIDES

1.3.1 Chemical Structures

Chemical Structures of peptides with fixed-chelators comprising HYNIC and N4, Tc-CXCR4- 13 to -16 are illustrated in the following

Table 14: Summary of structural modifications and assigned abbreviations for peptides with fixed chelators Tc-CXCR4-13 to -16.

1.3.2 In Vitro Data

Table 15: Inverse IC ₅₀ values, logD _7.4 and internalization of Tc-labeled compounds Tc-CXCR4- 13 to -16.

1.3.4 Mouse Imaging Studies

Figure 5 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-13 1h p.i. in female Jurkat tumor-bearing mice without competitor from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 6 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-14 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 7 shows the MIP of both CT and SPECT Scans of ^99m Tc-CXCR4-14 2h p.i. in female Jurkat tumor-bearing mice without competitor from 1 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver. 1.3.6 In vivo Studies

Due to the favorable preclinical data obtained for [ ^99m Tc]CXCR4-Tc-14, the ligand was selected for a first proof-of-concept study in patients suffering from multiple myeloma. The radiosynthesis of [ ^99m Tc]CXCR4-Tc-14 was performed manually, as described in chapter 4.2.3 above. Images were acquired after injection of 430-604 MBq of [ ^99m Tc]CXCR4-Tc-14 at 5 min to 21 h p.i. Details of the scanning procedure are set out below.

Figure 8 shows: maximum intensity projection (MIP) images obtained from SPECT and PET imaging of a female patient suffering from multiple myeloma; A) [ ¹⁸ F]FDG PET MIP (1h p.i., 189 MBq of [ ¹⁸ F]FDG), B) [ ^99m Tc]CXCR4-Tc-14 SPECT MIP (3h p.i., 604 MBq of [ ^99m Tc]CXCR4-Tc-14); straight arrows indicate tumor lesions, pointed arrows indicate physiological uptake of [ ¹⁸ F]FDG in the heart and of [ ^99m Tc]CXCR4-Tc-14 in the spleen.

The biodistribution of [ ^99m Tc]CXCR4-Tc-14 is similar to that of the established PET tracer [ ⁶⁸ 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 found in the kidneys. Relatively high uptake in the spleen reflects the known expression of CXCR4 and uptake of CXCR4-addressing ligands in humans. Significant uptake is also observed in liver and the bone marrow, both known to express CXCR4 under physiological but even more so under 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 blood pool uptake and the rapid clearance of the tracer from non-target tissues is most probably a result of the suitable hydrophilicity of [ ⁹⁹ⁿ Tc]CXCR4-Tc-14 (logD _7.4 = -1.75) combined with the outstanding targeting potential of the tracer determined in preclinical experiments (InvIC ₅₀ = 10.2 nM, Internalization (in % of ¹²⁵ I-FC-131) = 742%). The high uptake of the tracer in the tumor lesions together with low accumulation in background tissues enables visualization of multiple metastasis in high contrast (Figure 8, B). The SPECT/CT image of the axial plane further acknowledges the potential of the tracer by displaying high lesion uptake and beneficial resolution of [ ^99m Tc]CXCR4-Tc-14 in the osteolytic lesion in the pelvis in agreement with the PET/CT scan using [ ¹⁸ F]FDG (Fig. 9). No [ ¹⁸ F]FDG-positive lesion was found negative in the CXCR4-targeted SPECT scan with [ ^99m Tc]CXCR4-Tc-14. Fig. 9 A) shows SPECT/CT image of the axial plane 3h p.i. of 604 MBq [99mTc]CXCR4-Tc-14 in a patient suffering from multiple myeloma; Figure 9 B) shows PET/CT image of the axial plane 1h p.i. of 189 MBq [ ¹⁸ F]FDG in the same patient: straight arrows indicate the urinary bladder, dashed arrows indicate osteolytic lesions.

Opposed to the observations made in preclinical studies with tumor-bearing mice, no substantially elevated ligand uptake was detected in human liver and lung, confirming the elevated uptake in murine liver and lung to be caused by the mCXCR4 affinity of the ligand.

Physiological uptake in the stomach and thyroids was low and comparable to other normal organs indicating the absence of [ ^99m Tc]-pertechnetate contamination or in vivo loss of radiometal (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).

Figure 10 10 illustrates the biodistribution of [ ^99m Tc]CXCR4-Tc-14 in the same patient between 5 min and 21 h p.i. The specific doses were determined for [ ^99m Tc]CXCR4-Tc-14 in different tissues and organs of interest.

Figure 10 A) shows MIP images obtained from SPECT imaging at 5 min, 60 min, 120 min, 5h and 21 h p.i. of 604 MBq [ ^99m Tc]CXCR4-Tc-14 in a female patient suffering from multiple myeloma; Figure 10 B) shows specific doses [μGy/MBq] calculated for selected organs and tissues from the same patient.

The time-resolved biodistribution of [ ^99m Tc]CXCR4-Tc-14 reconfirms rapid background clearance and uptake in lesions as well as the spleen and the bone marrow, even at early time points such as 5 min p.i. No delayed clearance via the bile can be detected at later time points, indicating clearance of the tracer via the kidneys. However, uptake in the kidneys as well as the urinary bladder are found to be low above the entire observation period, suggesting prolonged retention of the ligand in CXCR4 expressing tissues (Figure 10 10, B). This might be a direct consequence of the high target affinity and the pronounced internalization into CXCR4 expressing cancer cells as determined in preclinical experiments.

The highest specific dose is determined for spleen (47 μGy/MBq), followed by liver (14 μGy/MBq), osteogenic cells (13 μGy/MBq) red bone marrow (11 μGy/MBq) and the kidneys (10 μGy/MBq) (Figure 10 10, B). The whole-body effective dose is calculated to be 6.3 μSv/MBq, with 604 MBq of [ ^99m Tc]CXCR4-Tc-14 injected, representing a delivered whole- body dose of 3.8 mSv. The specific doses delivered to organs and the whole-body effective dose is found to be similar to other technetium-99m-labeled ligands such as PSMA-targeted [99mTc]PSMA l&S or the CXCR4-trageted [99mTc]CXCR4-L (P. Vallejo-Armenta et al., Contrast Media and Molecular Imaging, 2020, Article 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 multiple myeloma patients, [99mTc]CXCR4-Tc-14 displays a higher whole-body absorbed dose but decreased specific organ doses (K. Herrmann et al., J. Nucl. Med. 2015, 56(3), 410-416).

This proof-of-concept study warrants further evaluation of [ ^99m Tc]CXCR4-Tc-14 in a clinical context.

Clinical SPECT/CT Imaging

Clinical evaluation of [ ^99m Tc]CXCR4-Tc-14 in patients was conducted under compassionate use in compliance with the German Medicinal Products Act, AMG §13 2b, and in accordance with the responsible authorities (Government of Oberbayern). First proof-of-concept studies were performed at the Universitatsklinikum Augsburg (Augsburg, Germany).

All subjects were examined on a Discovery MN CT 670 Pro equipped with an Optima 540 CT (GE Healthcare, Solingen, Germany). Full-body SPECT/CT scans at 5 and 60 min after tracer injection were acquired with a scanning speed of 30 cm/min, scans at 120 and 300 min were acquired with a scanning speed of 12 cm/min and scans at 21 h were acquired with a scanning speed of 5 cm/min. The scans were obtained in 60 subsets using the double-head technology in matrices of 128x128 (zoom 1). SPECT scans at 60 and 180 min after tracer injection were acquired with an exposure time of 8 sec per subset and scans at 21h were acquired with an exposure time of 16 sec per subset. Emission data were smoothed applying a Butterworthfilter (0.48) and reconstructed iteratively by an ordered-subsets expectation maximization algorithm (2 iterations, 10 subsets). Images were obtained after injection of 430-604 MBq of [ ^99m Tc]CXCR4-Tc-14 at 5 min to 21 h p.i.

The specific and effective doses in selected organs were analyzed. For calculation of the doses, voxels of interest were defined around areas with increased uptake in full-body images. Time-activity curves for the respective organs were automatically determined and the residence time of the tracer calculated using the specific calibration of the SPECT camera and a standard activity value. The residence time was used as the input data for the Olinda/EXM software. The output of this calculation was referred to the dosimetry guideline of the International Commission on Radiological Protection.

Clinical PET/CT Imaging

Clinical evaluation of [ ¹⁸ F]FDG in patients was conducted in accordance with the responsible authorities (Government of Oberbayern). The clinical studies were performed at the Universitatsklinikum Augsburg (Augsburg, Germany).

All subjects were examined on a Biograph mCT-S40 (Siemens Healthineers, Erlangen, Germany). Full-body PET/CT scans at 1h after injection of [ ¹⁸ F]FDG were acquired with a scanning speed of 2 min per bed position. Emission data were reconstructed iteratively. A low- dose CT was conducted for attenuation correction and anatomical correlation. Images were obtained after injection of 189 MBq of [ ¹⁸ F]FDG at 1 h p.i.

2. Lutetium-177/Gallium-68 theranostics

2.1 Chemical Structures

Chemical Structures of DOTA-bearing peptides CXCR4-DOTA-1 to -5 are illustrated in the following.

2.4 Mouse Imaging Studies

Figure 11 shows the MIP of both CT and SPECT Scans of ¹⁷⁷ Lu-CXCR4-DOTA-1 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 2 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 12 shows the MIP of both CT and SPECT Scans of ¹⁷⁷ Lu-CXCR4-DOTA-2 1h p.i. in female Jurkat tumor-bearing mice without competitor from 2- 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 13 shows the MIP of both CT and SPECT Scans of ¹⁷⁷ Lu-CXCR4-DOTA-3 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 2 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver. Figure 14 shows the MIP of both CT and SPECT Scans of ¹⁷⁷ Lu-CXCR4-DOTA-4 1h p.i. in female Jurkat tumor-bearing mice without (left) and with competitor (right, 100 nmol AMD 3100) from 2 - 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

Figure 15 shows the MIP of both CT and SPECT Scans of ¹⁷⁷ Lu-CXCR4-DOTA-4 6h p.i. in female Jurkat tumor-bearing mice without competitor from 2- 10%iD/mL; white arrows indicate organs of interest: solid = tumor, points = kidney, dashed = liver.

2.5 Internalization Studies

3. FLUORINE-18 PET TRACER/RADIOHYBRIDS

3.1 Chemical Structures

Chemical Structures of SiFA-bearing peptides and radiohybrids are illustrated below

Afterwards, two additional biodistribution studies were undertaken to review this proposition by co-injecting one and two nanomole of the cold substance, respectively. The aim of this approach was the partial and complete blockage of murine CXCR4 receptors by the cold substance.

Figure 16 shows the biodistribution profile of [ ¹⁸ F, ^nat Ga]CXCR4-SiFA-07 1h post injection in Jurkat tumor-bearing female CB-17 SCID mice; different amounts of radioligand were applied: 36 pmol, 1,000 pmol and 2,000 pmol; data are expressed as %iD/g values and are means ± SD of 5 animals for the experiment with 36 pmol; 1 animal each was used for both other experiments.

In order to spare animal lives, additional studies were conducted using only one mouse, respectively. Data obtained in these experiments is therefore not representative. However, a certain trend can be presumed. By co-injection of 1,000 pmol cold substance, elevated levels of circulating radioligand are observed. Blood activity level is raised by 310%, leading to increased uptake in CXCR4- tissues such as pancreas (196%) and muscle (226%). This extended blood circulation is particularly underrepresented in CXCR4 ⁺ organs liver (121 %) and spleen (114%) suggesting incipient receptor saturation. With more ligand circulating, a nearly 2-fold tumor uptake (180%) compared to the un-supplemented injection is observable.

When 2,000 pmol of cold substance are co-injected, significant receptor saturation is reached. Compared to the initial biodistribution with only 36 pmol ligand, most notable changes in organ uptake are found for CXCR4 ⁺ organs spleen (61%) and liver (70%) and even more pronounced, the tumor (40%). These sets of data endorse the above-described concept that uptake in tumor is dependent on the availability of mCXCR4 receptors in CXCR4 ⁺ organs.

3.4 Internalization Studies

10

Cytotoxic Effect

Evaluation of the cytotoxic efficacy was undertaken in collaboration with the group of Prof. Dr. med. Ulrich Keller, at this time leading the Myc associated cancer biology research at the Klinikum Rechts der Isar (TUM) in Munich. The group received CXCR4-MMAE-02 for an analysis of the compound’s cytotoxic capability in vitro.

Two cell lines were selected for the respective experiments. The B-cell lymphoma cell line U2932 and the higher CXCR4 expressing lymphoblast-like Raji cell line. Both cell lines were incubated with the PDC for 24, 48, 72 and 96 hours in varying concentrations of 0 (Blank), 10, 20, 40 and 100 nM. The cell viability was then assessed using propidium iodide as a marker for deceased cells in a flow cytometry experiment (Figure 17).

Figure 17 shows the results of the flow cytometry analysis of cell viabilities using Raji (left side) and U2932 (right side) cells: the cells were incubated for 0, 24, 48, 72 and 96h with 0 (Blank), 10, 20, 40 and 100 nM of CXCR4-MMAE-02 prior to staining with propidium iodide and flow cytometry analysis; the number of vital cells related to the total number of cells represents the cell viability.

Experiments with both cell lines show decreased cell viabilities for cells incubated with increasing concentrations of PDC. Furthermore, a time-dependent effect can be observed as cell viabilities generally drop with longer incubation times. In other words, cellular death induced by incubation with CXCR4-MMAE-02 is in accordance with the amount of PDC used and the incubation period. Deviating data points can be attributed to the low sample size and therefore experimental failure. Figure 18 shows the results of the 18 visualizes the amount of deceased cancer cells upon incubation with the PDC.

Figure 18 shows the results of the flow cytometry analysis of cell viabilities using Raji and U2932 cells: the cells were incubated for 96h with 0 (Blank), 10, 20, 40 and 100 nM of CXCR4-MMAE-02 prior to staining with propidium iodide and flow cytometry analysis of dead cells.

For concentrations of 10, 20 and 40 nM, a considerably higher fraction of dead Raji cells compared to the U2932 cells can be detected. This finding is in accordance with the higher CXCR4 expression of Raji cells and the supposedly more efficient PDC uptake compared to the U2932 cells. Again, deviating outcome for the 100 nM data point might be caused by the small sample size.

A cell cycle profiling experiment of U2932 cells after 72h incubation with varying concentrations of CXCR4-MMAE-02 was performed. For that purpose, cells were fixed, permeabilized and treated with propidium iodide. The number of cells in the G0/G1-, S- and G2/M-phase was then measured with regards to the amount of PDC used.

Figure 19 shows the results of cell cycle profiling experiments using U2932 cells: the cells were incubated for 72h with 0 (Blank), 10, 20, 40 and 100 nM of CXCR4-MMAE-02, fixed, permeabilized and stained with propidium iodide; the fraction of cells in the G0/G1 , S or G2/M phase was determined.

Figure 19 shows the results of19 shows a higher number of cells stuck in the G2/M-phase (Mitotic/cell dividing phase) by incubation with increasing amounts of PDC. This data suggests that MMAE is released inside the cells and prohibiting microtubule polymerization, which leads to an arrest of cells prior to their replication. This induced cell cycle arrest might then result in the activation of checkpoint sentinels such as kinases which operate the apoptosis of cancer cells.

5. OPTICAL IMAGING COMPOUNDS

5.1 Chemical Structures

Chemical Structures of optical imaging compounds CXCR4-OI-1 to -3 are illustrated in the following.

5.2 In Vitro Data

Table 41: IC ₅₀ values of cold compounds and logD _7.4 values of ¹²⁵ l- and ¹⁷⁷ Lu-labeled compounds CXCR4-OI-1 to -3.

How a partial blockage of CXCR4 ⁺ organs would benefit the tumor uptake was tested in another biodistribution study, employing the radioactive ligand in low molar activity. Only one mouse was used for this study in order to spare animal lives. The obtained biodistribution is therefore not representative, however, a certain trend can be presumed. Figure 20 summarizes the experimental outcome.

Figure 20 shows the biodistribution profile of [ ¹⁷⁷ Lu]CXCR4-OI-03 1h post injection in Jurkat tumor-bearing female CB-17 SCID mice; different amounts of radioligand were applied: 59 pmol and 1 ,000 pmol; data are expressed as %iD/g values and are means ± SD of 5 animals for the experiment with 59 pmol; 1 animal was used for the other experiment. Reduced uptake in CXCR4 ⁺ organs lung (-23%), liver (-10%) and spleen (-23%) is observed upon injection of 1 ,000 pmol of cold peptide. Hence, tumor uptake is 1.6-fold increased, which indicates that the higher amount of peptide in circulation accumulates in the tumor. Low tumor uptake might therefore be attributable to the trapping of ligand in CXCR4 ⁺ organs and not be a result of missing targeting potential.

5.4 Internalization Studies