Radiolabeled PSMA ligand compounds and precursors thereof

Abstract

# imgabs0 # The present application provides a PSMA-targeting ligand compound which is a compound of formula (1) or a salt thereof wherein RT is a PSMA binding group; l is a linking group; rC is a trivalent coupling group; r < S > is a silicon-containing moiety of formula-C (O)-R < S > 3-SiR < S > 1 R < S > 2OH wherein R < S > 1 and R < S > 2 are independently selected from C < 3 >-C < 10 > alkyl, and preferably tert-butyl, and R < S > 3 is a group comprising a 6-membered aromatic ring, and preferably phenyl < 2 >; rA is an amino acid unit; and RCH is selected from (i) a branched, acyclic chelating moiety having 4 amino groups, and (ii) a chelate moiety wherein a radioisotope selected from the group consisting of 99mTc, 94mTc, 186Re and 188Re is chelated by the branched, acyclic chelating moiety having 4 amino groups. The PSMA-targeting ligand compounds according to the present invention are suitable for use in radiopharmaceutical applications, such as radiation-guided surgery.

Description

[0001] The present invention relates to compounds suitable as PSMA-binding ligand compounds, which compounds can be radiolabeled and are suitable for use as radiotracers, for example, in the context of diagnostic radiology or radio-guided surgery, or for radiotherapy applications.

[0002] Over the past decade, radiopharmaceuticals targeting glutamate carboxypeptidase II (GCPII) (also known as prostate-specific membrane antigen (PSMA)) have become an integral part of the clinical management of prostate cancer (PCa) [1,2]. Almost in parallel with the clinical success of diagnostic tracers such as 68 Ga]Ga-PSMA-11 [3] and 18 F]DCFPyL [4] for positron emission tomography (PET) or 99m Tc]Tc-MIP-1404 [5] for single photon emission computed tomography (SPECT) imaging, several therapeutic compounds have entered this stage, including 177 Lu-labeled PSMA-I&T and PSMA-617 [6,7]. In addition, several different treatment modalities have entered preclinical and clinical studies, including long-acting albumin-bound PSMA-ligands [8,9], targeted alpha therapies

[10] , or the so-called tandem therapies combining 225 Ac]Ac- and 177 Lu]Lu-PSMA-617

[11] . The recently approved 177 Lu]Lu-PSMA-617 (Pluvicto TM , Novartis) for radionuclide ligand therapy (RLT) of metastatic castration-resistant PCa (mCRPC) represents a milestone in nuclear medicine, broadening the oncologist's armamentarium and potentially paving the way for further approved targeted therapeutic radiopharmaceuticals.

[0003] Radio-guided surgery (RGS) is another therapeutic intervention that successfully exploits the potential of radiolabeled PSMA-targeted probes

[12] . Patients with early biochemical recurrence who show only regional pelvic lymph node metastases (LNM) after radical prostatectomy (RP) can benefit from radio-guided salvage lymph node dissection (sLND) to delay disease progression and future systemic therapy

[13] . In contrast to conventional sLND, a gamma-emitting PSMA-targeted radioligand is injected intravenously up to 24 h before surgery. With the help of a gamma-probe, the localization and resection of metastatic lymph nodes are facilitated during surgery, which is particularly useful in the case of small or atypical local lesions. In addition, the excised tissue can be immediately identified by ex vivo gamma-probe measurement to confirm the successful removal of tumor-invaded tissue. In patients with 111After the initial proof-of-concept of In-PSMA-I&T

[14] , PSMA-RGS has been performed with 99m Tc]Tc-PSMA-I&S

[15] , thanks to its similar performance in vivo and more favorable radiation characteristics compared to 111 In]InCl3, the more commonly used 99m Tc tracer in the operating room, lower cost, and 99m Tc]TcO4 - higher availability. A recent study of 121 patients by Horn et al. described successful removal of preoperatively identified lesions in 99% of patients and complete biochemical response in 66% of patients

[16] , which confirmed the results of a smaller group of patients previously reported by Maurer et al.

[13] . In the latter study, additional lesions that had not been detected previously on PSMA-PET could even be removed, highlighting the potential of RGS for sLND

[13] . However, prospective clinical trials such as the TRACE study (NCT03857113) are needed to accurately evaluate the long-term outcomes and additional benefits of RGS for PCa patients.

[0004] Whether conventional or radioguided, the main limitation of sLND surgery is posed by incomplete resection of metastatic lesions. Even though the current state-of-the-art PSMA radioguiding using 99m Tc]Tc-PSMA-I&S showed excellent short-term efficacy in terms of biochemical response in the prospective study by Knipper et al.

[17] , the still insufficient sensitivity regarding micrometastatic lesions seems to be the main cause of recurrent disease [13, 18]. Technological developments such as robotic-assisted laparoscopic surgery incorporating drop-in or click-on γ-probes

[19] and more differentiated patient selection criteria

[20] will surely drive the application forward. However, at the radiopharmaceutical level, we identify a need for improvement, namely to provide radiotracers with higher contrast and lower background signal than the currently applied 99m Tc]Tc-PSMA-I&S. Compared to DOTA-based PSMA-radiolabeled chelator tracers, their relatively slow whole-body clearance and partial hepatobiliary excretion are caused by high plasma protein binding (PPB) of 94% and reduced hydrophilicity

[15] . Therefore, nonspecific background signals hamper contrast in early SPECT-imaging and accurate detection during surgery [21, 22]. Thus, generally 99mTc]Tc-PSMA-I&S is not used to reliably identify LNM in situ during surgery, but rather to confirm PSMA-positivity of excised tissue ex vivo. However, if the TBR is high enough, technically more accurate lesion detection should be feasible, even in the surgical area, and thus, even smaller lesions may become detectable by appropriate use of the radiotracer. Despite the 99m Tc]Tc-PSMA-I&S's known limitations, to our knowledge, no dedicated optimization of 99m Tc-labelled PSMA-ligands for RGS applications has been performed to date.

[0005] To address this medical need, the present invention provides a new series of radiolabelled PSMA-targeting ligand compounds and their precursors, which have improved pharmacokinetic properties and are suitable for use as radiotracers (or radio-targeting probes) in applications such as for radio-diagnosis or in the context of RGS or for radiotherapy applications. The optimized ligand structures are conceptually designed and contain a common PSMA-inhibitor motif derived from highly potent radiohybrid PSMA diagnostic agents

[23] and therapeutic agents

[24] , a tetraamine chelator for reliable complexation of a suitable radioisotope (such as technetium-99m), and variable amino acids for modulating the pharmacokinetic properties of the peptide. Such modifications have led to interesting PSMA radioligands for a variety of applications [25-27].

[0006] The PSMA-targeting ligand compound according to the invention is a compound of formula (1) or a salt thereof:

[0007]

[0008] wherein

[0009] R T is a PSMA-binding group;

[0010] L is a linking group;

[0011] R C is a trivalent coupling group;

[0012] R S is a silicon-containing moiety of the formula -C(O)-R S3 -SiR S1 R S2 OH, where R S1 and R S2 are independently selected from C3-C10 alkyl, and are preferably tert-butyl, and R S3 is a group containing a 6-membered aromatic ring, and is preferably benzenediyl;

[0013] RA is an amino acid unit; and

[0014] R CH is selected from

[0015] (i) a branched, acyclic chelating moiety having 4 amino groups, and

[0016] (ii) a chelate moiety, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the branched, acyclic chelating moiety having 4 amino groups.

[0017] It has been found that the compounds according to the invention combine a significant ability to bind to PSMA as inhibitors with general advantages compared to PSMA-I&S (such as reduced lipophilicity and reduced plasma protein binding (PPB)). Thus, the compounds according to the invention exhibit similar or even improved in vitro characteristics, while showing an increased tumor-to-organ ratio, and in particular an increased tumor-to-kidney ratio, in various organs, which is a key parameter for radioligand applications for the diagnosis and treatment of prostate cancer.

[0018] Thus, as a further aspect, the invention relates to a composition comprising a compound according to the invention, such as a therapeutic or diagnostic composition. Such a composition is suitable for the treatment and / or diagnosis of diseases associated with the overexpression of PSMA, such as prostate cancer.

[0019] As explained above, the compounds according to the invention encompass the compounds of formula (1). In addition, the invention encompasses salts of the compounds of formula (1), typically pharmaceutically acceptable salts. Thus, unless stated to the contrary, any reference herein to a compound according to the invention encompasses the compounds of formula (1) (and the preferred embodiments of these formulas disclosed herein) and their salts. Similarly, any racemate, enantiomer or diastereomer of any chiral compound of formula (1) and their salts are encompassed, unless the specific stereochemistry of the compound under consideration is indicated in the specific context. Due to their ability to bind and act as PSMA ligands, the compounds according to the invention may also be referred to as PSMA-targeting ligand compounds according to the invention, as PSMA-binding compounds according to the invention, or briefly as ligand compounds according to the invention.

[0020] In the following text, further descriptions of the compounds of formula (1), their salts, and their preferred embodiments will be provided. Based on the above description, it will be understood that the groups that can provide the salt forms of the compounds of formula (1) (such as acidic groups that can be deprotonated or basic groups that can be protonated) can be included in the compounds according to the invention in ionic (e.g., protonated or deprotonated) forms, even if the ionic forms of the relevant groups are not specifically stated herein.

[0021] R in formula (1) T represents a PSMA binding group. Due to its ability to bind to PSMA, it allows the compounds according to the invention to act as PSMA-targeting ligand compounds. A variety of PSMA binding groups are known in the art and are available to those skilled in the art for this purpose.

[0022] In formula (1), R T is preferably a group of the following structure (T-1):

[0023]

[0024] where the wavy line indicates the bond connecting the group R T to L in formula (1). More preferably, R T is a group of the following structure (T-2):

[0025]

[0026] where the wavy line indicates the bond connecting the group R T to L in formula (1).

[0027] As will be understood by those skilled in the art, depending on the storage conditions of the compounds according to the invention (such as the pH of their solutions), one or more of the carboxylic acid groups shown in the above formula may be deprotonated. The negative charge of the deprotonated carboxylic acid group can be balanced, for example, by the positive charge carried by other groups in the compounds according to the invention, or by a cationic counterion, as discussed below regarding the salt forms of the compounds according to the invention.

[0028] According to the above, the compounds of formula (1) are preferably compounds of formula (1A):

[0029]

[0030] where L, R C , R S , R A and R CH are as defined herein, including any preferred definitions thereof.

[0031] More preferably, the compound of formula (1) is a compound of formula (1AA):

[0032]

[0033] wherein L, R C , R S , R A and R CH are as defined herein, including any preferred definitions thereof.

[0034] It should be understood that unless otherwise stated, any reference herein to formula (1) also applies equally to formulas (1A) and (1AA) as its preferred variants.

[0035] The group L in formula (1) represents a linking group. As shown in formula (1), L is a divalent group which is connected to R T by a first covalent bond and to the coupling group R C by a second covalent bond. Thus, L usually contains a first functional group at the first end to which R T is attached, which is suitable for forming an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR- with a complementary group contained in R T , preferably an amide bond. Preferably, this first functional group in L is the group -NH-. Similarly, L usually contains a second functional group at the second end to which R C is attached, which is suitable for forming an amide bond or an alkylated amide bond with a complementary group contained in R C , preferably an amide bond. Preferably, this second functional group in L is also the group -NH-.

[0036] If an alkylated amide bond -C(O)-NR- (or -NR-C(O)-) is mentioned herein, the group R of the alkylated amide bond (also hereinafter) is a C1-C6 alkyl group and is preferably methyl.

[0037] In a preferred embodiment, L represents an oligomeric amide residue having a first end and a second end providing the above functional groups (i.e., a first end and a second end preferably providing the group -NH-). The oligomeric amide residue preferably contains 2 to 6, more preferably 2 to 4, and even more preferably 3 subunits. As implied by the name oligomeric amide residue, the adjacent subunits in the oligomeric amide residue are connected to each other by an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR-, preferably by an amide bond. Preferably, the backbone of the oligomeric amide unit extending from the first end to the second end contains 6 to 20, more preferably 8 to 18, and even more preferably 8 to 16 carbon atoms, excluding the carbon atoms in any optional substituents attached to the backbone.

[0038] As an example of the linking group L, the following group (L-1) may be illustrated:

[0039]

[0040] wherein the wavy line indicates the bonds formed by L with R T and R C respectively.

[0041] As will be understood by those skilled in the art, depending on the storage conditions of the compounds according to the invention (e.g., the pH of their solutions), one or more of the carboxylic acid groups shown in the above formula may be deprotonated. The negative charge of the deprotonated carboxylic acid group may be balanced, for example, by a positive charge carried by another group in the compounds according to the invention, or by a cationic counterion, as discussed below, for example, with respect to the salt forms of the compounds according to the invention.

[0042] R C in formula (1) is a trivalent coupling group which is used to couple the branched chain R T -L-, the branched chain -R A -R CH and the group -R S , as shown in formula (1). Thus, R C usually contains a functional group at its end to which L is attached, which is suitable for forming an amide bond (-C(O)-NH-) or an alkylated amide bond (-C(O)-NR-) with the complementary group contained in L, preferably an amide bond. Preferably, the functional group in R C is the group -C(O)-. Similarly, R C usually contains a functional group at the end or side chain to which R A is attached, which is suitable for forming an amide bond or an alkylated amide bond with the complementary group contained in R A , preferably an amide bond. Preferably, the coupling group in R C is the group -NH-. At the end to which R S is attached, R C usually contains a functional group at the end or side chain to which R S is attached, which is suitable for forming an amide bond or an alkylated amide bond with the complementary group -C(O)- contained in R S , preferably an amide bond. Preferably, the coupling group in R C is the group -NH-.

[0043] Preferably, R C is a trivalent amino acid unit. More preferably, R C is a trivalent amino acid unit which may be derived from an amino acid containing a carboxylic acid group and an amino group and a side chain carrying an additional functional group selected from a carboxylic acid group and an amino group. More preferably, R CSelected from 2,3-diaminopropionic acid (Dap) units, 2,4-diaminobutyric acid (Dab) units, ornithine (Orn) units, and lysine (Lys) units, most preferably Dap units. As will be understood by the reader skilled in the art, said amino acid units are units derived from the corresponding amino acids by using their functional groups to provide a bond (preferably an amide bond) to an adjacent group to which the amino acid unit is attached. With respect to their stereochemistry, these amino acid units are preferably D-amino acid units.

[0044] Said trivalent amino acid units are preferably linked by three amide bonds in the compounds of the present invention. Furthermore, more preferably, said amino acid units are oriented to provide: a -C(O)- functional group linked to L such that an amide bond is formed by the amino acid unit R C with L; an -NH- functional group linked to R A such that an amide bond is formed by the amino acid unit R C with R A ; and an -NH- functional group linked to R S such that an amide bond is formed by the amino acid unit R C with R S .

[0045] R S in formula (1) is a silicon-containing moiety of the formula -C(O)-R S3 -SiR S1 R S2 OH, which may also be represented by formula (S-1)

[0046]

[0047] wherein R S1 and R S2 are independently selected from C3-C10 alkyl groups, and are preferably tert-butyl groups, and R S3 is a group containing a 6-membered aromatic ring. Preferably, R S3 is phenylene, and more preferably a benzene-1,4-diyl group. The wavy line indicates the bond connecting the group R S to R C in formula (1).

[0048] As shown in the above formula, the moiety R S contains a -C(O)- functional group which is suitable for forming an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR- with a complementary group contained in R C , preferably an amide bond.

[0049] Thus, preferably, R S in formula (1) is a group of the following structure (S-2):

[0050]

[0051] wherein

[0052] R S3 is a group containing a 6-membered aromatic ring, and is preferably a phenylene group, more preferably a benzene-1,4-diyl group; and

[0053] The wavy line indicates the bond that connects the group R S to R C in formula (1).

[0054] According to the above, and most preferably, R in formula (1) S is part of the following structure (S-3):

[0055]

[0056] wherein

[0057] The wavy line indicates the bond that connects the group R S to R C in formula (1).

[0058] R in formula (1) A is an amino acid unit. As will be understood by those skilled in the art, an amino acid unit is a group that can be derived from an amino acid, that is, derived from a compound containing an amino group and a carboxylic acid group in the same molecule. Unless otherwise specified in a particular context, in addition to the amino group and the carboxylic acid group, one or more additional functional groups may also be present in the amino acid unit that can be derived from the amino acid. Specific amino acid units are usually identified by the name of the amino acid from which they can be derived, such as a glycine unit, an asparagine unit, etc. Unless otherwise specified in a particular context, the amino acid unit R A is preferably an α-amino acid from which it can be derived. If the amino acid unit R A can be derived from a chiral amino acid, the D-configuration is preferred.

[0059] Said amino acid unit R A is usually derived from an amino acid by using the amino group of the amino acid to provide the functional group -NH-, which forms an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR- with a complementary group contained in one of the adjacent groups in formula (1), preferably an amide bond, and by using the carboxylic acid group to provide the functional group -C(O)-, which forms an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR- with a complementary group contained in the other adjacent group in formula (1), preferably an amide bond. Preferably, said amino acid unit R A provides the functional group -C(O)- to form an amide bond with the group provided by R CThe provided group -NH- forms an amide bond and provides the functional group -NH- to form an amide bond with the group -C(O)- provided by R CH More preferably, the amino group from which the functional group -NH- provided by the amino acid unit R A to form an amide bond with R CH is derived is in the α-position relative to the carboxylic acid group that provides the functional group -C(O)- to form an amide bond with R C

[0060] Preferably, the amino acid unit R A has the following structure (A-1):

[0061]

[0062] Wherein

[0063] The wavy line at the -C(O)- group shown in the formula indicates the bond formed with R C The wavy line at the -NH- group shown in the formula indicates the bond formed with R CH And

[0064] R A1 is selected from hydrogen, -(CH2) k -COOH, -CH2-Ar and -Ar, where k is 1, 2 or 3, preferably 2,

[0065] And Ar is an optionally substituted phenyl group which may carry substituents selected from -OH and -NH2. If the phenyl group Ar is substituted, it preferably carries a single substituent selected from -OH and -NH2. Preferably, the -C(O)- group shown in formula (A-1) forms an amide bond with the group -NH- provided by R C and the -NH- group shown in formula (A-1) forms an amide bond with the group -C(O)- provided by R CH

[0066] More preferably, the amino acid unit R A has the following structure (A-1A):

[0067]

[0068] Wherein

[0069] The wavy line at the -C(O)- group shown in the formula indicates the bond formed with R C The wavy line at the -NH- group shown in the formula indicates the bond formed with R CH And R A1 is as defined in formula (A-1). Preferably, the -C(O)- group shown in formula (A-1A) forms an amide bond with the group -NH- provided by R​​C The provided group -NH- forms an amide bond, and the -NH- group shown in formula (A-1A) forms an amide bond with the group -C(O)- provided by R CH The provided group -C(O)- forms an amide bond with the group -C(O)- provided by R

[0070] In formula (A-1) and (A-1A) above, R A1 is preferably selected from -(CH2)2-COOH, -CH2-Ph, and -CH2-(p-NH2-Ph), where Ph is a phenyl group. Most preferably, R A1 is -(CH2)2-COOH.

[0071] As examples of suitable amino acid units R A units derived from Gly, Glu, Phe, (4-NH2)-Phe, Tyr, or phenylglycine can be mentioned, where the chiral amino acids are preferably in the D-configuration.

[0072] As will be understood by those skilled in the art, amino acid units containing side chains with basic groups such as -NH2 or acidic groups such as -COOH can be protonated or deprotonated respectively, depending on the storage conditions of the compounds according to the invention, such as the pH of their solutions. The positive charge of the protonated amino group can be balanced, for example, by the positive charge carried by another group in the compound according to the invention, or by an anionic counterion, as discussed below, for example, with respect to the salt forms of the compounds according to the invention. The negative charge of the deprotonated carboxylic acid group can be balanced, for example, by the positive charge carried by another group in the compound according to the invention, or by a cationic counterion, as discussed below, for example, with respect to the salt forms of the compounds according to the invention.

[0073] R CH is selected from

[0074] (i) a branched, acyclic chelating moiety having 4 amino groups, and

[0075] (ii) a chelate moiety, where a radioisotope selected from 99m Tc, 94m Tc, 186 Re, and 188 Re is chelated by the branched, acyclic chelating moiety having 4 amino groups.

[0076] Each of the four amino groups of the branched, acyclic chelating moiety having four amino groups provides a lone pair of electrons suitable for forming a metal / ligand coordination bond, and the branched, acyclic chelating moiety is generally a tetradentate chelating moiety. As will be understood by the reader skilled in the art, the chelating moiety is referred to as an acyclic chelating moiety because the coordinating nitrogen atoms of the four amino groups do not form ring members of a cyclic structure. As will be further understood, the reference to a chelating moiety as a branched chelating moiety requires the presence of at least one carbon atom in the chelating moiety that forms covalent bonds with at least three other carbon atoms. Different from other chelating moieties used to provide Tc-chelates, such as the mas3 chelating moiety used in PSMAI&S, the branched, acyclic chelating moiety having four amino groups defined above generally does not contain sulfur atoms.

[0077] Preferably, the branched, acyclic chelating moiety having four amino groups comprises an N4 chelating group (CH-1) of the following structure:

[0078]

[0079] where the wavy line indicates the bond connecting the group to the remainder of the compound according to the invention, and where optionally one or more (e.g., one, two or three) hydrogen atoms attached to the carbon atoms in the above formula may be replaced by substituents (e.g., methyl groups). However, such substituents are preferably absent.

[0080] In addition to the amino groups that allow the formation of a chelate complex, the chelating moiety R CH generally contains a functional group that is suitable for forming an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR- with a complementary group contained in R A , preferably an amide bond. Preferably, the functional group contained in R CH is the group -C(O)-.

[0081] Thus, more preferably, the branched, acyclic chelating moiety of R CH having four amino groups has the following structure (CH-2):

[0082]

[0083] where the wavy line indicates the bond connecting the group to the remainder of the compound according to the invention, and where optionally one or more (e.g., one, two or three) hydrogen atoms attached to the carbon atoms in the above formula may be replaced by substituents (e.g., methyl groups). However, such substituents are preferably absent.

[0084] According to option (ii) above, the branched, acyclic chelating moiety having four amino groups as discussed above can provide a chelate moiety, wherein selected from 99m Tc,94m Tc, 186 Re, and 188 radioisotopes of Re are chelated by a chelating moiety. Accordingly, the chelating moiety forms multiple (usually 4) coordination bonds with the chelated radioisotope. In addition, one or more additional ligands may coordinate with the radioisotope. Compounds according to the invention comprising a chelate moiety are also referred to herein as chelate compounds.

[0085] Preferably, the chelated radioisotope is 99m Tc.

[0086] In the chelate moiety and chelate compounds comprising it, the chelated radioisotope is generally cationic, such as 99m Tc, 94m Tc, 186 Re, or 188 Re in the +V oxidation state. The chelated radioisotope may carry one or more (e.g., one, two, or three) additional ligands other than the chelating moiety contained in the compound according to the invention. For example, in a chelate moiety comprising a Tc cation (i.e., 99m Tc cation or 94m Tc cation, preferably in the +V oxidation state), the Tc cation complexed by the chelating moiety as a chelating ligand may carry, for example, two or more anionic ligands as additional ligands, such as two oxo ligands. Similarly, in a chelate moiety comprising a Re cation (i.e., 186 Re cation or 188 Re cation, preferably in the +V oxidation state), the Re cation complexed by the chelating moiety as a chelating ligand may carry, for example, two or more anionic ligands as additional ligands, such as two oxo ligands.

[0087] Accordingly, 99m Tc, 94m Tc, 186 Re, or 188 Re radioisotopes are preferably included in the chelating moiety in a compound according to the invention in the form of dioxo species of the formula ( 99m Tc]TcO2) + , ( 94m Tc]TcO2) + , ( 186 Re]ReO2) + or ( 186 Re]ReO2) + .

[0088] Accordingly, according to the above R CHIn option (ii) of the definition, the preferred chelate moiety comprises a group having the following structure (CH-3):

[0089]

[0090] where the wavy line indicates the bond connecting the group to the remainder of the compound according to the invention, and wherein the Tc is 99m Tc or 94m Tc, and wherein optionally, one or more (e.g., one, two or three) hydrogen atoms attached to the carbon atom in the above formula may be replaced by substituents (e.g., methyl groups). However, such substituents are preferably absent.

[0091] According to the above, more preferably the chelate moiety of R CH has the following structure (CH-4):

[0092]

[0093] where the wavy line indicates the bond connecting the group to the remainder of the compound according to the invention, and wherein the Tc is 99m Tc or 94m Tc, and wherein optionally, one or more (e.g., one, two or three) hydrogen atoms attached to the carbon atom in the above formula may be replaced by substituents (e.g., methyl groups). However, substituents are preferably absent.

[0094] If the chelate moiety is a charged moiety, such as the cationic moieties shown in formulas (CH-3) and (CH-4), the positive charge can be balanced, for example, by a negative charge carried by another group in the compound according to the invention, or by an anion counterion, as discussed below, for example, with respect to the salt form of the compound according to the invention. As exemplary counterions, any anion in a sterile solution for injection can be mentioned, such as Cl - 、HPO4 2- 、PO4 3- 、H2PO4 - 、the citrate anion or the ascorbate anion.

[0095] A compound according to the invention comprising a chelate moiety (wherein the chelate is selected from the radioactive isotopes of 99m Tc, 94m Tc, 186 Re and 188 Re) may herein be referred to as a radiolabeled compound (or more specifically as 99m Tc-labeled, 94m Tc-labeled, 186 Re-labeled or 188Compounds labeled with Re). In contrast, compounds according to the invention that contain a chelating moiety without a chelated radioisotope may be referred to herein as unlabeled or non-labeled compounds.

[0096] As will be appreciated, the radiolabeled compounds according to the invention are suitable as radiopharmaceuticals, for example in radiotherapy, and / or as radioactive tracers in diagnosis or radiosurgery. The unlabeled compounds according to the invention provide useful precursors for radiolabeling, for example.

[0097] According to the above description, the compound of formula (1) is preferably a compound of the following formula (1B):

[0098]

[0099] wherein

[0100] The groups L, R C , R S1 , R S2 , R S3 and R A1 are as defined above, including any preferred definitions thereof, and

[0101] Optionally, one or more (e.g., one, two, or three) hydrogen atoms attached to the carbon atom in the chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 included in the above formula may be replaced by substituents (e.g., methyl groups);

[0102] or a chelate compound, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the optionally substituted chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 included in the above formula (1B).

[0103] More preferably, the compound of formula (1) is a compound of formula (1BB):

[0104]

[0105] wherein

[0106] The groups L, R C , R S1 , R S2 , R S3 and R A1 are as defined above, including any preferred definitions thereof, and

[0107] Optionally, one or more (e.g., one, two, or three) hydrogen atoms attached to the carbon atom in the chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 included in the above formula may be replaced by substituents (e.g., methyl groups);

[0108] or a chelate compound, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re, and 188 Re is chelated by the optionally substituted chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 included in the above formula.

[0109] Even more preferably, the compound of formula (1) is a compound of formula (1C):

[0110]

[0111] wherein

[0112] the group R A1 is as defined above, including any of its preferred definitions, and

[0113] Optionally, one or more (e.g., one, two, or three) hydrogen atoms attached to the carbon atom in the chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 included in the above formula may be replaced by substituents (e.g., methyl groups);

[0114] or a chelate compound, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re, and 188 Re is chelated by the optionally substituted chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 in the above formula (1C).

[0115] More preferably, the compound of formula (1) is a compound of formula (1CC):

[0116]

[0117] wherein

[0118] the group R A1 is as defined above, including any of its preferred definitions, and

[0119] Optionally, one or more (e.g., one, two, or three) hydrogen atoms attached to the carbon atom in the chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 included in the above formula may be replaced by substituents (e.g., methyl groups);

[0120] or a chelate compound, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the optionally substituted chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 in the above formula.

[0121] As specific examples of the compounds according to the present invention, the following compounds may be mentioned: the corresponding chelate compounds, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the chelating moiety -C(O)-CH(CH2NHCH2CH2NH2)2 in the formulae of these compounds, and salts of said compounds or chelate compounds:

[0122]

[0123]

[0124] As described above, the compounds according to the present invention encompass the compounds of formula (1) (including any preferred embodiments thereof, such as the compounds of formula (1A), (1AA), (1B), (1BB), (1C) or (1CC)) and their salts. The salts are preferably pharmaceutically acceptable salts, i.e., salts formed with pharmaceutically acceptable anions or cations. The salts can be formed, for example, by protonating an atom with an easily protonatable lone pair of electrons (such as a nitrogen atom) with an inorganic or organic acid or by separating a proton from an acidic group (such as a carboxylic acid group) (for example, by neutralization with a base). Other charged groups that can be present in the compounds according to the present invention and that can provide the compounds in salt form include groups that are always charged, such as a charged chelating moiety.

[0125] If the salt form comprises a positively charged form of the compound of formula (1), exemplary anions which can be present as counterions in the salt forms of the compounds of the invention may be mentioned, for example, anions selected from: chloride, bromide, iodide, sulfate, nitrate, phosphate (e.g., phosphate, hydrogen phosphate or dihydrogen phosphate), carbonate, hydrogencarbonate or perchlorate; acetate, trifluoroacetate, propionate, butyrate, valerate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, undecanoate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, nicotinate, benzoate, salicylate or ascorbate; sulfonate, such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate (tosylate), 2-naphthalenesulfonate, 3-phenylsulfonate or camphorsulfonate. Since trifluoroacetic acid is often used in the synthesis of peptides, trifluoroacetate is the representative salt provided if a compound comprising an oligomeric amide structure is formed. Such trifluoroacetate can be converted, for example, to acetate during its work-up.

[0126] If the salt form comprises a negatively charged form of the compound of formula (1), exemplary cations which can be present as counterions in the salt forms of the compounds of the invention may be mentioned, for example, cations 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).

[0127] In addition to the compounds according to the invention as discussed above, there is provided a method for preparing a radiolabeled compound according to the invention, the method comprising contacting a non-radiolabeled compound according to the invention as a precursor compound with a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re or with a compound comprising such a radioisotope. Preferably, the compound comprising the radioisotope is used in an environment selected from 99m Tc] pertechnetate, 94m Tc] pertechnetate, 186 Re] perrhenate and 188 Re] perrhenate.

[0128] For example, a non-radiolabeled compound according to the invention is used as a precursor compound and contacted with a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188The step of contacting a radioisotope of Re or a compound containing such a radioisotope can be carried out by dissolving a precursor compound and a compound containing a radioisotope in a common solvent, preferably in an aqueous solution.

[0129] As described above, the non-radioactively labeled compound according to the present invention is a compound according to the present invention that does not contain a chelated radioisotope. The radioactively labeled compound according to the present invention (or more specifically, respectively 99m Tc-labeled, 94m Tc-labeled, 186 Re-labeled or 188 Re-labeled compound) is a compound according to the present invention that contains a chelated radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re. According to the above, the chelated radioisotope contained in the radioactively labeled compound according to the present invention is contained in the chelate moiety, wherein the radioisotope is chelated by a branched, acyclic chelating moiety having 4 amino groups as defined above, including any preferred form of the chelating moiety, such as the moiety -C(O)-CH(CH2NHCH2CH2NH2)2.

[0130] As further described above, the PSMA-targeting ligand compound according to the present invention can be used for applications as a radioactive tracer, for example, in the context of radio-diagnosis or radio-guided surgery, or in radiotherapy applications. For example, the radioactively labeled compound according to the present invention (preferably the 99m Tc-labeled or 94m Tc-labeled compound according to the present invention) can be effectively used as a radioactive tracer in the diagnosis of diseases associated with overexpression of PSMA, or as a radioactive tracer in radio-guided surgery (also referred to herein as a radioactive targeting probe). Through radio-guided surgery assisted by the radioactively labeled compound according to the present invention, diseased tissues associated with overexpression of PSMA can be identified and removed.

[0131] Therefore, another aspect of the present invention is represented by a composition, such as a therapeutic composition or a diagnostic composition, which contains a compound according to the present invention, preferably a radioactively labeled compound according to the present invention. More specifically, preferably the therapeutic composition contains a 186 Re-labeled or 188 Re-labeled compound according to the present invention, and preferably the diagnostic composition contains a 99m Tc-labeled or 94m Tc-labeled compound according to the present invention.

[0132] In a first related aspect, the present invention provides a radiolabeled compound according to the present invention, preferably a 99m Tc-labeled or 94m Tc-labeled compound, or provides a diagnostic composition comprising such a compound for in vivo diagnosis of a disease associated with overexpression of PSMA. The disease associated with overexpression of PSMA is preferably cancer, and more preferably prostate cancer. The diagnosis preferably involves nuclear medicine tomography, and more preferably single photon emission computed tomography (SPECT).

[0133] In a further related aspect, the present invention provides a radiolabeled compound according to the present invention, preferably a 99m Tc-labeled or 94m Tc-labeled compound, or provides a diagnostic composition comprising such a compound for identifying diseased tissue in vivo associated with overexpression of PSMA. In a preferred embodiment, the compound or composition is provided for identifying diseased tissue in vivo associated with overexpression of PSMA in the context of radio-guided surgery for removing the diseased tissue. The diseased tissue associated with overexpression of PSMA is preferably cancer tissue, and more preferably prostate cancer tissue.

[0134] As a further related aspect, the present invention provides a radiolabeled compound according to the present invention (preferably a 99m Tc-labeled or 94m Tc-labeled compound) or a diagnostic composition comprising such a compound for ex vivo or in vitro use for identifying tissue or cells associated with overexpression of PSMA, wherein the tissue or cells are preferably cancer tissue or cells, and more preferably prostate cancer tissue or cells.

[0135] According to a further related aspect, there is provided an ex vivo or in vitro method for identifying whether tissue or cells overexpress PSMA, the method comprising contacting the tissue or cells with a radiolabeled compound according to the present invention (preferably a 99m Tc-labeled or 94m Tc-labeled compound) or with a diagnostic composition comprising such a compound, wherein the tissue or cells are preferably cancer tissue or cells, and more preferably prostate cancer tissue or cells.

[0136] In a further related aspect, the present invention provides a radiolabeled compound according to the present invention (preferably a 186 Re-labeled or 188 Re-labeled compound) or provides a therapeutic composition comprising such a compound for treating or preventing a disease associated with overexpression of PSMA, wherein the disease is preferably cancer, and more preferably prostate cancer.

[0137] A composition (e.g., a therapeutic composition or a diagnostic composition) comprising a compound according to the present invention 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, amino acid buffered solutions (with or without saline), water for injection, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by conventional methods well known in the art. These compositions can be administered to an individual in suitable doses. Administration of the suitable compositions can be achieved in different ways, such as by intravenous injection and / or delivery. The compositions can be administered directly to the target site.

[0138] The following entries summarize aspects of the present invention. It will be understood that these entries are closely related to the above portion of the specification, and the information provided in these entries can supplement the above portion of the specification and vice versa.

[0139] 1. A compound of formula (1) or a salt thereof:

[0140]

[0141] wherein

[0142] R T is a PSMA binding group;

[0143] L is a linking group;

[0144] R C is a trivalent coupling group;

[0145] R S is a silicon-containing moiety of the formula -C(O)-R S3 -SiR S1 R S2 OH, wherein R S1 and R S2 are independently selected from C3-C10 alkyl, and R S3 is a group containing a 6-membered aromatic ring;

[0146] R A is an amino acid residue; and

[0147] R CH is selected from

[0148] (i) a branched, acyclic chelating moiety having 4 amino groups, and

[0149] (ii) a chelate moiety, wherein selected from 99m Tc, 94m Tc, 186Re and 188 The radioisotope of Re is chelated by the branched, acyclic chelating moiety having 4 amino groups.

[0150] 2. The compound or salt according to item 1, wherein R in formula (1) T is a group of the following structure (T-1):

[0151]

[0152] where the wavy line indicates the bond connecting the group R T to the remainder of the compound of formula (1).

[0153] 2. The compound or salt according to item 2, wherein R in formula (1) T is a group of the following structure (T-2):

[0154]

[0155] where the wavy line indicates the bond connecting the group R T to the remainder of the compound of formula (1).

[0156] 3. The compound or salt according to item 1 or 2, wherein R in formula (1) S is a group of the following structure (S-2):

[0157]

[0158] where

[0159] R S3 is a group containing a 6-membered aromatic ring; and

[0160] the wavy line indicates the bond connecting the group R S to the remainder of the compound of formula (1).

[0161] 4. The compound or salt according to any one of items 1 to 3, wherein R in formula (1) S is a group of the following structure (S-3):

[0162]

[0163] where

[0164] the wavy line indicates the bond connecting the group R S to the remainder of the compound of formula (1).

[0165] 5. The compound or salt according to any one of items 1 to 4, wherein R in formula (1) Ais a group of the following structure (A-1):

[0166]

[0167] wherein

[0168] R A1 is selected from hydrogen, -(CH2) k -COOH, -CH2-Ar and -Ar,

[0169] where k is 1, 2 or 3, preferably 2, and

[0170] Ar is an optionally substituted phenyl group which may carry substituents selected from -OH and -NH2,

[0171] The wavy line at the -C(O)- group shown in the formula indicates the bond formed with R C and the wavy line at the -NH- group shown in the formula indicates the bond formed with R CH formed.

[0172] 6. The compound or salt according to item 5, wherein R in formula (1) A is a group of the following structure (A-1A):

[0173]

[0174] wherein

[0175] R A1 is selected from hydrogen, -(CH2) k -COOH, -CH2-Ar and -Ar,

[0176] where k is 1, 2 or 3, preferably 2, and

[0177] Ar is an optionally substituted phenyl group which may carry substituents selected from -OH and -NH2,

[0178] The wavy line at the -C(O)- group shown in the formula indicates the bond formed with R C and the wavy line at the -NH- group shown in the formula indicates the bond formed with R CH formed.

[0179] 7. The compound or salt according to item 5 or 6, wherein R A1 is selected from -(CH2)2-COOH, -CH2-Ph and -CH2-(p-NH2-Ph), where Ph is a phenyl group.

[0180] 8. The compound or salt according to item 7, wherein R A1 is -(CH2)2-COOH.

[0181] 9. A compound or salt according to any one of items 1 to 8, wherein R in formula (1) CH is selected from:

[0182] (i) an N4 chelating group of the following formula:

[0183]

[0184] wherein

[0185] the wavy line indicates the bond connecting the group R CH to the remainder of the compound of formula (1), and wherein optionally, one or more (e.g., one, two or three) hydrogen atoms attached to the carbon atom in the above formula may be substituted by substituents, and

[0186] (ii) a chelate moiety, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the N4 chelating group.

[0187] 10. A compound or salt according to any one of items 1 to 9, wherein L in formula (1) is an oligomeric amide residue comprising 2 to 6 subunits connected to each other by an amide bond -C(O)-NH- or by an alkylated amide bond -C(O)-NR-, wherein R is a C1-C6 alkyl group.

[0188] 11. A compound or salt according to item 10, wherein the oligomeric amide residue comprises 2 to 4 subunits.

[0189] 12. A compound or salt according to item 10 or 11, wherein the oligomeric amide residue comprises 6 to 20, preferably 8 to 18, and more preferably 8 to 16 carbon atoms, excluding the carbon atoms in any optionally present substituents attached to the backbone.

[0190] 13. A compound or salt according to any one of items 1 to 12, wherein R in formula (1) C is a trivalent amino acid unit derived from an amino acid comprising a carboxylic acid group and an amino group and a side chain carrying an additional functional group selected from a carboxylic acid group and an amino group.

[0191] 14. A compound or salt according to any one of items 1 to 13, wherein R in formula (1) C is selected from 2,3-diaminopropionic acid (Dap) unit, 2,4-diaminobutyric acid (Dab) unit, ornithine (Orn) unit and lysine (Lys) unit.

[0192] 15. A compound or salt according to any one of items 1 to 14, which is a radiolabeled compound comprising a chelated radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re.

[0193] 16. A compound or salt according to item 15, wherein the chelated radioisotope is in the +V oxidation state.

[0194] 17. A compound or salt according to item 15 or 16, wherein the chelated radioisotope is chelated as a dioxo species.

[0195] 18. A compound or salt according to any one of items 15 to 17, wherein the chelated radioisotope is selected from 99m Tc and 94m Tc.

[0196] 19. A compound or salt according to any one of items 15 to 17, wherein the chelated radioisotope is selected from 186 Re and 188 Re.

[0197] 20 A composition comprising a compound or salt according to any one of items 1 to 19.

[0198] 21. A composition according to item 20, which is a diagnostic composition.

[0199] 22 A diagnostic composition according to item 21, which comprises a compound or salt according to item 18.

[0200] 23. A compound or salt according to any one of items 1 to 19, preferably a compound or salt according to item 18, or a diagnostic composition according to item 21 or 22, for in vivo diagnosis of diseases associated with overexpression of PSMA.

[0201] 24. A compound or salt or diagnostic composition for the said use according to item 23, wherein the disease is cancer, and preferably prostate cancer.

[0202] 25. A compound or salt or diagnostic composition for the said use according to item 23 or 24, wherein the diagnosis involves nuclear medicine tomography, preferably single photon emission computed tomography (SPECT).

[0203] 26. A compound or salt according to any one of items 1 to 19, preferably a compound or salt according to item 18, or a diagnostic composition according to item 21 or 22, for in vivo identification of diseased tissue associated with overexpression of PSMA.

[0204] 27. A compound or salt or diagnostic composition for the use according to item 26, wherein the diseased tissue is cancer tissue, and preferably prostate cancer tissue.

[0205] 28. A compound or salt or diagnostic composition for the use according to item 26 or 27, wherein the diseased tissue is identified in the context of radioguided surgery for removing the diseased tissue.

[0206] 29. A compound or salt according to any one of items 1 to 19, preferably according to item 18, or a diagnostic composition according to item 21 or 22, for in vitro or ex vivo use in identifying a tissue or cell associated with overexpression of PSMA.

[0207] 30. The use according to item 29, wherein the tissue or cell is cancer tissue or cell, and preferably prostate cancer tissue or cell.

[0208] 31. An in vitro or ex vivo method for identifying whether a tissue or cell overexpresses PSMA, which comprises contacting the tissue or cell with a compound or salt according to any one of items 1 to 19, preferably according to item 18, or a diagnostic composition according to item 21 or 22.

[0209] 32. The method according to item 31, wherein the tissue or cell is cancer tissue or cell, and preferably prostate cancer tissue or cell.

[0210] 33. The composition according to item 20, which is a therapeutic composition.

[0211] 34. The therapeutic composition according to item 33, which comprises a compound or salt according to item 19.

[0212] 35. A compound or salt according to any one of items 1 to 19, preferably according to item 19, or a therapeutic composition according to item 33 or 34, for treating or preventing a disease associated with overexpression of PSMA.

[0213] 36. The compound or salt or therapeutic composition according to item 35, wherein the disease is cancer, and preferably prostate cancer.

[0214] In this specification, many documents are cited, including patent applications and manufacturer's manuals. Although the disclosures of these documents are considered not relevant to the patentability of the present invention, they are incorporated herein by reference in their entirety. More specifically, all cited documents are incorporated by reference to the extent as if each individual document was specifically and individually stated to be incorporated by reference.

[0215] The following examples further illustrate the invention without limiting it to the embodiments described. Example

[0216] Materials and Methods

[0217] General Information

[0218] The protected amino acids for peptide synthesis were purchased from Carbolution (St. Ingbert, Germany) and Iris Biotech (Marktredwitz, Germany). 2-Chlorotrityl chloride polystyrene (TCP) resin was obtained from Sigma-Aldrich (Steinheim, Germany). Solvents and all other organic and inorganic reagents were purchased from Alfa Aesar (Karlsruhe, Germany), Fluorochem (Hadfield, UK), Sigma-Aldrich (Steinheim, Germany) or VWR (Darmstadt, Germany) and were used without further purification. Radioactive 99m Tc]TcO4 - was obtained from an Ultra-Technekow FM 2 (15 - 43.00 GBq) generator (Curium, Petten, Netherlands).

[0219] Solid-phase peptide synthesis (SPPS) was carried out manually in a syringe reactor for peptide synthesis (Carl Roth, Karlsruhe, Germany), using an MX-RD-Pro syringe oscillator from SCILOGEX (Rocky Hill, USA). Analytical and preparative reversed-phase high-performance liquid chromatography (RP-HPLC) were performed using a Shimadzu gradient system (Shimadzu, Neufahrn, Germany) equipped with an SPD-20A UV / Vis detector (detecting at λ = 220 nm) and an LC-20AD solvent pump, respectively. The eluents for all chromatographic procedures were water (solvent A, 0.1% TFA (v / v)) and acetonitrile (solvent B, 0.1% TFA (v / v), 2% or 5% water (v / v) in analytical or preparative operation, respectively). For analytical measurements, a MultoKrom 100-5C18 column (150 mm x 4.6 mm, CS Chromatographie-Service, Langerwehe, Germany) was used at a constant flow rate of 1 mL / min. Preparative RP-HPLC was carried out on a MultoKrom 100-5C18 column (250 mm x 20 mm, CS Chromatographie-Service), applying a constant flow rate of 10 mL / min. Reversed-phase high-performance fast chromatography (RP-HPFC) was performed on an SP HPFC system using a SNAP cartridge (KP-C18-HS, 12 g) from Biotage (Charlottesville, USA), with water (solvent A, 0.1% TFA (v / v)) and acetonitrile (solvent B, 0.1% TFA (v / v)) as eluents. Electrospray ionization (ESI) mass spectra and atmospheric pressure chemical ionization (APCI) mass spectra for compound characterization were acquired on an expression L CMS mass spectrometer from Advion (Harlow, UK). 1H-NMR spectra were obtained at 300 K on an AVHD 400 from Bruker (Billerica, USA). 1 Chemical shifts (δ) were given in parts per million (ppm), and the spectra were calibrated to the residual 1 1H solvent signal of DMSO-d6 at 2.50 ppm, and the signal multiplicity was described as: s = singlet, m = multiplet.

[0220] Analytical and preparative radio-RP-HPLC were performed on the same Shimadzu system as above and additionally equipped with a SIL-20A HAT autosampler, using a MultoKrom100-5 C18 column (125 mm x 4.6 mm) from CS Chromatographie-Service, with a constant flow rate of 1 mL / min. A HERM LB 500 NaI scintillation detector (Berthold Technologies, Bad Wildbad, Germany) was connected to the outlet of the UV photometer for the detection of radioactivity. Radioactive thin-layer chromatography (TLC) was performed on iTLC-SG strips (Agilent Technologies, Waldbronn, Germany) using butanone or NH4OAc (1 M in water) with DMF (1 / 1 (v / v)) as the mobile phase (for the quantification of free 99m Tc]TcO4 - or colloidal technetium-99m, respectively). Radioactive-TLC strips were analyzed using a Scan-RAM radio-TLC detector from Lablogic Systems (Sheffield, UK). The activity of the radioactive probes was quantified using a 2480 WIZARD 2 automated gamma counter (PerkinElmer, Waltham, USA).

[0221] The centrifuges used for the determination of lipophilicity and binding to human plasma were a HERAEUS Pico 17 and a HERAEUS Megafuge 16R (Thermo Scientific, Osterode, Germany), respectively.

[0222] Solution-phase synthesis of the structural units for SPPS

[0223] (tBu)2EuE(tBu):

[0224] The tert-butyl-protected Glu-urea-Glu binding motive was synthesized analogously to the synthesis of tert-butyl-protected Lys-urea-Glu reported in the literature [28, 35].

[0225] 4-(Di-tert-butylhydroxysilyl)benzoic acid (SiOH-BA):

[0226] 4-(Di-tert-butylhydroxysilyl)benzoic acid (SiOH-BA) was obtained by hydrolysis of 4-(di-tert-butylfluorosilyl)benzoic acid (SiFA-BA). The latter was synthesized according to the published procedure

[36] . At room temperature, a solution of KOH (113 mg, 2013 μmol, 5.0 equiv) in TP-water (1 mL) was added to a stirred solution of SiFA-BA (114 mg, 403 μmol, 1.0 equiv) in DMF (4 mL), and the mixture was stirred for 1 h. The solution was acidified (pH 4-5) by addition of 2.013 mL of 1 M HCl (aq) and extracted with diethyl ether (5 x 5 mL). The combined organic phases were dried over MgSO4 and the solvent was evaporated in vacuo. Residual DMF was removed by freeze-drying to give the product as a colorless amorphous solid (96%).

[0227] RP-HPLC (50-100% B in 15 min): t R = 5.8 min, K' = 2.90. Calculated monoisotopic mass (C 15 H 24 O3Si): 280.2; found: m / z (APCI) = 279.0 [M-H] - .

[0228] N,N',N',N”-Tetra(tert-butoxycarbonyl)-6-carboxy-1,4,8,11-tetraazoundecane ((tBu)4N4):

[0229] N-Boc-ethylenediamine (4.0 equiv) was slowly added to a solution of 3-bromo-2-(bromomethyl)-propionic acid (1.0 equiv) in THF (25 mL / mmol), and the mixture was stirred at room temperature for 24 h. The solvent was removed in vacuo and the crude residue was dissolved in acetone / H2O (1 / 1 (v / v), 25 mL / mmol). The solution was cooled to 0 °C and triethylamine (3.0 equiv) was added. After 5 min, di-tert-butyl dicarbonate (4.0 equiv) was added and the stirred mixture was allowed to warm to room temperature over 15 h. The solvent was removed in vacuo and the crude product was purified by RP-HPFC (35-71% B in 15 min) to give the desired product as a colorless amorphous solid.

[0230] ESI-MS: Calculated monoisotopic mass (C 28 H 52 N4O 10 ): 604.4; found: m / z (ESI) = 605.5 [M+H] + .

[0231] 1H-NMR (400 MHz, DMSO-d6) δ = 7.17 - 6.19 (m, 2H, NH), 3.28 - 3.17 (m, 6H, CH2), 3.10 - 2.95 (m, 6H, CH2), 2.94 - 2.90 (m, 1H, CH), 1.38 (s, 18H, CH3), 1.36 (s, 18H, CH3).

[0232] Synthesis of PSMA ligands

[0233] The chemical synthesis of a novel N4-bearing PSMA-ligand was carried out by Fmoc-based standard solid-phase peptide synthesis (SPPS). The reference compound PSMA-I&S

[15] was synthesized as previously described. The purity of all labeled precursors was determined by RP-HPLC (UV detection at 220 nm) and was >98% in all cases.

[0234] General procedure (GP) for solid-phase peptide synthesis:

[0235] All synthesis steps were carried out at room temperature in a syringe reactor for peptide synthesis. After each coupling or deprotection step, the resin was washed thoroughly six or eight times with DMF (5 mL / g resin), respectively.

[0236] TCP-resin loading (GP1): The amino acid (2.0 equiv) and DIPEA (3.75 equiv) were dissolved in DMF (5 mL / g resin) and added to the TCP resin. After 2.5 h, methanol (2 mL / g resin) was added for capping of the remaining trityl chloride groups. Subsequently, the resin was washed thoroughly with DMF (6 x 5 mL / g resin), DCM (3 x 5 mL / g resin), and methanol (3 x 5 mL / g resin) and dried in vacuo. The loading l of the amino acid was determined by the following equation:

[0237]

[0238] where n1 = weight of the unloaded resin [g], m2 = weight of the loaded resin [g], M AA = molecular weight of the amino acid [g / mol], and M HCl = molecular weight of HCl [g / mol].

[0239] Amide bond formation on resin (GP2): To conjugate Fmoc-protected amino acids and other structural units, their carboxylic acid functional groups were pre-activated by adding TBTU (2.0 equiv), HOAt (2.0 equiv), and DIPEA (6.0 equiv) in DMF. After 5 minutes, the solution was added to the resin and allowed to react for 2.5 hours (different coupling times are mentioned in the synthesis protocol). The coupling of Fmoc-D-Dap(Dde)-OH was carried out using 2,4,6-trimethylpyridine (6.7 equiv) as the base instead of DIPEA to prevent racemization.

[0240] Fmoc-deprotection on resin (GP3): Deprotection of the Fmoc protecting group was achieved by adding DMF containing 20% piperidine (8 mL / g resin) within 5 minutes and then holding for 15 minutes.

[0241] N4-PSMA ligand:

[0242] Load Fmoc-D-Orn(Dde)-OH as the first building block onto TCP resin according to GP1, and after subsequent Fmoc cleavage (GP3), conjugate (tBuO)EuE(OtBu)2 for 4.5 h (GP2). Deprotect Dde using a solution of 2% hydrazine monohydrate in DMF (5 mL / g resin) for 20 min. Subsequently, add a solution of succinic anhydride (7 equiv) and DIPEA (7 equiv) in DMF and allow to react for 2.5 h. Then preactivate the resin-bound carboxylate for 30 min by adding TBTU (2.0 equiv), HOAt (2.0 equiv), and DIPEA (6.0 equiv) in DMF, and add Fmoc-D-Lys-OtBu (2.0 equiv) in DMF for conjugation for 2.5 h. Subsequently, perform Fmoc deprotection (GP3), and then conjugate Fmoc-D-Dap(Dde)-OH (GP2). Perform orthogonal deprotection of Dde using hydroxylamine hydrochloride (1.26 g / g resin) and imidazole (0.92 g / g resin) in a mixture of DMF (1 mL / g resin) and NMP (5 mL / g resin) for 3.5 h. Subsequently, conjugate SiOH-BA (GP2), and cleave the remaining Fmoc protecting groups according to GP3. In the following steps, couple Fmoc-D-Glu(OtBu)-OH (in N4-PSMA-12), Fmoc-D-Phe-OH (in N4-PSMA-13), or Fmoc-D-Phe(4-NHBoc)-OH (in N4-PSMA-21) according to GP2. After subsequent Fmoc deprotection (GP3), conjugate the tert-butyl-protected N4-chelator according to GP2. Perform cleavage from the resin and simultaneous ligand deprotection in TFA (+2.5% TIPS, +2.5% H2O) for 1 h. After purification by semi-preparative RP-HPLC, obtain N4-PSMA-12, N4-PSMA-13, and N4-PSMA-21 as colorless amorphous solids in yields of 29%, 25%, and 21% respectively (yield reference to the amount of substance of resin-bound Fmoc-D-Orn(Dde) at the start of solid-phase synthesis).

[0243] N4-PSMA-12: RP-HPLC (10 - 60% B in 15 min): t R = 9.3 min, K' = 3.72. Calculate the monoisotopic mass (C 57 H 95 N 13 O 21 Si): 1325.7; found: m / z (ESI) = 1326.5 [M+H] + , 664.0 [M+2H] 2+ .

[0244]

[0245] N4 - PSMA - 13: RP - HPLC (10 - 60% B in 15 minutes): t R = 10.2 minutes, K' = 4.08. Calculated monoisotopic mass (C 61 H 97 N 13 O 19 Si): 1343.7; Found: m / z (ESI) = 1344.4 [M + H] + , 673.0 [M + 2H] 2+ .

[0246]

[0247] N4 - PSMA - 21: RP - HPLC (10 - 60% B in 15 minutes): t R = 9.0 minutes, K' = 3.60. Calculated monoisotopic mass (C 61 H 98 N 14 O 19 Si): 1358.7; Found: m / z (ESI) = 1359.7 [M + H] + , 680.5 [M + 2H] 2+ .

[0248]

[0249] The following ligands were prepared according to the corresponding procedures.

[0250] N4 - PSMA - 11:

[0251]

[0252] N4 - PSMA - 11: RP - HPLC (10 - 60% B in 15 minutes): t R = 8.8 minutes, K' = 3.52. Calculated monoisotopic mass (C 54 H 91 N 13 O 19 Si): 1253.6; Found: m / z (ESI) = 1255.0 [M + H] + , 627.8 [M + 2H] 2+ .

[0253] N4 - PSMA - 32:

[0254]

[0255] N4 - PSMA - 32: RP - HPLC (10 - 60% B within 15 minutes): t R = 9.2 minutes, K' = 3.68. Calculated monoisotopic mass (C 61 H 97 N 13 O 20 Si): 1359.7; Found: m / z (ESI) = 681.6 [M + 2H] 2+ , 454.7 [M + 3H] 3+ .

[0256] N4 - PSMA - 33:

[0257]

[0258] N4 - PSMA - 33: RP - HPLC (10 - 60% B within 15 minutes): t R = 9.4 minutes, K' = 3.76. Calculated monoisotopic mass (C 60 H 95 N 13 O 19 Si): 1329.7; Found: m / z (ESI) = 665.0 [M + 2H] 2+ , 443.6 [M + 3H] 3+ .

[0259] PSMA - I&S (reference compound):

[0260]

[0261] PSMA - I&S: RP - HPLC (10 - 70% B within 15 minutes): t R = 8.8 minutes, K' = 5.02. Calculated monoisotopic mass (C 59 H 82 N 10 O 21 S): 1298.5; Found: m / z (ESI) = 1299.0 [M + H] + , 650.6 [M + 2H] 2+ .

[0262] IBA - KuE:

[0263] To synthesize IBA-KuE, the protected binding motif (OtBu)KuE(OtBu)2 was synthesized as described previously

[28] . 4-Iodobenzoic acid (6.1 mg, 24.6 μmol, 1.2 eq.) was preactivated by the addition of TBTU (7.9 mg, 24.6 μmol, 1.2 eq.), HOAt (3.3 mg, 24.6 μmol, 1.2 eq.) and DIPEA (12.9 μL, 73.8 μmol, 3.6 eq.) in DMF (1 mL). After 5 min of preactivation at room temperature, (OtBu)KuE(OtBu)2 (10.0 mg, 20.5 μmol, 1.0 eq.) in DMF (2 mL) was added and the solution was stirred at room temperature overnight (21 h). The solvent was evaporated and after the addition of TFA (+2.5% TIPS, +2.5% H2O), the solution was stirred for 1 h. TFA was evaporated and the crude product was dissolved in DMF. After purification by semi-preparative RP-HPLC (30-45% B in 20 min), the product was obtained as a colorless amorphous solid (48%).

[0264] RP-HPLC (20-40% B in 20 min): t R = 11.0 minutes, K' = 7.68. Calculate the monoisotopic mass (C 19 H 24 IN3O8): 549.1; Found: m / z (ESI) = 550.2 [M+H] + .

[0265] Radiolabeling

[0266] Radiolabeling of N4-PSMA ligand

[0267] By adding 0.05M Na2HPO4 (12.5 μL, -water, pH 9.25) and 0.1 M disodium citrate sesquihydrate (1.5 μL, in To the mixture in saline (0.5 mM in DMSO) was added 1 nmol of peptide precursor (0.5 mM in DMSO) for the N4-PSMA ligand. 99m Tc-labeling. After adding freshly prepared SnCl2 solution (2.5 μL, 1 mg / mL in ethanol), [ 99m Tc]TcO4 - (40 MBq / nmol) and the labeling solution (final volume 250 μL) was heated to 95°C for 15 min. Subsequently, 10 μL of 1 M sodium ascorbate (in PBS) was added and quality control was performed using radio-TLC and radio-RP-HPLC (UV detection at 220 nm).

[0268] 99m Radiosynthesis of [Tc]Tc-N4-PSMA-12 at the patient-level scale

[0269] In a 10 mL glass vial, 15 nmol of N4-PSMA-12 (20 μg, 0.5 mM in DMSO) was added to a mixture of 0.05 M Na2HPO4 (250 μL, in TP-water, pH 9.25) and 0.1 M sodium citrate sesquihydrate (30 μL, in TP-water) in saline. After addition of freshly prepared SnCl2 solution (10 μL, 1 mg / mL, in ethanol), [Tc]TcO4 in saline was added 99m [Tc]TcO4 - , and the labeling solution (final volume 2 - 5 mL) was heated to 95 °C for 15 minutes. The labeling solution was cooled for 10 minutes, followed by quality control using radio-TLC and radio-RP-HPLC.

[0270] 99m Radiosynthesis of [Tc]Tc-PSMA-I&S (reference compound)

[0271] As described by Robu et al.

[15] , 2 nmol of peptide precursor was used for the labeling of PSMA-I&S in a kit formulation. [Tc]TcO4 (40 MBq / nmol) was added to 500 μL of saline 99m [Tc]TcO4 - after which the solution was heated to 95 °C for 20 minutes. Subsequently, 10 μL of 1 M sodium ascorbate (PBS) was added, and quality control was performed using radio-TLC and radio-RP-HPLC.

[0272] 125 Radioiodination of [I]IBA-KuE

[0273] Synthesis, radioiodination, and deprotection of the protected stannyl-precursor were performed as described previously

[28] to obtain [I]IBA-KuE. The crude labeled product was purified using preparative radio-RP-HPLC with a gradient of 20 - 40% B in 20 minutes. 125 [I]IBA-KuE

[0274] Analytical data of PSMA inhibitors labeled with technetium-99m or iodine-125

[0275] 99m [Tc]Tc-N4-PSMA-12: Radio-RP-HPLC (10 - 70% B in 15 minutes): t R = 9.1 minutes, K' = 6.36.

[0276] 99m ​​​​​Tc]Tc-N4-PSMA-13: Radioactive-RP-HPLC (10 - 70% B within 15 minutes): t R = 10.0 minutes, K' = 6.98.

[0277] 99m Tc]Tc-N4-PSMA-21: Radioactive-RP-HPLC (10 - 70% B within 15 minutes): t R = 8.6 minutes, K' = 5.99.

[0278] 99m Tc]Tc-PSMA-I&S: Radioactive-RP-HPLC (10 - 70% B within 15 minutes): t R = 8.2 minutes, K' = 5.76.

[0279] 125 I]IBA-KuE: Radioactive-RP-HPLC (20 - 40% B within 20 minutes): t R = 11.0 minutes, K' = 7.68.

[0280] Lipophilicity and binding to human plasma

[0281] The lipophilicity of the Tc-labelled PSMA ligands, expressed as the distribution coefficient (logD 7.4 ), was determined using the shake-flask method. Approximately 1 MBq of the radioligand was vortexed vigorously for 3 minutes in a 1:1 mixture of 1 mL of PBS (pH = 7.4) and 1-octanol (n = 8). After centrifugation at 9000 rpm for 5 minutes, aliquots of the two phases (200 μL of 1-octanol, 50 μL of PBS) were collected and the activity was quantified in a γ-counter. The logD 99m value was calculated as the base-10 logarithm of the ratio of the activity concentration in the 1-octanol phase to that in the aqueous phase. Data are given as mean ± standard deviation (SD). 7.4 Binding to human plasma was determined by incubating the radioligand (2 nM concentration, 37 °C, 30 minutes) in human plasma and subsequent ultrafiltration (250 μL aliquots, 3200 rpm, 40 minutes) in an ultrafiltration device (Merck Millipore, Cork, Ireland). The fraction bound to human plasma proteins was calculated as the ratio of the unfiltered activity to the total activity in the ultrafiltration device. The determination was performed in three independent experiments, each with two replicates (n = 6, data are given as mean ± SD). All values were corrected for non-specific binding (control experiment in PBS).

[0282] The binding to human plasma was determined by incubating the radioligand (2 nM concentration, 37 °C, 30 minutes) in human plasma and subsequent ultrafiltration (250 μL aliquots, 3200 rpm, 40 minutes) in an ultrafiltration device (Merck Millipore, Cork, Ireland). The fraction bound to human plasma proteins was calculated as the ratio of the unfiltered activity to the total activity in the ultrafiltration device. The determination was performed in three independent experiments, each with two replicates (n = 6, data are given as mean ± SD). All values were corrected for non-specific binding (control experiment in PBS). The binding to human plasma was determined by incubating the radioligand (2 nM concentration, 37 °C, 30 minutes) in human plasma and subsequent ultrafiltration (250 μL aliquots, 3200 rpm, 40 minutes) in an ultrafiltration device (Merck Millipore, Cork, Ireland). The fraction bound to human plasma proteins was calculated as the ratio of the unfiltered activity to the total activity in the ultrafiltration device. The determination was performed in three independent experiments, each with two replicates (n = 6, data are given as mean ± SD). All values were corrected for non-specific binding (control experiment in PBS).

[0283] ​​​Determination of human serum albumin (HSA) binding by high-performance affinity chromatography (HPAC)

[0284] The HSA binding of the uncomplexed PSMA ligand was determined by HPLC according to the previously published procedure (Valko, K.; Nunhuck, S.; Bevan, C.; Abraham, M.H.; Reynolds, D.P., Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol / water and immobilized artificial membrane lipophilicity. J Pharm Sci. 2003, 92, 2236-2248). A Chiralpak HSA column (50 x 3 mm, 5 μm, H13H-2433, Daicel, Tokyo, Japan) was used at a constant flow rate of 0.5 mL / min at room temperature. Mobile phase A was freshly prepared 50 mM aqueous NH4OAc solution (pH 6.9), and mobile phase B was isopropanol (HPLC grade, VWR). The applied gradient for all experiments was 100% A (0 to 3 minutes), followed by 80% A (3 to 40 minutes). Before the experiment, the column was calibrated using nine reference substances with a known HSA binding range of 13 to 99% in the literature (Valko, K.; Nunhuck, S.; Bevan, C.; Abraham, M.H.; Reynolds, D.P., J Pharm Sci. 2003, 92, 2236-2248; Yamazaki, K.; Kanaoka, M., Computational prediction of the plasma protein-binding percent of diverse pharmaceutical compounds. J Pharm Sci. 2004, 93, 1480-1494). All substances (including the PSMA ligand being examined) were dissolved in a 1:1 (v / v) mixture of isopropanol and 50 mM aqueous NH4OAc solution (pH 6.9) at a final concentration of 0.5 mg / mL. Nonlinear regression was established using OriginPro 2016G software (Northampton, USA).

[0285] Affinity (IC 50 ) and internalization studies

[0286] Competitive binding studies were performed similar to the previously reported procedure

[28] . As a modification, the non-radioactive labeled standard competitor ((S)-1-carboxy-5-(4-(iodo)benzamido)pentyl)carbamoyl)-L-glutamic acid (IBA-KuE) was applied at increasing concentrations (10 -5 -10 -11 m / well, each n = 3), while the novel PSMA-binding compound of interest was applied as a 99m Tc-labeled radioligand (0.2 nM / well). In this inversed experimental approach, higher values correspond to higher affinity and are referred to as IC 50 inverse IC 50 ). Poly-L-lysine-coated 24-well plates (n = 3) were used. Data are given as mean ± SD. To determine the cellular uptake of the 99m Tc-labeled PSMA ligand in LNCaP cells at 1 h via PSMA-mediated internalization, the previously reported protocol

[28] was applied at assay concentrations of 1.0 nM and 0.2 nM for the 99m Tc-labeled PSMA ligand and the reference compound 125 I]IBA-KuE, respectively. Data were corrected for non-specific binding and normalized to the specific internalization of the reference. Results are given as mean ± SD.

[0287] In Vivo Experiments

[0288] All animal experiments were conducted in accordance with the German General Animal Welfare Regulations (German animal protection act, official version of May 18, 2006, Article 280 amended on June 19, 2020, approval number ROB-55.2-1-2532.Vet_02-18-109 of the General Administration of Upper Bavaria) and the institutional guidelines for animal care and use. Male CB17-SCID mice were purchased from Charles River (Sulzfeld, Germany) and arrived at the in-house animal facility at least 1 week before the start of the experiment to acclimatize. LNCaP cells were inoculated subcutaneously (approx. 2 × 10 7Tumor xenografts were established by inoculating cells in 200 μL of a 1:1 mixture of Cultrex BME (R&D Systems, Minneapolis, USA) and DMEM / Ham's F-12 onto the right shoulder of 6-8 week old male CB17-SCID mice. Animals were used for experiments when tumors grew to a size of 5-10 mm in diameter. Criteria for excluding animals from the experiment were weight loss greater than 20%, tumor size greater than 1.5 cm 3 , tumor ulceration, respiratory distress, or behavioral changes. These criteria were not applied to any mice. Neither randomization nor blinding was applied in the allocation of experiments. Quarterly health monitoring was performed according to FELASA recommendations.

[0289] Biodistribution study. Under isoflurane anesthesia, 99m Tc-labeled radioligand (2.7 ± 0.7 MBq, 82 ± 20 pmol) was injected into the lateral tail vein of LNCaP tumor-bearing mice (n = 4-5). Animals were sacrificed by carbon dioxide inhalation and blood extraction via cardiac puncture 6 hours after injection (pi). Blood and tissues of interest were collected, weighed and activity was determined in a gamma counter. Radioligand uptake is given as a percentage of injected dose per gram of tissue (% ID / g), and the results are presented as mean ± SD.

[0290] According to the above, n = 5 animals ("Dataset I") were used for [ 99m [Tc] Biodistribution study of Tc-N4-PSMA-21. All animals were healthy and did not show any signs of pain, distress or behavioral changes throughout the experiment. However, a significant deviation in renal uptake and elevated tumor uptake was observed for one particular animal (mouse #3) compared to the other four animals. The renal uptake of mouse #3 (36.17% ID / g) exceeded the values ​​obtained from the other four animals (4.58±1.44% ID / g, mean±SD) by 7.9 times or more than 20 standard deviations. In addition, the tumor uptake of mouse #3 (21.92% ID / g) exceeded the values ​​obtained from the other four animals (11.02±2.22% ID / g, mean±SD) by 2.0 times or more than 4 standard deviations. Because it is uncertain whether these large deviations are due to experimental error or may be related to strongly deviated metabolic processes (e.g., abnormal renal function in mouse #3), the study mainly presents and discusses the data set based on the other four animals (n=4, "Dataset II"). However, a comprehensive evaluation and comparison including mouse #3 was also performed. For both, "Dataset I" (n=5, including mouse #3) and "Dataset II" (n=4, without mouse #3) [ 99m Tc]Tc-N4-PSMA-21 renal uptake and [ 99mCompared with Tc-PSMA-I&S, it was significantly reduced (P < 0.001 for both), but there was no statistical difference from other 99m Tc-labeled N4-PSMA ligands (P > 0.92 and P > 0.81 for "Dataset I" and "Dataset II" respectively). Although mouse #3 also showed increased tumor uptake (see above), there was no statistically significant difference in tumor uptake between the 99m Tc-labeled PSMA ligands evaluated in this article when comparing either "Dataset I" (P > 0.69) or "Dataset II" (P > 0.27) with the biodistribution data of other radioligands.

[0291] μSPECT / CT imaging. After blood collection, static imaging of sacrificed animals was directly performed on a VECTor4 small animal SPECT / PET / CT / OI scanner from MILabs (Utrecht, Netherlands) using an HE-GP-RM collimator and stepwise multi-planar bed movement through the MILabs acquisition software (v11.00 and v12.26) for 45 minutes. The imaging data was reconstructed using MILabs-Reconstruction software (v12.00) and image analysis was performed using PMOD4.0 (PMOD technologies LLC, Zurich, Switzerland). Biodistribution studies were performed on the animals after imaging.

[0292] Data analysis

[0293] The acquired data was statistically analyzed. After performing one-way analysis of variance (ANOVA) using OriginPro software (version 9.7) from OriginLab Corporation (Northampton, USA), Tukey's multiple comparison post hoc test was performed. A two-sample student t-test was applied in Microsoft Excel (Redmond, USA) for paired statistical comparisons. A P-value < 0.05 obtained was considered statistically significant.

[0294] Results

[0295] Synthesis and radiolabeling

[0296] Novel PSMA ligands were synthesized using a mixed solution-phase / solid-phase synthesis method and obtained in yields of 29%, 25%, and 21% respectively, with chemical purity > 98%. The compound identity was confirmed by mass spectrometry. Using 99m Tc]TcO4 - for radiolabeling resulted in radiochemical purity (RCP) > 95% as determined by radio-TLC and radio-RP-HPLC. It was found that for 99mScaling up of the reported labeling protocol for patient-level production of Tc]Tc-N4-PSMA-12 is feasible, but a scaled-down amount of stannous chloride is applied to prevent the formation of colloidal technetium species. Considering the requirements of the clinical workflow for routine radiopharmaceutical synthesis, labeling with N4-PSMA-12 (20 μg, 15 nmol) with an activity of 194 - 810 MBq (553 ± 187 MBq, mean ± SD, n = 10) reproducibly yields the desired radiopharmaceutical with an RCP of 98.5 ± 0.6% (range, 97.6 - 99.2%, n = 10). Details on single radiolabeling are provided in Table 1 below.

[0297] Table 1: 99m Detailed data for the Tc]Tc-labeling of N4-PSMA-12 at the patient level. Given are the volume (V) of the labeling solution, the applied activity (A), and the amounts of free 99m Tc]TcO4 - and colloidal technetium-99m, as well as the resulting radiochemical purity (RCP), for the single-labeling experiments. Mean values and standard deviations (SD, n = 10) are given for the entire data set.

[0298]

[0299] In vitro characterization

[0300] 99m Tc]Tc-labeled N4-PSMA ligands and the reference 99m Tc]Tc-PSMA-I&S are summarized in Figure 1 and Tables 2a and 2b below. PSMA affinity was evaluated by determining the IC 50 reciprocal (IC 50,inv ). Independent of the variable amino acid, all radiopharmaceuticals showed high PSMA affinity (as indicated by low nanomolar IC 50,inv. values (range, 10.0 - 11.8 nM)) with no statistically significant differences (P > 0.66). In contrast, PSMA-mediated internalization, expressed as a percentage of the reference compound 125 I]IBA-KuE, was significantly affected by the variable amino acid. 99m Tc]Tc-N4-PSMA-12 (311 ± 16%) showed 1.3-fold higher internalization than 99m Tc]Tc-PSMA-I&S (240 ± 13%) and even higher than 99m Tc]Tc-N4-PSMA-13 (164 ± 15%) and 99m ​Tc]Tc-N4-PSMA-21 (180±4%) were 1.9 and 1.7 times higher, respectively. 99m Compared with Tc]Tc-PSMA-I&S, all novel [ 99m Tc]Tc-N4-PSMA compounds showed increased hydrophilicity, as indicated by the distribution coefficient (logD 7.4 ) indicates. In the group of radioligand with N4, [ 99m Tc]Tc-N4-PSMA-13(logD 7.4 = -2.78 ± 0.05) (which contains aromatic D-Phe residues) has the highest lipophilicity, followed by [ 99m Tc]Tc-N4-PSMA-21(logD 7.4 =-3.13±0.05, D-(4-NH2)-Phe) and [ 99m Tc]Tc-N4-PSMA-12(logD 7.4 =-3.35±0.05, D-Glu). 99m Tc]Tc-PSMA-I&S compared to all [ 99m Binding to human plasma was significantly reduced for the [Tc]Tc-N4-PSMA tracer (94.4% vs 55.1-88.5%) and followed the same trend as described for lipophilicity. 99m Tc]Tc-N4-PSMA-12 was found to have the highest hydrophilicity (logD 7.4 =-3.35±0.05) and minimal binding to human plasma proteins (55.1±2.5%) (P<0.001 for both parameters).

[0301] Table 2a: Using the n-octanol / PBS (pH 7.4) partition system (n=8), the distribution coefficient (logD 7.4 ) 99m Tc-labeled N4-PSMA compounds and 99m Lipophilicity of Tc-labeled PSMA-I&S; Binding affinity to PSMA (IC 50 Reciprocal (nM), 1 hour, 4°C, n=3); PSMA-mediated internalization in LNCaP cells (1 hour, 37°C, n=3), with radiolabeled reference ([ 125 I] in the form of a percentage of IBA)KuE); and 99m Tc-labeled N4-PSMA compounds and [ 99m Plasma protein binding (PPB, determined by ultrafiltration, n=6) of [Tc]Tc-PSMA-I&S; and HSA binding of the uncomplexed compound (determined by HPAC, n=1).

[0302]

[0303] Table 2b: Using the n-octanol / PBS (pH 7.4) partition system (n=8), the distribution coefficient (logD 7.4 ) 99m Lipophilicity of Tc-labeled N4-PSMA compounds; Binding affinity to PSMA (IC 50 Reciprocal (nM), 1 hour, 4°C, n=3); and 99m PSMA-mediated LNCaP cell internalization (1 hour, 37°C, n=3) of Tc-labeled N4-PSMA compounds as a percentage of the radiolabeled reference ([125I]IBA)KuE); and HSA binding of uncomplexed compounds (as determined by HPAC, n=1).

[0304]

[0305] In vivo characterization

[0306] Biodistribution studies. Comparative biodistribution studies of all four radiotracers were performed in LNCaP tumor-bearing mice 6 hours after injection ( Figure 2 , Table 3).

[0307] Among the three N4-bearing radioligands, similar distribution characteristics were observed: high uptake in tumor (11.0-13.0% ID / g), varying but moderate levels of activity in the kidney, and efficient clearance from blood and background tissues (see also Figure 3 μSPECT / CT scan in 99m Tc]Tc-N4-PSMA-12 showed a 99m Tc]Tc-N4-PSMA-13 ​​and [ 99m Tc]Tc-N4-PSMA-21 (6.6±4.8% ID / g and 4.6±1.4% ID / g, respectively), but this was not statistically significant (P>0.81). In sharp contrast to the N4-PSMA radioligand, for [ 99m Tc]Tc-PSMA-I&S (191±26% ID / g, than [ 99m Tc]Tc-N4-PSMA-12) found very high activity retention in the kidney. 99m Tc]Tc-PSMA-I&S 6 hours later, the activity retention in some other organs (e.g., lung, spleen, adrenal gland, and parotid gland) was also significantly higher (P < 0.001 for each). 99mThe relatively high activity uptake of [Tc]Tc-PSMA-I&S in tumors was less prominent (15.6 ± 2.8% ID / g). The corresponding differences for the novel N4-PSMA radioligands were not statistically significant (P > 0.27). For all radioligands, effective activity clearance from the blood pool was observed. For 99m Tc]Tc-N4-PSMA-12, the lowest blood activity level (0.0200 ± 0.0044% ID / g) was found 6 h after injection, which was associated with its low logD 7.4 and low PPB. Compared with 99m Tc]Tc-N4-PSMA-12, 99m Tc]Tc-N4-PSMA-13, 99m Tc]Tc-N4-PSMA-21, and 99m Tc]Tc-PSMA-I&S had 1.6-fold, 5.4-fold, and 3.6-fold higher blood activity levels, respectively.

[0308] Tumor-to-background ratio (TBR). As Figure 4 shown, 99m Tc]Tc-N4-PSMA-12 showed the highest TBR among all radioligands in most organs. Notably, in direct comparison with 99m Tc]Tc-PSMA-I&S, 99m Tc]Tc-N4-PSMA-12 showed increased TBR for each analyzed organ and blood, thus demonstrating its significantly superior clearance properties. In particular, a 3-fold T / blood ratio (658 ± 147 vs 219 ± 62) and even a 20-fold T / kidney ratio (1.64 ± 1.29 vs 0.08 ± 0.01) represent important progress in optimized pharmacokinetics. Although less prominent, improved TBR was also found for 99m Tc]Tc-N4-PSMA-13 and 99m Tc]Tc-N4-PSMA-21. A tabular overview of the TBR is provided in Table 4 below.

[0309]

[0310]

[0311] Discussion

[0312] Using N4-PSMA ligand design, a versatile structural platform is provided that ensures high PSMA affinity and reliable complexation of radioisotopes such as technetium-99m, while also being able to be flexibly modified to regulate the pharmacokinetic characteristics of the entire ligand. To maintain high affinity while ensuring low lipophilicity of the new tracer, a less lipophilic SiOH-group (formally hydrolyzed SiFA) is used. For complexation of radioisotopes, a tetraamine chelator is selected because this chelation system exhibits excellent in vivo stability and confers high hydrophilicity to the peptide radioligand [29, 30]. Compared to N3S-based chelation systems such as mercaptoacetyltris serine, an additional advantage is the absence of chemically reactive thiol groups, which may limit the shelf life of the radioligand precursor, an aspect often overlooked in early radioligand development. Finally, the incorporation of variable amino acids provides novel ligands with different properties in vitro and in vivo.

[0313] Compared with other PSMA-radiometal chelates, 99m The slow whole-body clearance and partial hepatobiliary excretion of [99mTc]Tc-PSMA-I&S are thought to be caused by high PPB and increased lipophilicity

[15] . Therefore, the main focus in the design of novel ligands is set on reduced lipophilicity and PPB. These objectives are met by N4-PSMA compounds. The inherent lipophilicity of variable amino acids (logP: Phe > (4-NH2)-Phe > Glu)

[31] translates into a similar trend in the lipophilicity and PPB of the corresponding radioligands (logD 7.4 and PPB: 99m [99mTc]Tc-PSMA-I&S > 99m [99mTc]Tc-N4-PSMA-13 > 99m [99mTc]Tc-N4-PSMA-21 > 99m [99mTc]Tc-N4-PSMA-12). In particular, 99m [99mTc]Tc-N4-PSMA-12 combines favorable lipophilicity, comparable to diagnostic rhPSMA compounds

[23] , and significantly lower PPB than 99m [99mTc]Tc-PSMA-I&S, 68 [68Ga]Ga / 177 [177Lu]Lu-PSMA-I&F

[32] or 99m [99mTc]Tc-EuK-(SO3)Cy5-mas3

[25] (all ligands developed for PSMA-guided surgery), and is comparable to the rapidly cleared 177 [177Lu]Lu-PSMA-617

[33] . Consistent with many previous studies [25-27], these findings emphasize how the pharmacokinetic-related properties of radioligands can be shaped through deliberate structural modification.

[0314] Although 99m Tc]Tc-PSMA-I&S has shown a favorable dose distribution in patients

[21] , and its application in RGS has shown advantages over conventional salvage surgery

[17] , we must admit that 99m Tc]Tc-PSMA-I&S is not yet the optimal radiopharmaceutical for RGS. This is illustrated by the study of Maurer et al. in 31 patients, who reported successful resection of all lesions detected on previous PSMA-PET, as well as additional lesions as small as 3 mm

[13] . However, the same study revealed that in 12 of 86 resected tissue specimens classified as PSMA-negative based on γ-probe measurements, histochemical analysis found previously undetected metastatic lesions.

[0315] From the perspective of radiopharmaceuticals, the obvious need for higher sensitivity in RGS is an optimized radiotracer that provides a higher TBR during surgery. Therefore, to evaluate the TBR of our novel 99m Tc-labeled N4-PSMA ligand, biodistribution studies were performed 6 hours after injection. This rather long distribution time poses a limitation in terms of lack of comparability with the literature, where most time points studied were 1 hour or 4 hours. However, RGS is performed approximately 20 - 24 hours after injection in patients [13, 19, 34], and based on the rule of thumb of approximately 4 times faster metabolism in mice compared to men, we chose a distribution time of 6 hours to simulate the TBR at surgery as accurately as possible. In this context, our preclinical findings on the in vivo performance of 99m Tc]Tc-N4-PSMA ligand represent a significant development. Although similar high uptake was observed in LNCaP-xenografts 6 hours after injection, compared to 99m Tc]Tc-PSMA-I&S, the clearance of the novel ligand from most background tissues was significantly improved. In addition, compared to 99m Tc]Tc-PSMA-I&S, a significantly and favorably reduced renal retention was found to be a common feature of the new ligand, which may be attributed to its shared molecular scaffold derived from the rhPSMA-compound [23, 24], while being significant through the use of the hydrolyzed SiFA moiety. This finding is of particular interest because high renal activity accumulation may interfere with accurate lesion detection during RGS

[34] .

[0316] To an even greater extent, 99mIncomplete clearance of [[Tc]]Tc-PSMA-I&S from the blood pool and thus suboptimal T / Blood ratios may affect the accuracy of RGS

[15] . In this regard, the reduction of blood activity 6 hours post injection represents a further distinct advantage of the preferred PSMA ligand compounds according to the invention, especially for the 3.6-fold reduction observed for 99m [[Tc]]Tc-N4-PSMA-12. Compared to 99m [[Tc]]Tc-N4-PSMA-12, slightly higher uptake in blood and some background organs such as the heart, lungs, liver, parotid and submandibular glands was observed for 99m [[Tc]]Tc-N4-PSMA-13 and 99m [[Tc]]Tc-N4-PSMA-21, which can be attributed to the incorporation of the aromatic amino acids D-Phe and D-(4-NH2)Phe (conferring higher lipophilicity and plasma protein binding to these compounds compared to 99m [[Tc]]Tc-N4-PSMA-12 which contains the negatively charged amino acid D-Glu). 99m Another possible limitation of [[Tc]]Tc-PSMA-I&S is that background activity in the intestine hampers the detection of lesions with low signal intensity during surgery. In this regard, when completely disregarding the significantly improved T / Kidney ratio of 99m [[Tc]]Tc-N4-PSMA-12 and when only considering the liver and intestinal uptake of 99m [[Tc]]Tc-PSMA-I&S and 99m [[Tc]]Tc-N4-PSMA-12 (no statistically significant differences were observed, P > 0.19 and P > 0.36 respectively), we expected the performance of 99m [[Tc]]Tc-PSMA-I&S and 99m [[Tc]]Tc-N4-PSMA-12 to be at least similar in males. In addition to this, the finding that the TBR of blood and all analyzed organs obtained with 99m [[Tc]]Tc-N4-PSMA-12 (see Figure 4 ) is higher than that of 99m [[Tc]]Tc-PSMA-I&S clearly confirms the excellent pharmacokinetic profile of the radiopharmaceuticals according to the invention.

[0317] To facilitate future clinical translation, we developed a GMP-production protocol for 99m [[Tc]]Tc-N4-PSMA-12 (see Figure 5 ). Since it is a simple, reliable, one-step method that conforms to the basic nuclear medicine infrastructure and established clinical workflows and procedures, it provides a 99mThe widespread availability of [[Tc]]Tc-N4-PSMA-12 provides an important foundation and can help minimize the logistical barriers to its first clinical application.

[0318] Abbreviations

[0319] HPLC: high performance liquid chromatography; IBA-KuE: (((S)-1-carboxy-5-(4-(iodo)benzamido)pentyl)carbamoyl)-L-glutamic acid; LNM: lymph node metastasis; mCRPC: metastatic castration-resistant prostate cancer; N4: tetraamine / 6-carboxy-1,4,8,11-tetraazoundecane; PBS: phosphate buffered saline; PCa: prostate cancer; PET: positron emission tomography; PPB: plasma protein binding; PSMA: prostate-specific membrane antigen; RCP: radiochemical purity; RGS: radio-guided surgery; RP: reverse phase; SD: standard deviation; SiFA: silicon fluoride receptor; sLND: sentinel lymph node dissection; SPECT: single photon emission computed tomography; TBR: tumor-to-background ratio; TLC: thin layer chromatography; UV: ultraviolet.

[0320] Figure 1 : 99m [[Tc]]Tc-N4-PSMA-12, 99m [[Tc]]Tc-N4-PSMA-13, 99m [[Tc]]Tc-N4-PSMA-21 and 99m In vitro characterization of [[Tc]]Tc-PSMA-I&S: A) Binding affinity to PSMA (IC 50,inv. (nM), 1 h, 4 °C, n = 3); B) PSMA-mediated internalization (1 h, 37 °C, (% of 125 [[I]]IBA-KuE), n = 3); C) Lipophilicity expressed as logD 7.4 (n-octanol / PBS, pH 7.4, n = 8); D) Binding to human plasma (PPB) (incubated at 37 °C for 30 min, determined by ultrafiltration (%), n = 6).

[0321] Figure 2 : 99m [[Tc]]Tc-labeled N4-PSMA derivatives and 99m [[Tc]]Tc-PSMA-I&S ex vivo biodistribution data at 6 h after injection in male CB17-SCID mice bearing LNCaP tumors. Data are presented as percentage of injected dose per gram (% ID / g), mean ± standard deviation (n = 4-5). gl.: gland; submand.: submandibular.

[0322] Figure 3 : In mice bearing LNCaP tumors99m Static μSPECT / CT images (maximum intensity projection) of 99m 99mTc-labelled N4-PSMA-derivatives and

[0323] Figure 4 : 99m 99mTc-labelled N4-PSMA compounds and 99m 99mTc]99mTc-PSMA-I&S at 6 h post-injection in male CB17-SCID mice bearing LNCaP tumours. Data are given as mean ± standard deviation (n = 4-5). The mean was determined from the TBR calculated for individual animals. gl.: gland; submand.: submandibular.

[0324] Figure 5 : For 99m Suggested workflow for the clinical preparation of

[0325] References

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Claims

1. A compound of formula (1) or a salt thereof: Wherein R T is a PSMA binding group; L is a linking group; R C is a trivalent coupling group; R S is the silicon-containing moiety of formula -C(O)-R S3 -SiR S1 R S2 OH, where R S1 and R S2 are independently selected from C3-C10 alkyl, and R S3 is a group containing a 6-membered aromatic ring; R A is an amino acid residue; and R CH Selected from (i) A branched, acyclic chelating moiety having 4 amino groups, and (ii) A chelate moiety, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the branched, acyclic chelating moiety having 4 amino groups.

2. The compound or salt according to claim 1, wherein R in formula (1) T is a group having the following structure: wherein the wavy line indicates the bond connecting the group R T to the remainder of the compound of formula (1).

3. The compound or salt according to claim 1 or 2, wherein R in formula (1) S is a group of the following structure (S-2): Wherein R S3 is a group containing a 6-membered aromatic ring; and The wavy line indicates the bond connecting the group R S to the remainder of the compound of formula (1).

4. The compound or salt according to any one of claims 1 to 3, wherein R in formula (1) S is a group of the following structure (S-3): Wherein The wavy line indicates the bond connecting the group R S to the remainder of the compound of formula (1).

5. The compound or salt according to any one of claims 1 to 4, wherein R in formula (1) A is a group having the following structure: Wherein R A1 selected from hydrogen, -(CH2) k -COOH, -CH2-Ar and -Ar, Where k is 1, 2 or 3, preferably 2, and Ar is an optionally substituted phenyl group which may carry substituents selected from -OH and -NH2, The wavy line at the -C(O)- group shown in the formula indicates the bond formed with R C and the wavy line at the -NH- group shown in the formula indicates the bond formed with R CH to form a bond.

6. The compound or salt according to any one of claims 1 to 5, wherein R in formula (1) CH is selected from: (i) An N4 chelating group of the following formula: Wherein The wavy line indicates the bond connecting the group R CH to the remainder of the compound of formula (1), and optionally, one or more hydrogen atoms attached to a carbon atom in the above formula, such as one, two or three hydrogen atoms, may be substituted by substituents, and (ii) Chelate moiety, wherein a radioisotope selected from 99m Tc, 94m Tc, 186 Re and 188 Re is chelated by the N4 chelating group.

7. The compound or salt according to any one of claims 1 to 6, wherein L in formula (1) is an oligomeric amide residue comprising 2 to 6 subunits linked to each other by an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR-, wherein R is a C1-C6 alkyl group.

8. The compound or salt according to any one of claims 1 to 7, wherein R in formula (1) C is a trivalent amino acid unit derived from an amino acid comprising a carboxylic acid group and an amino group and a side chain carrying an additional functional group selected from the carboxylic acid group and the amino group.

9. The compound or salt according to any one of claims 1 to 8, which is a radiolabeled compound comprising a chelating radioisotope selected from 99m Tc, 94m Tc, 186 Re, and 188 Re.

10. The compound or salt according to claim 9, which is used for in vivo diagnosis of a disease associated with overexpression of PSMA, wherein the disease is preferably cancer, and more preferably prostate cancer, and the diagnosis preferably involves nuclear medicine tomography, more preferably single photon emission computed tomography (SPECT).

11. The compound or salt according to claim 9, which is used for identifying diseased tissue in vivo associated with overexpression of PSMA, wherein the diseased tissue is preferably cancer tissue, and more preferably prostate cancer tissue.

12. The compound or salt according to claim 11, wherein the diseased tissue is identified in the context of a radio-guided surgery for removing the diseased tissue.

13. Use of the compound or salt according to claim 9 for identifying tissues or cells in vitro or ex vivo associated with overexpression of PSMA, wherein the tissues or cells are preferably cancer tissues or cells, and more preferably prostate cancer tissues or cells.

14. An in vitro or ex vivo method for identifying whether a tissue or cell overexpresses PSMA, which comprises contacting the tissue or cell with the compound or salt according to claim 9, wherein the tissue or cell is preferably cancer tissue or cell, and more preferably prostate cancer tissue or cell.

15. The compound or salt according to claim 9, which is used for treating or preventing a disease associated with overexpression of PSMA, wherein the disease is preferably cancer, and more preferably prostate cancer.

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