Radiolabeled PSMA Ligand Compounds and Their Precursors

Abstract

Formula (1) (where R T is a PSMA binding group, L is a linking group, and R C is a trivalent coupling group, and R S is the formula -C(O)-R S3 -SiR S1 R S2 is the silicon-containing portion of OH, where R S1 and R S2 are independently selected from C3 to C10 alkyl, preferably tert-butyl, and R S3 is a group containing a 6-membered aromatic ring, preferably benzenediyl), R A is an amino acid unit, and R CH is a compound that contains (i) a branched, acyclic chelating moiety having four amino groups, and (ii) 99m Tc, 94m Tc, 186 Re, and 188 and a chelating moiety in which a radioisotope selected from Re is chelated by a branched, acyclic chelating moiety having four amino groups, or a salt thereof. The PSMA targeting ligand compound according to the present invention is suitable for radiopharmaceutical applications such as radiation-guided surgery. JPEG2025542269000051.jpg2477

Description

[Technical Field]

[0001] The present invention relates to compounds that can be radiolabeled and are suitable as PSMA binding ligand compounds, for example, for use as radiotracers in radiodiagnostic or radioguided surgical settings or for use in radiotherapy. [Background technology]

[0002] Throughout the past decade, radiopharmaceuticals targeting glutamate carboxypeptidase II (GCPII), also referred to as prostate-specific membrane antigen (PSMA), have become an essential part of the clinical management of prostate cancer (PCa) [1, 2]. For positron emission tomography (PET) imaging [ 68 Ga]Ga-PSMA-11[3] and [ 18 F]DCFPyL[4] or for single photon emission computed tomography (SPECT) imaging [ 99m Almost in parallel with the clinical success of diagnostic tracers such as [Tc]Tc-MIP-1404 [5], several therapeutic compounds have entered the field, including 177 These included Lu-labeled PSMA-I&T and PSMA-617 [6, 7]. Furthermore, several different therapeutic approaches have progressed into preclinical and clinical studies, including long-acting albumin-binding PSMA-ligands [8, 9], targeted alpha therapy

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

[11] . 177 The recent approval of [Lu]Lu-PSMA-617 (Pluvicto™, Novartis) represents a milestone in nuclear medicine, expanding the armamentarium of oncologists and potentially paving the way for further approval of radiopharmaceuticals for targeted therapeutics.

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

[12] . Patients with early biochemical recurrence after RP and who demonstrate only localized pelvic lymph node metastases (LNM) on PSMA-PET imaging can benefit from radiation-guided salvage lymph node dissection (sLND), delaying disease progression and future systemic treatment

[13] . In contrast to conventional sLND, a gamma-emitting PSMA-targeted radioligand is injected intravenously up to 24 hours before surgery. Utilizing the gamma-probe facilitates intraoperative localization and resection of metastatic lymph nodes, which is particularly useful in cases of small or abnormally localized lesions. Furthermore, resected tissue can be immediately identified by ex vivo gamma-probe measurement to confirm successful removal of tumor-affected tissue.

[14] 111 After the initial proof of concept using In]In-PSMA-I&T

[14] , PSMA-RGS was 111 In]InCl3, its comparable performance in vivo and 99m Tc]TcO4 - more favorable radiation properties in the operating room 99m Due to the more general tolerance, lower cost, and higher availability of Tc tracers, 99m This study was performed using [Tc]Tc-PSMA-I&S

[15] . A recent study by Horn et al. in 121 patients noted successful removal of preoperatively identified lesions in 99% of patients and complete biochemical responses in 66% of patients

[16] , confirming the results of a smaller patient group previously reported by Maurer et al.

[13] . In the latter study, even additional lesions not previously detected by PSMA-PET were removed, highlighting the potential of RGS in sLND. However, prospective clinical trials such as the TRACE trial (NCT03857113) are needed to accurately evaluate the long-term outcomes and additional benefits of RGS for PCa patients.

[0004] The main limitation of sLND procedures, whether conventional or radiation-guided, consists of incomplete resection of metastatic lesions. 99m Although state-of-the-art PSMA radiological guidance using [Tc]Tc-PSMA-I&S showed excellent short-term efficacy in terms of biochemical response in a prospective study by Knipper et al.

[17] , its still insufficient sensitivity for micrometastatic lesions seems to be the main reason for disease recurrence [13, 18]. Technological developments such as robotic-assisted laparoscopy, meaning drop-in or click-on γ-probes

[19] and more differentiated criteria for patient selection

[20] could certainly promote its application. However, we note the unmet need for improvement in the radiopharmaceutical level, i.e., the current application [ 99m We identified the need for a radiotracer that provides higher contrast and lower background signal than [Tc]Tc-PSMA-I&S. Its relatively slow systemic clearance and partial hepatobiliary excretion are due to its high plasma protein binding (PPB) of 94% and reduced hydrophilicity compared to DOTA-based PSMA-radioactive chelated tracers

[15] . As a result, nonspecific background signal interferes with both contrast in initial SPECT images and accurate detection during surgery [21, 22]. For this reason, [ 99m [Tc]Tc-PSMA-I&S is not used to definitively identify LNM in situ during surgery, but is used to confirm ex vivo that excised tissue is PSMA-positive. However, if the TBR is sufficiently high so that even smaller lesions may be detectable with a properly functioning radiotracer, more accurate lesion detection should be technically possible, even in the surgical field. 99m Despite the known limitations of [Tc]Tc-PSMA-I&S, to our knowledge, there are no other methods for the application of RGS. 99m Dedicated optimization of Tc-labeled PSMA ligands has not been performed to date. Summary of the Invention [Problem to be solved by the invention]

[0005] To address this medical need, the present invention provides a novel series of radiolabeled PSMA-targeting ligand compounds and their precursors with improved pharmacokinetic behavior, suitable for use as radiotracers (or radioactive targeting probes) for radiodiagnosis, in the setting of RGS, or for radiotherapy applications. The optimized ligand structures were conceptually designed and contain a common PSMA inhibitory motif derived from highly potent radiohybrid PSMA diagnostic agents

[23] and therapeutic agents

[24] , a tetraamine chelator for reliable complexation of suitable radioisotopes such as technetium-99m, and variable amino acids for tuning the pharmacokinetic properties of the peptides. Such modifications have yielded interesting PSMA radioligands for diverse applications [25-27]. [Means for solving the problem]

[0006] The PSMA targeting ligand compound according to the present invention has the formula (1):

[0007] [ka]

[0008] (In the formula, R T is the PSMA binding group, L is a linking group, R C is a trivalent coupling group, R S is the formula -C(O)-R S3 -SiR S1 R S2 is the silicon-containing portion of OH, where R S1 and R S2 are independently selected from C3 to C10 alkyl, preferably tert-butyl, and R S3 is a group containing a 6-membered aromatic ring, preferably benzenediyl), R A is an amino acid unit, and R CH teeth, (i) a branched, acyclic chelating moiety having four amino groups, and (ii) 99m Tc, 94m Tc, 186 Re, and 188 a chelating moiety in which a radioisotope selected from Re is chelated by a branched acyclic chelating moiety having four amino groups; or a salt thereof.

[0009] It has been found that the compounds of the present invention combine outstanding inhibitory binding capacity to PSMA with general advantages over PSMA-I&S, such as reduced lipophilicity and reduced plasma protein binding (PPB), thus exhibiting comparable or even improved in vitro properties and increased tumor-to-organ ratios in various organs, in particular increased tumor-to-kidney ratios, which are crucial parameters for the application of radioligands in the diagnosis and treatment of prostate cancer.

[0010] Thus, in a further aspect, the present invention relates to compositions, e.g., therapeutic or diagnostic compositions, comprising compounds according to the invention, which are suitable for the treatment and / or diagnosis of diseases associated with overexpression of PSMA, such as prostate cancer. DETAILED DESCRIPTION OF THE INVENTION

[0011] As explained above, compounds according to the present invention include compounds of formula (1). Furthermore, salts of compounds of formula (1), typically pharmaceutically acceptable salts, are also encompassed by the present invention. Thus, unless otherwise indicated, all references to compounds according to the present invention encompass compounds of formula (1) (and 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 context indicates the specific stereochemistry of the compound under consideration. Due to their ability to bind to PSMA and act as ligands for PSMA, compounds according to the present invention may also be referred to as PSMA-targeting ligand compounds according to the present invention, PSMA-binding compounds according to the present invention, or, simply, ligand compounds according to the present invention.

[0012] In the following, further description of the compounds of formula (1) and their salts and preferred embodiments thereof will be provided. In accordance with the above description, it will be understood that the groups discussed in this context that can provide a salt form of the compounds of formula (1), such as deprotonable acidic groups or protonable basic groups, can be contained in the compounds according to the present invention in ionic (e.g., protonated or deprotonated) form, even if this ionic form is not specifically exemplified for the relevant group herein.

[0013] R in formula (1) T represents a PSMA-binding group. Its ability to bind to PSMA allows compounds according to the invention to function as PSMA-targeting ligand compounds. A variety of PSMA-binding groups are known in the art and available to those skilled in the art for this purpose.

[0014] In formula (1), R T preferably has the following structure (T-1):

[0015] [ka]

[0016] (In the formula, the wavy line represents R T (depicts the bond connecting the group to L in formula (1)) More preferably, R T has the following structure (T-2):

[0017] [ka]

[0018] (In the formula, the wavy line represents R T (depicts the bond connecting the group to L in formula (1)) is the basis of As will be understood by those skilled in the art, one or more of the carboxylic acid groups shown in the formula above may be deprotonated depending on the conditions under which the compound according to the invention is maintained, for example, the pH of the solution. The negative charge of the deprotonated carboxylic acid group may be balanced, for example, by a positive charge carried by another group in the compound according to the invention, or by a cationic counterion, for example, as described below with respect to salt forms of the compound according to the invention.

[0019] Consistent with the above, the compound of formula (1) preferably has the formula (1A):

[0020] [ka]

[0021] (In the formula, L, R C , R S , R A , and R CH is as defined herein, including any preferred definitions thereof) is a compound of

[0022] Even more preferably, the compound of formula (1) has the formula (1AA):

[0023] [ka]

[0024] (In the formula, L, R C , R S , R A , and R CH is as defined herein, including any preferred definitions thereof) is a compound of

[0025] It should be understood that all references herein to formula (1) apply equally to formulas (1A) and (1AA), as preferred variants thereof, unless indicated to the contrary.

[0026] The L group in formula (1) represents a linking group. As illustrated in formula (1), L is connected to R via a first covalent bond. T and via a second covalent bond to a coupling group R C Therefore, L is typically a divalent group bonded to R T a first functional group at a first end to which R T L contains a first functional group suitable for forming an amide bond, -C(O)-NH-, or an alkylated amide bond, -C(O)-NR-, preferably an amide bond, with a complementary group contained in R. Preferably, this first functional group of L is an -NH- group. Similarly, L typically contains a first functional group 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 a second functional group at the second end to which R C Preferably, L contains a second functional group suitable for forming an amide bond or an alkylated amide bond, preferably an amide bond, with the complementary group contained in L. Preferably, this second functional group of L is also an —NH— group.

[0027] When an alkylated amide bond -C(O)-NR- (or -NR-C(O)-) is referred to herein, the R group of the alkylated amide bond, as will be seen hereinafter, is a C1-C6 alkyl group, preferably methyl.

[0028] In a preferred embodiment, L represents an oligoamide residue having first and second ends bearing the above-described functional groups, i.e., preferably -NH- groups. This oligoamide residue preferably consists of 2 to 6, more preferably 2 to 4, and even more preferably 3 subunits. As the name "oligoamide residue" suggests, adjacent subunits of the oligoamide residue are linked to each other via an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR-, preferably an amide bond. The backbone of the oligoamide unit extending from the first to the second end preferably consists of 6 to 20, more preferably 8 to 18, and even more preferably 8 to 16 carbon atoms, not including carbon atoms in any substituents attached to the backbone.

[0029] Examples of the linking group L include the following group (L-1):

[0030] [ka]

[0031] (In the formula, the wavy lines represent L to R, respectively. T and R C (The figure shows the bond formed by can be exemplified.

[0032] As will be understood by those skilled in the art, one or more of the carboxylic acid groups shown in the formula above may be deprotonated depending on the conditions under which the compound according to the invention is maintained, for example, the pH of the solution. The negative charge of the deprotonated carboxylic acid group may be balanced, for example, by a positive charge carried by another group in the compound according to the invention, or by a cationic counterion, for example, as described below with respect to salt forms of the compound according to the invention.

[0033] R in formula (1) C As shown in equation (1), the branch R T -L-, branching -RA -R CH , and -R S It is a trivalent coupling group that serves to couple groups. C is typically a functional group at the end to which L is attached, and contains a functional group suitable for forming an amide bond (-C(O)-NH-) or an alkylated amide bond (-C(O)-NR-), preferably an amide bond, with a complementary group contained in L. Preferably, R C This functional group in R is a -C(O)- group. C is typically R A A functional group at either the end or side chain to which R A Preferably, R contains a functional group suitable for forming an amide bond or an alkylated amide bond, preferably an amide bond, with a complementary group contained in R C The coupling group in R is an -NH- group. S At the end where R C is typically R S A functional group at either the end or side chain to which R S Preferably, R contains a functional group suitable for forming an amide bond or an alkylated amide bond, preferably an amide bond, with the complementary group —C(O)— contained in R. C The coupling group in is an -NH- group.

[0034] R C It is preferred that R is a trivalent amino acid unit. C is a trivalent amino acid unit that may be derived from an amino acid consisting of a carboxylic acid group and an amino group, with a side chain bearing an additional functional group selected from a carboxylic acid group and an amino group. More preferably, R Cis selected from 2,3-diaminopropionic acid (Dap) unit, 2,4-diaminobutanoic acid (Dab) unit, ornithine (Orn) unit, and lysine (Lys) unit, and is most preferably a Dap unit. As will be understood by those skilled in the art, this amino acid unit is a unit obtained from each amino acid by using its functional group to form a bond, preferably an amide bond, to the 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.

[0035] The trivalent amino acid unit is preferably linked to the compound of the present invention by three amide bonds. Furthermore, this amino acid unit, together with L, is linked to the amino acid unit R C a —C(O)— functional group, R, which is bonded to L such that an amide bond is formed by A together with the amino acid unit R C R A and an —NH— functional group bonded to R S together with the amino acid unit R C R S More preferably, the aryl group is arranged to provide an -NH- functional group to be bonded to the aryl group.

[0036] R in formula (1) S is the formula -C(O)-R S3 -SiR S1 R S2 OH, a silicon-containing moiety of formula (S-1)

[0037] [ka]

[0038] (In the formula, R S1 and R S2 are independently selected from C3 to C10 alkyl, preferably tert-butyl, and R S3 is a group containing a six-membered aromatic ring It can also be exemplified by RS3 is preferably a benzenediyl group, even more preferably a benzene-1,4-diyl group. S group in formula (1) C The bond that binds to is shown.

[0039] As shown in the above formula, R S The part is R C together with the complementary group contained in, an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR-, preferably a functional group -C(O)- suitable for forming an amide bond.

[0040] Therefore, R in Eq. (1) S However, the following structure (S-2):

[0041] [ka]

[0042] (In the formula, R S3 is a group containing a 6-membered aromatic ring, preferably benzenediyl, more preferably benzene-1,4-diyl, and The wavy line is R S group in formula (1) C (The bond connecting to the It is preferred that the group is:

[0043] Consistent with the above, most preferably, R in formula (1) S has the following structure (S-3):

[0044] [ka]

[0045] (In the formula, The wavy line is R S group in formula (1) C (The bond connecting to the This is the part.

[0046] 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, i.e., a compound that contains an amino group and a carboxylic acid group in the same molecule. Unless the specific context dictates otherwise, one or more further functional groups in addition to the amino and carboxylic acid groups may be present in the amino acid from which the amino acid unit can be derived. A specific amino acid unit is typically identified by the name of the amino acid from which it can be derived, e.g., a glycine unit, an asparagine unit, etc. Unless the specific context dictates otherwise, an amino acid unit R A The amino acids from which the amino acid unit R can be obtained are preferably α-amino acids. A When can be obtained from a chiral amino acid, the D-configuration is preferred.

[0047] Amino acid unit R A is typically obtained from an amino acid by using the amino group of the amino acid to provide a functional group -NH- that forms an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR-, preferably an amide bond, with a complementary group contained in one of the adjacent groups of formula (1), and by using the carboxylic acid group to provide a functional group -C(O)-NH- or an alkylated amide bond -C(O)-NR-, preferably an amide bond, with a complementary group contained in the other adjacent group of formula (1). Preferably, the amino acid unit R A is R C functional groups -C(O)- and R for forming an amide bond together with the -NH- group provided by CH More preferably, R provides a functional group -NH- for forming an amide bond with the -C(O)- group provided by CH to form an amide bond with the amino acid unit R A The functional group -NH- provided by R C It is obtained from an amino group alpha to a carboxylic acid group, which provides the functional group -C(O)- to form an amide bond with

[0048] Preferably, the amino acid unit R A has the following structure (A-1):

[0049] [ka]

[0050] (In the formula, The wavy line in the -C(O)- group shown in the formula represents R C The wavy line in the -NH- group shown in the formula represents the bond formed with R CH and R A1 is hydrogen, -(CH2) k -COOH, -CH-Ar, and -Ar, where k is 1, 2, or 3, preferably 2; and Ar is an optionally substituted phenyl group, which may have a substituent selected from —OH and —NH. When the phenyl group Ar is substituted, it is preferred that it has a single substituent selected from -OH and -NH2. The -C(O)- group shown in formula (A-1) has R C and the —NH— group shown in formula (A-1) is formed into an amide bond together with the —NH— group provided by R CH It is preferred to form an amide bond with the -C(O)- group provided by:

[0051] More preferably, the amino acid unit R A has the following structure (A-1A):

[0052] [ka]

[0053] (In the formula, The wavy line in the -C(O)- group shown in the formula represents R CThe wavy line in the -NH- group shown in the formula represents the bond formed with R CH and R A1 is defined in formula (A-1)) The —C(O)— group shown in formula (A-1A) is R C and the —NH— group shown in formula (A-1A) forms an amide bond with the —NH— group provided by R CH It is preferred to form an amide bond with the -C(O)- group provided by:

[0054] In the above formulas (A-1) and (A-1A), R A1 are preferably selected from -(CH2)2-COOH, -CH2-Ph, and -CH2-(p-NH2-Ph), respectively, where Ph is a phenyl group. Most preferably, R A1 is -(CH2)2-COOH.

[0055] Appropriate amino acid unit R A As examples, reference may be made to units derived from Gly, Glu, Phe, (4-NH2)-Phe, Tyr, or phenylglycine, where the chiral amino acid is preferably in the D-configuration.

[0056] As will be understood by those skilled in the art, amino acid units containing a side chain bearing a basic group such as -NH or an acidic group such as -COOH can be protonated or deprotonated, respectively, depending on the conditions under which the compound according to the invention is maintained, for example, at the pH of its solution. The positive charge of the protonated amino group can be balanced, for example, by a positive charge carried by another group in the compound according to the invention, or by an anionic counterion, for example, as described below with respect to salt forms of the compound according to the invention. The negative charge of the deprotonated carboxylic acid group can be balanced, for example, by a positive charge carried by another group in the compound according to the invention, or by a cationic counterion, for example, as described below with respect to salt forms of the compound according to the invention.

[0057] R CH teeth, (i) a branched, acyclic chelating moiety having four amino groups, and (ii) 99m Tc, 94m Tc, 186 Re, and 188 a chelating moiety in which a radioisotope selected from Re is chelated by a branched acyclic chelating moiety having four amino groups; is selected from.

[0058] Each of the four amino groups of a branched-chain acyclic chelating moiety having four amino groups provides a lone pair of electrons suitable for forming a metal / ligand coordinate bond, and the branched-chain acyclic chelating moiety is typically a tetradentate chelating moiety. As will be understood by those 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 atoms of a cyclic structure. As will be further understood, the term branched-chain chelating moiety requires the presence of at least one carbon atom in the chelating moiety that forms covalent bonds to at least three other carbon atoms. Unlike other chelating moieties used to deliver Tc-chelates, such as the mas3 chelating moiety used in PSMA I&S, the branched-chain acyclic chelating moiety having four amino groups as defined above typically does not have a sulfur atom.

[0059] Preferably, the branched acyclic chelating moiety having four amino groups has the following structure (CH-1):

[0060] [ka]

[0061] wherein the wavy line marks the bond connecting this group to the remainder of the compound according to the invention, and optionally one or more hydrogen atoms, such as one, two, or three, attached to the carbon atoms of the above formula can be replaced by a substituent, such as a methyl group. However, it is preferred that such substituents are absent.

[0062] In addition to the amino group, which allows for the formation of a chelate complex, a chelating moiety R CH is R A Generally, R 1 contains a functional group suitable for forming an amide bond, —C(O)—NH—, or an alkylated amide bond, —C(O)—NR—, preferably an amide bond, with a complementary group contained in R 2 . CH The functional group contained in is a —C(O)— group.

[0063] For this reason, R CH The branched acyclic chelating moiety having four amino groups has the following structure (CH-2):

[0064] [ka]

[0065] wherein the wavy line marks the bond connecting this group to the remainder of the compound according to the invention, and optionally one or more hydrogen atoms, such as one, two, or three, attached to the carbon atoms of the above formula can be replaced by a substituent, such as a methyl group. However, it is preferred that such substituents are absent.

[0066] According to option (ii) above, a branched acyclic chelating moiety having four amino groups as defined above is 99m Tc, 94m Tc, 186 Re, and 188A radioisotope selected from Re can be provided as a chelating moiety, which is chelated by the chelating moiety. Thus, the chelating moiety forms multiple (usually four) coordinate bonds with the chelated radioisotope. In addition, one or more additional ligands may be coordinated to the radioisotope. Compounds according to the present invention that include a chelating moiety are also referred to herein as chelating compounds.

[0067] Preferably, the chelated radioisotope is 99m Tc. In the chelating moiety and chelating compounds containing it, the chelated radioisotope is typically a cationic species in the +V oxidation state, e.g., 99m Tc, 94m Tc, 186 Re, or 188 The chelated radioisotope may have one or more additional ligands, such as one, two, or three, other than the chelating moiety contained in the compound according to the invention. For example, a Tc cation (i.e., preferably in the oxidation state +V) may be used. 99m Tc cation or 94m In a chelating moiety containing a Tc cation (i.e., preferably in the oxidation state +V), the Tc cation complexed by the chelating moiety as a chelating ligand may have, for example, two or more anionic ligands as additional ligands, such as two oxo ligands. 186 Re cation or 188 In a chelating moiety containing a Re cation, the Re cation complexed by the chelating moiety as a chelating ligand may have, for example, two or more anionic ligands as additional ligands, such as two oxo ligands.

[0068] For this reason, 99m Tc, 94m Tc, 186 Re, or 188 The Re radioisotopes are the dioxo species of this formula, respectively ([ 99m Tc]TcO2)+ , ([ 94m Tc]TcO2) + , ([ 186 Re]ReO2) + ,or([ 186 Re]ReO2) + As such, it is preferably chelated by a chelating moiety contained in the compound according to the invention.

[0069] For this reason, R CH A preferred chelating moiety according to option (ii) above in the definition of

[0070] [ka]

[0071] where the wavy line marks the bond connecting this group to the remainder of the compound according to the invention, and Tc is 99m Tc or 94m Tc, and optionally, one or more hydrogen atoms, such as one, two, or three, bonded to the carbon atoms of the above formula may be replaced by a substituent, such as a methyl group. However, it is preferred that such substituents are absent.

[0072] Consistent with the above, R CH The chelating portion of the compound has the following structure (CH-4):

[0073] [ka]

[0074] where the wavy line marks the bond connecting this group to the remainder of the compound according to the invention, and Tc is 99m Tc or 94m Tc, and optionally, one or more hydrogen atoms, such as one, two, or three, bonded to the carbon atoms of the above formula may be replaced by a substituent, such as a methyl group. However, it is preferred that this substituent is absent.

[0075] When the chelating moiety is a charged moiety, e.g., a cationic moiety, as exemplified in formulas (CH-3) and (CH-4), the positive charge can be balanced, e.g., by a negative charge carried by another group of the compound according to the invention, or by an anionic counterion, e.g., as described below with respect to salt forms of the compound according to the invention. Exemplary counterions include Cl. - , HPO4 2- , PO4 3- , H2PO4 - Reference may be made to any anion used in sterile solutions for injection, such as citric acid, citrate, or ascorbic acid.

[0076] 99m Tc, 94m Tc, 186 Re, and 188 Compounds according to the invention comprising a chelating moiety to which a radioisotope selected from Re is chelated may be used as radiolabeled compounds (or more specifically, 99m Tc label, 94m Tc label, 186 Re sign, or 188 Re-labeled compounds), whereas compounds according to the present invention that include a chelating moiety that does not have a chelated radioisotope may be referred to herein as unlabeled or unlabeled compounds.

[0077] As will be appreciated, radiolabeled compounds according to the invention are suitable, for example, as radiopharmaceuticals in radiotherapy and / or as radiotracers in diagnosis or radiosurgery. Unlabeled compounds according to the invention, for example, provide useful precursors for radiolabeling.

[0078] Consistent with the above description, the compound of formula (1) preferably has the following formula (1B):

[0079] [ka]

[0080] (In the formula, L, R C , R S1 , R S2 , R S3 , and R A1 The group is as defined above, including any preferred definitions thereof, and Optionally, one or more hydrogen atoms, such as one, two, or three, attached to a carbon atom of the chelating moiety -C(O)-CH(CHNHCHCHNH) in the above formula may be replaced by a substituent, such as a methyl group. The compound or 99m Tc, 94m Tc, 186 Re, and 188 a chelating compound in which a radioisotope selected from Re is chelated by an optionally substituted chelating moiety, -C(O)-CH(CHNHCHCHNH) and which is included in formula (1B) above; is.

[0081] More preferably, the compound of formula (1) has the formula (1BB):

[0082] [ka]

[0083] (In the formula, L, R C , R S1 , R S2 , R S3 , and R A1 The group is as defined above, including any preferred definitions thereof, and Optionally, one or more hydrogen atoms, such as one, two, or three, attached to a carbon atom of the chelating moiety -C(O)-CH(CHNHCHCHNH) in the above formula may be replaced by a substituent, such as a methyl group. The compound or 99m Tc, 94m Tc, 186 Re, and 188 A chelating compound in which a radioisotope selected from Re is chelated by an optionally substituted chelating moiety -C(O)-CH(CHNHCHCHNH) and which is included in the formula above. is.

[0084] Even more preferably, the compound of formula (1) has the formula (1C):

[0085] [ka]

[0086] (In the formula, R A1 The group is as defined above, including any preferred definitions thereof, and Optionally, one or more hydrogen atoms, such as one, two, or three, attached to a carbon atom of the chelating moiety -C(O)-CH(CHNHCHCHNH) in the above formula may be replaced by a substituent, such as a methyl group. The compound or 99m Tc, 94m Tc, 186 Re, and 188 a chelating compound in which a radioisotope selected from Re is chelated by an optionally substituted chelating moiety, -C(O)-CH(CHNHCHCHNH) and which is included in formula (1C) above; is.

[0087] Even more preferably, the compound of formula (1) has the formula (1CC):

[0088] [ka]

[0089] (In the formula, R A1 The group is as defined above, including any preferred definitions thereof, and Optionally, one or more hydrogen atoms, such as one, two, or three, attached to a carbon atom of the chelating moiety -C(O)-CH(CHNHCHCHNH) in the above formula may be replaced by a substituent, such as a methyl group. The compound or 99m Tc, 94m Tc, 186 Re, and 188 A chelating compound in which a radioisotope selected from Re is chelated by an optionally substituted chelating moiety -C(O)-CH(CHNHCHCHNH) and which is included in the formula above. is.

[0090] Specific examples of the compounds according to the present invention include the following compounds: 99m Tc, 94m Tc, 186 Re, and 188 Reference can be made to the corresponding chelate compounds, and salts of the compounds or chelate compounds, in which a radioisotope selected from Re is chelated by a chelating moiety, —C(O)—CH(CHNHCHCHNH)—, which formulas of these compounds include:

[0091] [ka]

[0092] [ka]

[0093] [ka]

[0094] As noted above, compounds according to the present invention include compounds of formula (1) (including any preferred embodiments thereof, such as compounds of formula (1A), (1AA), (1B), (1BB), (1C), or (1CC)) and salts thereof. The salts are preferably pharmaceutically acceptable salts, i.e., formed with a pharmaceutically acceptable anion or cation. Salts can be formed, for example, by protonation of an atom having a lone pair of electrons that is susceptible to protonation, such as a nitrogen atom, with an inorganic or organic acid, or by removing a proton from an acidic group, such as a carboxylic acid group, by neutralization, for example, with a base. Other charged groups that may be present in compounds according to the present invention and provide the compounds in salt form include permanently charged groups, such as charged chelating moieties.

[0095] When a salt form of a compound of the present invention comprises a positively charged form of a compound of Formula (1), exemplary anions that may be present as a counterion in the salt form include, for example, chloride, bromide, iodide, sulfate, nitrate, phosphate (e.g., phosphate, hydrogen phosphate, or dihydrogen phosphate), carbonate, bicarbonate, or perchlorate; acetate, trifluoroacetate, propionate, butyrate, valerate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, uranium, thiazolinone ... Mention may be made of an anion selected from the group consisting of decanoate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, nicotinate, benzoate, salicylate, or ascorbate; sulfonates such as methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-toluenesulfonate (tosylate), 2-naphthalenesulfonate, 3-phenylsulfonate, or camphorsulfonate. Because trifluoroacetic acid is often used in the synthesis of peptides, trifluoroacetate is a typical salt obtained when a compound consisting of an oligoamide structure is formed. Such trifluoroacetate can be exchanged, for example, for acetate during their testing.

[0096] When a salt form of a compound of the present invention comprises a negatively charged form of a compound of formula (1), exemplary cations that may be present as counterions in the salt form include, 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 with an organic group).

[0097] In addition to the compounds according to the invention as described above, there is also provided a method for the preparation of radiolabeled compounds according to the invention, which comprises using a non-radiolabeled compound according to the invention as a precursor compound, 99m Tc, 94m Tc, 186 Re, and 188Preferably, the method comprises contacting a compound containing a radioisotope selected from Re with a compound containing such a radioisotope. 99m Tc]pertechnetate, [ 94m Tc]pertechnetate, [ 186 Re] perrhenate, and [ 188 Compounds containing a radioactive isotope selected from [Re] perrhenate are used in connection with this method.

[0098] For example, a non-radiolabeled compound according to the present invention can be used as a precursor compound, 99m Tc, 94m Tc, 186 Re, and 188 The step of contacting with a radioisotope selected from Re or a compound containing such a radioisotope can be carried out by dissolving the precursor compound and the compound containing the radioisotope in a common solvent, preferably an aqueous solution.

[0099] As explained above, a non-radiolabeled compound according to the present invention is a compound according to the present invention that does not contain a chelated radioisotope. 99m Tc label, 94m Tc label, 186 Re sign, or 188 Re-labeled compounds) 99m Tc, 94m Tc, 186 Re, and 188 and Re. Consistent with the above, the chelated radioisotope contained in the radiolabeled compound of the present invention is contained in a chelating moiety in which the radioisotope is chelated by a branched, acyclic chelating moiety having four amino groups, as defined above, which chelating moiety includes any of the preferred forms of chelating moieties, such as a -C(O)-CH(CHNHCHCHNH) moiety.

[0100] As further explained above, the PSMA targeting ligand compounds according to the present invention are useful, for example, for use as radiotracers in the context of radiodiagnosis or radiation-guided surgery, or in radiotherapy. For example, the radiolabeled compounds according to the present invention, preferably the PSMA targeting ligand compounds according to the present invention, are useful. 99m Tc-labeled or 94m The Tc-labeled compound can be effectively used, for example, as a radiotracer in the diagnosis of diseases associated with PSMA overexpression, or as a radiotracer (also referred to in this context as a radioactive targeting probe) in radiation-guided surgery.Radio-guided surgery assisted by the radiolabeled compound according to the present invention enables the identification and removal of diseased tissues associated with PSMA overexpression.

[0101] Thus, a further aspect of the present invention is represented by a composition, e.g., a therapeutic or diagnostic composition, comprising a compound according to the present invention, preferably a radiolabeled compound according to the present invention. More specifically, a therapeutic composition comprises a compound according to the present invention 186 Re sign or 188 The diagnostic composition according to the present invention comprises a Re-labeled compound. 99m Tc-labeled or 94m It is preferred to include a Tc-labeled compound.

[0102] In a first related aspect, the present invention provides a radiolabeled compound according to the invention, preferably a compound according to the invention, for use in diagnosing in vivo a disease associated with PSMA overexpression. 99m Tc labeling or 94m The present invention provides a Tc-labeled compound or a diagnostic composition comprising such a compound.The disease associated with PSMA overexpression is preferably cancer, more preferably prostate cancer.The diagnosis is preferably related to nuclear medicine tomography, more preferably single photon emission computed tomography (SPECT).

[0103] In a further related aspect, the present invention provides a radiolabeled compound according to the present invention, preferably a compound according to the present invention, for use in identifying diseased tissue with PSMA overexpression in vivo. 99m Tc labeling or 94m Provided is a Tc-labeled compound or a diagnostic composition comprising such a compound.In a preferred embodiment, this compound or composition is provided for use in identifying diseased tissue with PSMA overexpression in vivo, so as to remove the diseased tissue under the circumstances of radiation-guided surgery.The diseased tissue with PSMA overexpression is preferably cancer tissue, more preferably prostate cancer tissue.

[0104] In yet a further related aspect, the present invention provides a radiolabeled compound according to the present invention, preferably a radiolabeled compound according to the present invention, for the identification of tissues or cells with PSMA overexpression, which tissues or cells are preferably cancer tissues or cells, more preferably prostate cancer tissues or cells. 99m Tc labeling or 94m The present invention provides ex vivo or in vitro uses of Tc-labeled compounds or diagnostic compositions containing such compounds.

[0105] According to another related aspect, there is provided an ex vivo or in vitro method for identifying whether a tissue or cell overexpresses PSMA, comprising: subjecting the tissue or cell to a radiolabeled compound according to the invention, preferably a compound according to the invention. 99m Tc labeling or 94m The method includes a step of contacting a tissue or cell with a Tc-labeled compound or a diagnostic composition containing such a compound, wherein the tissue or cell is preferably a cancer tissue or cell, more preferably a prostate cancer tissue or cell.

[0106] In a still further related aspect, the present invention provides a radiolabeled compound according to the present invention, preferably a compound according to the present invention, for use in the treatment or prevention of a disease associated with overexpression of PSMA, which disease is preferably cancer, more preferably prostate cancer.186 Re sign or 188 Re-labeled compounds or therapeutic compositions comprising such compounds are provided.

[0107] Compositions containing compounds according to the present invention, such as therapeutic or diagnostic compositions, may further contain 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, amino acid buffer (with or without saline), water, for injection, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These compositions can be administered to the subject at an appropriate dose. Administration of suitable compositions can be achieved by different methods, for example, intravenous injection and / or intravenous delivery. The composition can be administered directly to the target site.

[0108] The following sections summarize aspects of the present invention. It will be understood that these sections are closely related to the sections of the description above, and that the information provided in these sections may supplement the sections of the description above, and vice versa. 1.Equation (1):

[0109] [ka]

[0110] (In the formula, R T is the PSMA binding group, L is a linking group, R C is a trivalent coupling group, R S is the formula -C(O)-R S3 -SiR S1 R S2 is the silicon-containing portion of OH, where R S1 and R S2 are independently selected from C3 to C10 alkyl, and RS3 is a group containing a 6-membered aromatic ring), R A is an amino acid residue, and R CH teeth, (i) a branched, acyclic chelating moiety having four amino groups, and (ii) 99m Tc, 94m Tc, 186 Re, and 188 a chelating moiety in which a radioisotope selected from Re is chelated by a branched acyclic chelating moiety having four amino groups; (selected from or a salt thereof. 2. R in Equation (1) T But the following structure (T-1):

[0111] [ka]

[0112] (In the formula, the wavy line represents R T (depicts the bond connecting the group to the remainder of the compound of formula (1)) Item 1. The compound or salt according to item 1, wherein the compound or salt is a group represented by the formula: 2. R in Equation (1) T But the following structure (T-2):

[0113] [ka]

[0114] (In the formula, the wavy line represents R T (depicts the bond connecting the group to the remainder of the compound of formula (1)) Item 3. The compound or salt according to item 2, wherein the compound or salt is a group represented by the formula: 3. R ​​in Equation (1) S However, the following structure (S-2):

[0115] [ka]

[0116] (In the formula, R S3 is a group containing a 6-membered aromatic ring, and The wavy line is R S (depicts the bond connecting the group to the remainder of the compound of formula (1)) 3. The compound or salt according to item 1 or 2, wherein 4. R in Equation (1) S However, the following structure (S-3):

[0117] [ka]

[0118] (In the formula, The wavy line is R S (depicts the bond connecting the group to the remainder of the compound of formula (1)) 4. A compound or salt according to any one of items 1 to 3, wherein the compound or salt is a group represented by the formula: 5. R in Equation (1) A However, the following structure (A-1):

[0119] [ka]

[0120] (In the formula, R A1 is hydrogen, -(CH2) k -COOH, -CH2-Ar, and -Ar wherein k is 1, 2, or 3, preferably 2; and Ar is an optionally substituted phenyl group, optionally bearing a substituent selected from —OH and —NH; The wavy line in the -C(O)- group in the formula represents R C The wavy line in the -NH- group in the formula indicates the bond formed by R CH (The bond formed is shown below.) 5. A compound or salt according to any one of items 1 to 4, wherein 6. R in Equation (1) A However, the following structure (A-1A):

[0121] [ka]

[0122] (In the formula, R A1 is hydrogen, -(CH2) k -COOH, -CH-Ar, and -Ar; wherein k is 1, 2, or 3, preferably 2; and Ar is an optionally substituted phenyl group, optionally bearing a substituent selected from —OH and —NH; The wavy line in the -C(O)- group in the formula represents R C The wavy line in the -NH- group in the formula indicates the bond formed by R CH (The bond formed is shown below.) Item 6. The compound or salt according to item 5, wherein the compound or salt is a group represented by the formula: 7.R A1 7. The compound or salt according to item 5 or 6, wherein is selected from —(CH 2 ) 2 —COOH, —CH 2 —Ph, and —CH 2 —(p-NH 2 —Ph), where Ph is a phenyl group. 8.R A1 8. The compound or salt according to item 7, wherein is —(CH 2 ) 2 —COOH. 9. R in Equation (1) CH but: (i) an N4 chelating group of the formula:

[0123] [ka]

[0124] (In the formula, The wavy line is R CHdepicts a bond attaching the group to the remainder of the compound of formula (1), and optionally one or more hydrogen atoms, such as one, two, or three, attached to the carbon atoms of the above formula may be replaced by a substituent), and (ii) 99m Tc, 94m Tc, 186 Re, and 188 a chelating moiety in which a radioisotope selected from Re is chelated by an N4 chelating group; 9. The compound or salt according to any one of items 1 to 8, selected from: 10. The compound or salt according to any one of items 1 to 9, wherein L in formula (1) is an oligoamide residue comprising 2 to 6 subunits linked together via an amide bond -C(O)-NH- or an alkylated amide bond -C(O)-NR- (wherein R is a C1-C6 alkyl group). 11. The compound or salt according to item 10, wherein the oligoamide residue consists of 2 to 4 subunits. 12. The compound or salt according to item 10 or 11, wherein the oligoamide residue contains 6 to 20, preferably 8 to 18, more preferably 8 to 16 carbon atoms, and does not include carbon atoms in any substituents attached to the backbone. 13. R in Equation (1) C is a trivalent amino acid unit derived from an amino acid comprising a carboxylic acid group and an amino group as well as a side chain bearing an additional functional group selected from a carboxylic acid group and an amino group. 14. R in Equation (1) C is selected from a 2,3-diaminopropionic acid (Dap) unit, a 2,4-diaminobutanoic acid (Dab) unit, an ornithine (Orn) unit, and a lysine (Lys) unit. 15. 99m Tc, 94m Tc, 186 Re, and 188 15. The compound or salt according to any of items 1 to 14, which is a radiolabeled compound comprising a chelated radioisotope selected from Re. 16. The compound or salt according to item 15, wherein the chelated radioisotope is in the oxidation state +V. 17. The compound or salt according to item 15 or 16, wherein the chelated radioisotope is chelated as a dioxo species. 18. Chelated radioisotopes are 99m Tc and 94m 18. The compound or salt according to any of items 15 to 17, selected from: Tc. 19. Chelated radioisotopes are 186 Re and 188 18. The compound or salt according to any of items 15 to 17, wherein Re is selected from 20. A composition comprising a compound or salt according to any one of items 1 to 19. 21. The composition according to item 20, which is a diagnostic composition. 22. The diagnostic composition according to item 21, comprising the compound or salt according to item 18. 23. A compound or salt according to any of items 1 to 19, preferably item 18, or a diagnostic composition according to item 21 or 22, for use in diagnosing in vivo a disease associated with overexpression of PSMA. 24. The compound or salt or diagnostic composition for use according to item 23, wherein the disease is cancer, preferably prostate cancer. 25. The compound or salt or diagnostic composition for use according to item 23 or 24, wherein the diagnosis relates to nuclear medicine tomography, preferably single photon emission computed tomography (SPECT). 26. A compound or salt according to any of items 1 to 19, preferably item 18, or a diagnostic composition according to item 21 or 22, for use in identifying diseased tissue with overexpression of PSMA in vivo. 27. The compound or salt or diagnostic composition for use according to item 26, wherein the diseased tissue is cancer tissue, preferably prostate cancer tissue. 28. The compound or salt or diagnostic composition for use according to item 26 or 27, wherein diseased tissue is identified for removal of the diseased tissue in the context of radiation-guided surgery. 29. Use of any of items 1 to 19, preferably a compound or salt according to item 18 or a diagnostic composition according to item 21 or 22, ex vivo or in vitro, for the identification of tissues or cells with PSMA overexpression. 30. The use according to item 29, wherein the tissue or cell is a cancer tissue or cell, preferably a prostate cancer tissue or cell. 31. An ex vivo or in vitro method for identifying whether a tissue or cell overexpresses PSMA, comprising the step of contacting the tissue or cell with a compound or salt according to any of items 1 to 19, preferably with item 18, or with a diagnostic composition according to item 21 or 22. 32. The method according to item 31, wherein the tissue or cell is a cancer tissue or cell, preferably a prostate cancer tissue or cell. 33. The composition according to item 20, which is a therapeutic composition. 34. A therapeutic composition according to item 33, comprising a compound or salt according to item 19. 35. A compound or salt according to any of items 1 to 19, preferably item 19, or a therapeutic composition according to item 33 or 34, for use in treating or preventing a disease associated with overexpression of PSMA. 36. The compound or salt or therapeutic composition according to item 35, wherein the disease is cancer, preferably prostate cancer.

[0125] In this specification, many documents are cited, including patent applications and manufacturer's instructions.The disclosures of these documents are not considered to be related to the patent right of this invention, but are incorporated herein by reference in their entirety.More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0126] The following examples further illustrate the invention without limiting it to the exemplified embodiments. [Example]

[0127] material and method General information 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, United Kingdom), Sigma-Aldrich (Steinheim, Germany), or VWR (Darmstadt, Germany) and used without further purification. Radioactive [ 99m Tc]TcO4 - was obtained from an Ultra-Technekow FM 2 (15–43.00 GBq) generator (Curium, Petten, Netherlands).

[0128] Solid-phase peptide synthesis (SPPS) was performed manually in a syringe reaction vessel for peptide synthesis (Carl Roth, Karlsruhe, Germany) using an MX-RD-Pro syringe shaker from SCILOGEX (Rocky Hill, United States). Analytical and preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using a Shimadzu gradient system (Shimadzu, Neufahrn, Germany) equipped with an SPD-20A UV / Vis-detector (detection 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 procedures, respectively). For analytical measurements, a MultoKrom 100-5 C18 column (150 mm × 4.6 mm, CS Chromatographie-Service, Langerwehe, Germany) was used at a constant flow rate of 1 mL / min. Preparative RP-HPLC was performed on a MultoKrom 100-5 C18 column (250 mm × 20 mm, CS Chromatographie-Service) at a constant flow rate of 10 mL / min. Reversed-phase high-performance flash chromatography (RP-HPFC) was performed on a SP HPFC system equipped with a SNAP cartridge (KP-C18-HS, ​​12 g) from Biotage (Charlottesville, United States) and using 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 obtained from Advion (Harlow, United Kingdom). L Obtained on a CMS mass spectrometer. 1H-NMR-spectra were acquired on an AVHD 400 from Bruker (Billerica, United States) at 300 K. Chemical shifts (δ) are given in parts per million (ppm), and the spectrum shows a residual DMSO-d6 at 2.50 ppm. 1 Calibrated to the H solvent signal, signal multiplicities are given as: s = singlet, m = multiplet.

[0129] Analytical and preparative radioactive RP-HPLC were performed on a Shimadzu equivalent system, as described above, additionally equipped with a SIL-20A HAT autosampler, using a MultoKrom 100-5 C18 column (125 mm × 4.6 mm) from CS Chromatographie-Service at a constant flow rate of 1 mL / min. A HERM LB 500 Nal scintillation detector (Berthold Technologies, Bad Wildbad, Germany) was connected to the UV-photometer outlet for radioactivity detection. Radioactive thin-layer chromatography (TLC) was performed on iTLC-SG strips (Agilent Technologies, Waldbronn, Germany) using butanone or NHOAc (1 M in water) containing DMF (1 / 1 (v / v)) as the mobile phase, respectively, with free [ 99m Tc]TcO4 - The radioactive TLC strips were analyzed using a Scan-RAM radioactive TLC detector from LabLogic Systems (Sheffield, United Kingdom). The radioactivity of the radioactive probes was determined using a 2480 WIZARD 2 This was done using an automated gamma counter (PerkinElmer, Waltham, United States).

[0130] The centrifuges used for measuring lipophilicity and binding to human plasma were HERAEUS Pico 17 and HERAEUS Megafuge 16R (Thermo Scientific, Osterode, Germany), respectively. Solution-State Synthesis of Building Blocks for SPPS (tBu)2EuE(tBu): The tert-butyl protected Glu-urea-Glu binding motif was synthesized in analogy to the synthesis of tert-butyl protected Lys-urea-Glu reported in the literature [28, 35]. 4-(Di-tert-butylhydroxysilyl)benzoic acid (SiOH-BA): 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 a published procedure

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

[0131] RP-HPLC (50–100% B in 15 min): R = 5.8 min, K' = 2.90. The calculated monoisotopic mass (C 15 H 24 0Si): 280.2; Found: m / z (APCI) = 279.0 [MH] - . N,N',N'',N'''-Tetrakis(tert-butyloxycarbonyl)-6-carboxy-1,4,8,11-tetraazaundecane ((tBu)N): 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 stirred at room temperature for 24 h. The solvent was removed under reduced pressure, and the crude residue was dissolved in acetone / HO (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 within 15 h. The solvent was removed under reduced pressure, and the crude product was purified via RP-HPFC (35–71% B in 15 min) to give the desired product as a colorless amorphous solid.

[0132] ESI-MS: Calculated monoisotopic mass (C 28 H 52 N4O 10 ): 604.4; Found: m / z (ESI) = 605.5 [M+H] + . 1 H-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). Synthesis of PSMA ligands Chemical synthesis of the novel PSMA-ligands bearing N4 was performed via Fmoc-based standard solid-phase peptide synthesis (SPPS). The reference compound PSMA-I&S was synthesized as previously described

[15] . The purity of all labeled precursors was determined via RP-HPLC (UV detection at 220 nm) and was >98% for all samples. General Procedure (GP) for Solid Phase Peptide Synthesis: All synthesis steps were carried out at room temperature in syringe reaction vessels for peptide synthesis. After each coupling or deprotection step, the resin was thoroughly washed with DMF (5 mL / g resin) six or eight times, respectively.

[0133] TCP-resin loading (GP1): Amino acid (2.0 equiv.) and DIPEA (3.75 equiv.) are dissolved in DMF (5 mL / g resin) and added to the TCP resin. After 2.5 h, methanol (2 mL / g resin) is added to cap the remaining trityl chloride groups. The resin is then thoroughly washed with DMF (6 × 5 mL / g resin), DCM (3 × 5 mL / g resin), and methanol (3 × 5 mL / g resin) and dried under reduced pressure. The amino acid loading, l, is given by m1 = mass of unloaded resin [g], m2 = mass of loaded resin [g], M AA = molecular weight of amino acid [g / mol], and M HCl = molecular weight of HCl [g / mol]

[0134]

number

[0135] is calculated by Resin-attached amide bond formation (GP2): For conjugation of Fmoc-protected amino acids with other building blocks, their carboxylic acid functions are preactivated by adding TBTU (2.0 equiv.), HOAt (2.0 equiv.), and DIPEA (6.0 equiv.) in DMF. After 5 min, this solution is added to the resin and allowed to react for 2.5 h (various coupling times are described in the synthesis protocol). Coupling of Fmoc-D-Dap(Dde)-OH is carried out using 2,4,6-trimethylpyridine (6.7 equiv.) as the base instead of DIPEA to prevent racemization.

[0136] Resin-attached Fmoc-deprotection (GP3): Deprotection of the Fmoc protecting group is achieved by adding 20% ​​piperidine in DMF (8 mL / g resin) for 5 min followed by 15 min. N4-PSMA Ligand: Fmoc-D-Orn(Dde)-OH was loaded onto TCP resin as the first building block via GP1. After subsequent Fmoc cleavage (GP3), (tBuO)EuE(OtBu)2 was conjugated for 4.5 h (GP2). Deprotection of Dde was carried out using 2% hydrazine monohydrate in DMF (5 mL / g resin) for 20 min. Subsequently, a DMF solution of succinic anhydride (7 equiv.) and DIPEA (7 equiv.) was added and allowed to react for 2.5 h. Next, the resin-bound carboxylate was preactivated by the addition of TBTU (2.0 equiv.), HOAt (2.0 equiv.), and DIPEA (6.0 equiv.) in DMF for 30 min, followed by the addition of Fmoc-D-Lys-OtBu (2.0 equiv.) in DMF for 2.5 h. Subsequent Fmoc deprotection (GP3) was followed by conjugation of Fmoc-D-Dap(Dde)-OH (GP2). Orthogonal deprotection of Dde was carried out 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, SiOH-BA was conjugated (GP2), and the remaining Fmoc protecting groups were cleaved according to GP3. In the next step, either 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) was coupled according to GP2. After subsequent Fmoc deprotection (GP3), the tert-butyl-protected N4-chelator was conjugated according to GP2. Cleavage from the resin and simultaneous deprotection of the ligand were carried out in TFA (+2.5% TIPS, +2.5% HO) for 1 h. After purification by semi-preparative RP-HPLC, N4-PSMA-12, N4-PSMA-13, and N4-PSMA-21 were obtained as colorless amorphous solids in 29%, 25%, and 21% yields, respectively (yield refers to the amount of resin-bound Fmoc-D-Orn(Dde) material at the start of solid-phase synthesis).

[0137] N4-PSMA-12: RP-HPLC (10–60% B in 15 min): R= 9.3 min, K' = 3.72. The calculated 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+ .

[0138] [ka]

[0139] N4-PSMA-13: RP-HPLC (10–60% B in 15 min): R = 10.2 min, K' = 4.08. The 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+ .

[0140] [ka]

[0141] N4-PSMA-21: RP-HPLC (10–60% B in 15 min): R = 9.0 min, K' = 3.60. The 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+ .

[0142] [ka]

[0143] The following ligands were prepared according to the corresponding procedures: N4-PSMA-11:

[0144] [ka]

[0145] N4-PSMA-11: RP-HPLC (10–60% B in 15 min): R = 8.8 min, K' = 3.52. The 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+ . N4-PSMA-32:

[0146] [ka]

[0147] N4-PSMA-32: RP-HPLC (10–60% B in 15 min): R = 9.2 min, K' = 3.68. The calculated monoisotopic mass (C 61 H 97 N 13 O 20 Si):1359.7;Actual value:m / z(ESI)=681.6[M+2H] 2+ , 454.7[M+3H] 3+ . N4-PSMA-33:

[0148] [ka]

[0149] N4-PSMA-33: RP-HPLC (10–60% B in 15 min): R = 9.4 min, K' = 3.76. The calculated monoisotopic mass (C 60 H 95 N 13O 19 Si):1329.7;Actual value:m / z(ESI)=665.0[M+2H] 2+ , 443.6[M+3H] 3+ . PSMA-I&S (reference compound):

[0150] [ka]

[0151] PSMA-I&S: ​​RP-HPLC (10-70% B in 15 min): R = 8.8 min, K' = 5.02. The 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+ . IBA-KuE: For the synthesis of IBA-KuE, the protected binding motif (OtBu)KuE(OtBu)2 was synthesized as previously described

[28] . 4-Iodobenzoic acid (6.1 mg, 24.6 μmol, 1.2 equiv.) was preactivated by the addition of TBTU (7.9 mg, 24.6 μmol, 1.2 equiv.), HOAt (3.3 mg, 24.6 μmol, 1.2 equiv.), and DIPEA (12.9 μL, 73.8 μmol, 3.6 equiv.) in DMF (1 mL). After 5 min of preactivation at room temperature, (OtBu)KuE(OtBu)2 (10.0 mg, 20.5 μmol, 1.0 equiv.) in DMF (2 mL) was added, and the solution was stirred overnight (21 h) at room temperature. The solvent was evaporated, TFA (+2.5% TIPS, +2.5% H2O) was added, and the solution was stirred for 1 h. The 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%).

[0152] RP-HPLC (20–40% B in 20 min): R=11.0 min, K'=7.68. Calculated monoisotopic mass (C 19 H 24 IN3O8): 549.1; Found: m / z (ESI) = 550.2 [M+H] + . radioactive label Radiolabeling of N4-PSMA ligand N4-PSMA ligand 99m Tc-labeling was performed by adding 1 nmol of peptide precursor (0.5 mM in DMSO) to a mixture of 0.05 M NaHPO in saline (12.5 μL, Tracepur®-water, pH 9.25) and 0.1 M disodium citrate sesquihydrate (1.5 μL, Tracepur®-water). After addition of a freshly prepared solution of SnCl (2.5 μL, 1 mg / mL in ethanol), [ 99m Tc]TcO4 - (40 MBq / nmol) was added 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). In the patient scale 99m Radiosynthesis of [Tc]Tc-N4-PSMA-12 In a 10 mL glass vial, 15 nmol N4-PSMA-12 (20 μg, 0.5 mM in DMSO) was added to a mixture of 0.05 M Na2HPO4 in saline (250 μL, TP-water, pH 9.25) and 0.1 M disodium citrate sesquihydrate (30 μL, TP-water). After the addition of a freshly prepared solution of SnCl2 (10 μL, 1 mg / mL in ethanol), [ 99m Tc]TcO4 - was added and the labeling solution (final volume 2-5 mL) was heated to 95°C for 15 min. The labeling solution was allowed to cool for 10 min, after which quality control was performed using radio-TLC and radio-RP-HPLC. [ 99mRadiosynthesis of [Tc]Tc-PSMA-I&S (reference compound) Labeling of PSMA-I&S was performed as described by Robu et al.

[15] using 2 nmol of peptide precursor in the kit formulation. 99m Tc]TcO4 - After the addition of (40 MBq / nmol), the solution was heated to 95° C. for 20 min. Subsequently, 10 μL of 1 M sodium ascorbate (PBS) was added and quality control was performed using radio-TLC and radio-RP-HPLC. [ 125 Radioiodination of [I]IBA-KuE Synthesis of protected stannyl precursors, radioiodination, and 125 Deprotection to give [I]IBA-KuE was performed as previously described

[28] . Purification of this crude labeled product was performed using preparative radio-RP-HPLC with a gradient of 20–40% B in 20 min. Analytical data for PSMA inhibitors labeled with technetium-99m or iodine-125 [ 99m Tc]Tc-N4-PSMA-12: Radioactive RP-HPLC (10~70%B in 15 minutes):t R =9.1 min, K'=6.36.

[0153] [ 99m Tc]Tc-N4-PSMA-13: Radioactive RP-HPLC (10~70%B in 15 minutes):t R =10.0 min, K'=6.98. [ 99m Tc]Tc-N4-PSMA-21: Radioactive RP-HPLC (10~70%B in 15 minutes):t R =8.6 minutes, K'=5.99.

[0154] [ 99m Tc]Tc-PSMA-I&S: ​​Radioactive RP-HPLC (10~70%B in 15 minutes):t R =8.2 minutes, K'=5.76. [ 125 [I]IBA-KuE: Radioactive RP-HPLC (20–40% B in 20 min): tR =11.0 min, K'=7.68. Lipophilicity and binding to human plasma Partition coefficient (logD 7.4 ) 99m The lipophilicity of the Tc-labeled PSMA ligand was measured using the shake-flask method. A solution of approximately 1 MBq of radioactive ligand in 1 mL of a 1:1 mixture of PBS (pH = 7.4) and n-octanol (n = 8) was vigorously stirred by vortexing for 3 min. After centrifugation at 9000 rpm for 5 min, aliquots of both phases (200 μL n-octanol, 50 μL PBS) were taken and their radioactivity was quantified in a γ-counter. LogD 7.4 Values ​​were calculated as the common logarithm of the radioactivity concentration ratio between the n-octanol and aqueous phases. Data are presented as mean ± standard deviation (SD). Binding to human plasma was measured by incubating the radioligand in human plasma (2 nM concentration, 37°C for 30 min) followed by ultrafiltration (250 μL volume, 3200 rpm, 40 min) using a Centrifree® ultrafiltration device (Merck Millipore, Cork, Ireland). The fraction bound to human plasma proteins was calculated as the ratio of unfiltered radioactivity to total radioactivity in the ultrafiltration device. Measurements were performed in three independent experiments, each with two replicates (n = 6, data presented as mean ± SD). All values ​​were corrected for nonspecific binding (control experiment in PBS). Measurement of human serum albumin (HSA) binding by high performance affinity chromatography (HPAC) HSA binding of uncomplexed PSMA ligand was measured according to a previously published HPLC-mediated 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 × 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 a freshly prepared 50 mM aqueous solution of NHOAc (pH 6.9), and mobile phase B was isopropanol (HPLC grade, VWR). The gradient applied in all experiments was 100% A (0–3 min), followed by 80% A (3–40 min). Prior to the experiment, the column was calibrated using nine reference substances with known HSA binding properties ranging from 13 to 99% (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 ligands studied, were dissolved in a 1:1 mixture (v / v) of isopropanol and a 50 mM aqueous solution of NHOAc (pH 6.9) to a final concentration of 0.5 mg / mL. Nonlinear regression was established using OriginPro 2016G software (Northampton, United States). Affinity (IC 50 ) and internalization measurement tests Competitive binding studies were performed similarly to previously reported procedures

[28] . As a modification, novel PSMA-binding compounds of interest were tested against 99m Tc-labeled radioligand (0.2 nM / well) was applied, whereas the non-radiolabeled 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, n=3 each). In this reversed experimental approach, higher values ​​correspond to higher affinities, which are the reversed IC 50 (inverse IC 50 Poly-L-lysine coated 24-well plates were used (n=3). Data are given as mean ± SD. PSMA-mediated internalization at 1 hour was used to 99m To measure the cellular uptake of Tc-labeled PSMA ligand into LNCaP cells, 99m Tc-labeled PSMA ligand and reference compound [ 125 A previously reported protocol was applied using assay concentrations of 1.0 nM and 0.2 nM for [I]IBA-KuE, respectively

[28] . Data are corrected for nonspecific binding and normalized to the specific internalization of the reference compound. Results are given as mean ± SD. In vivo experiments All animal experiments were performed in accordance with the German general animal welfare regulations (German Animal Protection Act, version published on May 18, 2006, as amended by Article 280 of June 19, 2020, approval number ROB-55.2-1-2532.Vet_02-18-109 by the General Affairs Department of Upper Bavaria) and the institutional guidelines for the care and use of animals. Male CB17-SCID mice were purchased from Charles River (Sulzfeld, Germany) and delivered to the company's in-house animal facility at least 1 week before the start of the experiments for acclimatization. Tumor xenografts were implanted into the right shoulder of 6- to 8-week-old male CB17-SCID mice using LNCaP cells (approximately 2 × 10 cells in 200 μL of a 1:1 mixture of Cultrex BME (R&D Systems, Minneapolis, United States) and DMEM / Ham's F-12). 7 The tumors were established by subcutaneous inoculation of 1000 cells. Animals were used for the experiment when the tumors reached a size of 5-10 mm in diameter. The criteria for the exclusion of animals from the experiment were: weight loss of more than 20%, a tumor size of 1.5 cm 3 The following criteria were observed for the animals: tumor size exceeding 100 μg / mL, tumor ulceration, respiratory distress, or behavioral changes. These criteria were not met for all mice. No randomization or blinding was applied to experimental assignment. Quarterly health monitoring was performed according to FELASA recommendations.

[0155] Biodistribution studies. 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) under isoflurane anesthesia. At 6 h postinjection (pi), animals were sacrificed by carbon dioxide inhalation and blood collection via cardiac puncture. Blood and tissues were collected, weighed, and their radioactivity was measured in a γ-counter. Radioligand uptake was expressed as a percent of the injected dose per gram of tissue (%ID / g), and results are presented as the mean ± SD.

[0156] Consistent with the above, 99mBiodistribution studies of [Tc]Tc-N4-PSMA-21 were performed on n = 5 animals ("Dataset I"). All animals were healthy and did not show any signs of pain, distress, or behavioral changes throughout the experiment. However, significant deviations in kidney uptake and increased tumor uptake were observed in one particular animal (mouse #3) compared to the other four animals. The kidney uptake of mouse #3 (36.17%ID / g) exceeded the value obtained from the other four animals (4.58 ± 1.44%ID / g, mean ± SD) by 7.9-fold or more than 20 standard deviations. Furthermore, the tumor uptake of mouse #3 (21.92%ID / g) exceeded the value obtained from the other four animals (11.02 ± 2.22%ID / g, mean ± SD) by 2.0-fold or more than 4 standard deviations. Because it is uncertain whether these significant deviations result from experimental error or may be related to significantly deviated metabolic processes, e.g., abnormal renal function in mouse #3, this study primarily presents and discusses datasets based on four other animals (n = 4, "Dataset II"). However, a thorough evaluation and comparison including mouse #3 was also performed. In both "Dataset I" (n = 5, including mouse #3) and "Dataset II" (n = 4, excluding mouse #3), [ 99m The renal uptake of ]Tc-N4-PSMA-21 was [ 99m Tc]Tc-PSMA-I&S (both P<0.001), but 99m The Tc-labeled N4-PSMA-ligand was not statistically different (P>0.92 and P>0.81 for "Dataset I" and "Dataset II," respectively). Mouse #3 also showed increased tumor uptake (see above), but this was not statistically different when comparing "Dataset I" (P>0.69) or "Dataset II" (P>0.27) with the biodistribution data of other radioligands evaluated herein. 99m No statistically significant differences in tumor uptake were observed between the Tc-labeled PSMA ligands.

[0157] μSPECT / CT Imaging. Immediately after blood collection, static imaging of sacrificed animals was performed on a VECTor4 small-animal SPECT / PET / CT / OI scanner from MILabs (Utrecht, Netherlands) using a 45-minute acquisition time with a HE-GP-RM collimator and stepwise multiplanar bed movement via MILabs acquisition software (v11.00 and v12.26). Image data were reconstructed using MILabs reconstruction software (v12.00), and image analysis was performed using PMOD4.0 (PMOD technologies LLC, Zurich, Switzerland). After imaging, animals were subjected to biodistribution studies. Data analysis The acquired data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparisons using OriginPro software (version 9.7) from OriginLab Corporation (Northampton, United States). Pairwise statistical comparisons were performed using a two-sample Student's t-test in Microsoft Excel (Redmond, United States). A P value of <0.05 was considered statistically significant. result Synthesis and radiolabeling The novel PSMA ligands were synthesized using a mixed solution / solid-phase synthesis approach and were obtained in yields of 29%, 25%, and 21%, respectively, with chemical purity of >98%. The identity of the compounds was confirmed by mass spectrometry. 99m Tc]TcO4 - The radiolabel with was obtained in >95% radiochemical purity (RCP) as determined by radioTLC and radioRP-HPLC. 99mScaling up the reported labeling protocol for patient-scale production of [Tc]Tc-N4-PSMA-12 has been found to be feasible, although a relative reduction in the amount of stannous chloride was applied to prevent the formation of colloidal technetium species. During consideration of the clinical workflow requirements for routine radiosynthesis, labeling of N4-PSMA-12 (20 μg, 15 nmol) with radioactivities ranging from 194 to 810 MBq (553 ± 187 MBq, mean ± SD, n = 10) reproducibly yielded the desired radioligand with an RCP of 98.5 ± 0.6% (range, 97.6–99.2%, n = 10). Detailed information regarding single radiolabeling is presented in Table 1 below.

[0158] [Table 1]

[0159] In vitro characterization [ 99m Tc]Tc-labeled N4-PSMA ligand and reference [ 99m The results of the in vitro characterization of [Tc]Tc-PSMA-I&S are summarized in Figure 1 and Tables 2a and 2b below. PSMA affinity was measured using the reverse IC 50 (I C 50,inv Regardless of the variable amino acid, all radioligands showed low nanomolar IC50, although the difference was not statistically significant (P > 0.66). 50 The compound showed high PSMA affinity as indicated by the inv. values ​​(range, 10.0–11.8 nM). In contrast, PSMA-mediated internalization was significantly higher than that of the reference compound [ 125 [I]IBA-KuE and were significantly affected by the variable amino acids. 99m Tc]Tc-N4-PSMA-12 (311±16%) is [ 99m Tc]Tc-PSMA-I&S (240±13%) showed 1.3-fold higher internalization than Tc-PSMA-I&S (240±13%), and 99m Tc]Tc-N4-PSMA-13 ​​(164 ± 15%) and [ 99mThese values ​​were 1.9 and 1.7 times higher than those of [Tc]Tc-N4-PSMA-21 (180±4%), respectively. 99m Compared with Tc]Tc-PSMA-I&S, all novel [ 99m The partition coefficient (logD 7.4 ) showed enhanced hydrophilicity. 99m Tc]Tc-N4-PSMA-13(logD 7.4 =-2.78±0.05), the highest lipophilicity was observed within the group of radioligands with N4, [ 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) followed. 99m Compared with [Tc]Tc-PSMA-I&S, the binding to human plasma was 99m The Tc-N4-PSMA tracer showed a significant decrease in the α-lipophilicity (94.4% vs. 55.1-88.5%), a trend similar to that described for lipophilicity. 99m Tc]Tc-N4-PSMA-12 exhibited the highest hydrophilicity (logD 7.4 =-3.35±0.05) and the lowest binding to human plasma proteins (55.1±2.5%) was observed (P<0.001 for both parameters).

[0160] [Table 2]

[0161] [Table 3]

[0162] In vivo characterization Biodistribution Studies. Relative biodistribution studies in LNCaP tumor-bearing mice at 6 hours pi were performed for all four radiotracers (Figure 2, Table 3).

[0163] Similar distribution profiles were observed among the three radioligands with N4, with high uptake in tumors (11.0–13.0% ID / g), variable but moderate radioactivity levels in the kidneys, and efficient clearance from blood and background tissues (see also μSPECT / CT scans in Figure 3). 99m Tc]Tc-N4-PSMA-12 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) showed slightly higher kidney retention (12.3±8.0%ID / g), but this was not statistically significant (P>0.81). In stark contrast to the N4-PSMA radioligand, extremely high radioactivity retention in the kidney was observed with [ 99m Tc]Tc-PSMA-I&S(191±26%ID / g, [ 99m 15-fold higher than that of [Tc]Tc-N4-PSMA-12). 99m At 6 hours after injection of [Tc]Tc-PSMA-I&S, significantly higher radioactivity retention was also present in several other organs, such as the lung, spleen, adrenal gland, and parotid gland (P<0.001 for each). However, [Tc]Tc-PSMA-I&S in the tumors was significantly higher than in the control group. 99m The higher radioactivity uptake of [Tc]Tc-PSMA-I&S was less pronounced (15.6 ± 2.8% ID / g). The corresponding difference with the novel N4-PSMA radioligand was not statistically significant (P > 0.27). Efficient radioactivity clearance from the blood pool was observed with all radioligands. The lowest blood radioactivity levels were observed at 6 hours p.i. with [ 99m Tc]Tc-N4-PSMA-12 (0.0200 ± 0.0044%ID / g), which is due to its low log D 7.4 and correlates with low PPB. 99mTc]Tc-N4-PSMA-13, [ 99m Tc]Tc-N4-PSMA-21, and [ 99m The blood radioactivity levels of Tc]Tc-PSMA-I&S were [ 99m Tc]Tc-N4-PSMA-12 were 1.6-, 5.4-, and 3.6-fold higher, respectively.

[0164] Tumor to background ratio (TBR). As shown in Figure 4, [ 99m [Tc]Tc-N4-PSMA-12 showed the highest TBR among all radioligands throughout most organs. 99m By direct comparison with ]Tc-PSMA-I&S, [ 99m It is noteworthy that [Tc]Tc-N4-PSMA-12 showed increased TBR in all analyzed organs and blood, thus revealing its particularly superior clearance characteristics. In particular, the 3-fold increase in the T / blood ratio (658±147 vs. 219±62) and the even 20-fold increase in the T / kidney ratio (1.64±1.29 vs. 0.08±0.01) represent an important step toward optimized pharmacokinetics. Although less pronounced, [ 99m Tc]Tc-N4-PSMA-13 ​​and [ 99m Improvements in the TBR of [Tc]Tc-N4-PSMA-21 were also observed. A tabular summary of the TBR is presented below in Table 4.

[0165] [Table 4]

[0166] [Table 5]

[0167] Consideration The versatile structural platform used in the N4-PSMA ligand design ensured high PSMA affinity and reliable complexation of radioisotopes such as technetium-99m, while also allowing for flexible modifications to tailor the overall pharmacokinetic profile of the ligand. To maintain high affinity while ensuring low lipophilicity of the novel tracer, a less lipophilic SiOH moiety (formally hydrolyzed SiFA) was used. A tetraamine chelator was selected for radioisotope complexation because this chelating system exhibits excellent in vivo stability and confers high hydrophilicity to peptide radioligands [29, 30]. An additional advantage over N3S-based chelating systems, such as mercaptoacetyltricerin, is the absence of chemically reactive thiol groups, which can limit the shelf-life of radioligand precursors, an aspect often neglected in early radioligand development. Finally, the incorporation of variable amino acids yielded novel ligands with distinct in vitro and in vivo properties.

[0168] [ 99m The slow systemic clearance and partial hepatobiliary excretion of [Tc]Tc-PSMA-I&S are postulated to be due to its high PPB and enhanced lipophilicity compared to other PSMA-designated radiometal chelates

[15] . Therefore, the primary focus in the design of novel ligands was placed on attenuated lipophilicity and PPB. These goals were achieved by the N4-PSMA compound. The inherent lipophilicity of the variable amino acids (logP: Phe > (4-NH2)-Phe > Glu)

[31] significantly increased the lipophilicity and PPB (logD) of the corresponding radioligand. 7.4 and PPB:[ 99m Tc]Tc-PSMA-I&S>[ 99m Tc]Tc-N4-PSMA-13>[ 99m Tc]Tc-N4-PSMA-21>[ 99m Tc]Tc-N4-PSMA-12) showed similar trends, especially [ 99mTc]Tc-N4-PSMA-12 has desirable lipophilicity comparable to the diagnostic rhPSMA compound

[23] and 99m Tc]Tc-PSMA-I&S,[ 68 Ga]Ga / [ 177 Lu]Lu-PSMA-I&F

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

[25] (all ligands developed for PSMA-induced surgery) [ 177 Lu]Lu-PSMA-617

[33] . Consistent with many previous studies [25-27], these findings clearly demonstrate how the pharmacokinetically relevant properties of radioligands can be tailored by deliberate structural modifications.

[0169] [ 99m Even though Tc]Tc-PSMA-I&S has shown favorable dosimetry in patients

[21] and its use in RGS suggests superiority over conventional salvage surgery

[17] , we [ 99m It must be acknowledged that [Tc]Tc-PSMA-I&S is not yet the optimal radioligand for RGS. This was demonstrated by a study conducted in 31 patients by Maurer et al., who reported successful resection of all lesions detected by previous PSMA-PET, even additional lesions as small as 3 mm.

[13] However, the same study revealed that histochemical analysis uncovered previously unidentified metastatic lesions in 12 of 86 resected tissue specimens classified as PSMA-negative by γ-probe measurement.

[0170] The obvious need for higher sensitivity in RGS is the need for optimized radioactive probes that, from a radiopharmaceutical perspective, result in higher TBR during the procedure. 99mTo evaluate the TBR of the Tc-labeled N4-PSMA ligand, a biodistribution study was performed at 6 hours postoperatively. This somewhat long distribution time limits comparability with the literature, which primarily examines time points of 1 or 4 hours. However, RGS is performed approximately 20–24 hours postoperatively in patients [13, 19, 34], and based on the empirical rule of approximately four times faster metabolism in mice compared to humans, we chose a 6-hour distribution time to simulate as accurately as possible the TBR at the time of surgery. Given these circumstances, [ 99m Our preclinical findings on the in vivo performance of the [Tc]Tc-N4-PSMA ligand represent a surprising development. While similarly high uptake in LNCaP xenografts was observed at 6 hours pi, clearance of the novel ligand from most background tissues was significantly slower than [Tc]Tc-N4-PSMA. 99m Tc]Tc-PSMA-I&S showed a significant improvement. 99m Dramatically favorably reduced kidney retention compared to [Tc]Tc-PSMA-I&S was found to be a common feature of the novel ligands, likely due to their shared molecular scaffold, derived from the rhPSMA compound [23, 24], and further accentuated by the use of a hydrolyzed SiFA moiety. This finding is particularly interesting because high radioactivity accumulation in the kidney can potentially interfere with accurate lesion detection during RGS

[34] .

[0171] Even more broadly, [ 99m Incomplete clearance of [Tc]Tc-PSMA-I&S from the blood pool and the resulting suboptimal T / blood ratio may affect the accuracy of RGS

[15] . In this regard, the reduced blood radioactivity at 6 hours pi represents another distinct advantage of the preferred PSMA ligand compounds according to the present invention, in particular [ 99m A 3.6-fold reduction was observed with [Tc]Tc-N4-PSMA-12. 99mCompared with [Tc]Tc-N4-PSMA-12, slightly higher uptake was observed in the blood and in some background organs such as the heart, lung, liver, parotid gland, and submandibular gland. 99m Tc]Tc-N4-PSMA-13 ​​and [ 99m Tc]Tc-N4-PSMA-21, which contains the negatively charged amino acid D-Glu [ 99m This may be due to the incorporation of aromatic amino acids D-Phe and D-(4-NH2)Phe, which confers higher lipophilicity compared to [Tc]Tc-N4-PSMA-12, and plasma protein binding to these compounds. 99m A further possible limitation of [Tc]Tc-PSMA-I&S is the background radioactivity in the intestine that prevents the detection of lesions with low signal intensity during surgery. 99m Tc]Tc-N4-PSMA-12 without fully considering the significantly improved T / kidney ratio and [ 99m Tc]Tc-PSMA-I&S and [ 99m When only the liver and intestinal uptake of [Tc]Tc-N4-PSMA-12 was considered (no statistically significant difference, P > 0.19 and P > 0.36, respectively), we found that the [ 99m Tc]Tc-PSMA-I&S and [ 99m One would expect at least equivalent performance of [Tc]Tc-N4-PSMA-12. 99m TBRs of blood and all analyzed organs obtained with [Tc]Tc-N4-PSMA-12 (see Figure 4) were 99m The finding that the pharmacokinetic profile of the radioligand according to the present invention is higher than that of the radioligand [Tc]Tc-PSMA-I&S clearly demonstrates the superior pharmacokinetic profile of the radioligand according to the present invention.

[0172] To facilitate future clinical transition, we are 99m We have developed a protocol for GMP manufacturing of [Tc]Tc-N4-PSMA-12 (see Figure 5). This is a simple, robust, one-step process that follows basic nuclear medicine infrastructure and established clinical workflow and procedures. 99mThis may provide an important foundation for the widespread availability of [Tc]Tc-N4-PSMA-12 and contribute to minimizing logistical obstacles for its initial clinical use. abbreviation 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-tetraazaundecane; PBS: phosphate-buffered saline; PCa: prostate cancer; PET: positron emission tomography; PPB: plasma protein binding; PSMA: prostate-specific membrane antigen; RCP: radiochemical purity; RGS: radiation-guided surgery; RP: reversed phase; SD: standard deviation; SiFA: silicon fluoride acceptor; sLND: sentinel lymph node dissection; SPECT: single-photon emission computed tomography; TBR: tumor to background ratio; TLC: thin-layer chromatography; UV: ultraviolet. [Brief explanation of the drawings]

[0173] [Figure 1] Figure 1 shows in vitro characterization of [99mTc]Tc-N4-PSMA-12, [99mTc]Tc-N4-PSMA-13, [99mTc]Tc-N4-PSMA-21, and [99mTc]Tc-PSMA-I&S: ​​A) Binding affinity to PSMA (IC50, inv. (nM), 1 h, 4°C, n=3); B) PSMA-mediated internalization (1 h, 37°C, (% of reference [125I]IBA-KuE), n=3); C) Lipophilicity expressed as logD7.4 (n-octanol / PBS, pH 7.4, n=8); D) Binding to human plasma (PPB) (30 min incubation at 37°C, measured via ultrafiltration (%), n=6). [Figure 2]Figure 1 shows ex vivo biodistribution data of 99mTc-labeled N4-PSMA derivatives and [99mTc]Tc-PSMA-I&S in male LNCaP tumor-bearing CB17-SCID mice at 6 hours post-injection. Data are expressed as percentage of injected dose per gram (%ID / g), mean ± standard deviation (n=4-5). gl.: gland; submandibular. [Figure 3] Figure 1 shows static μSPECT / CT images (maximum intensity projection images) of 99mTc-labeled N4-PSMA derivatives and [99mTc]Tc-PSMA-I&S in LNCaP tumor-bearing mice. Animals were sacrificed at 6 hours post-transplant and immediately imaged for 45 minutes on a VECTor4 small animal SPECT / PET / OI / CT. Tracer uptake (percent of injected dose / gram, (%ID / g)) in tumor and kidney was determined from subsequent biodistribution studies. [Figure 4] Figure 1 shows tumor-to-background ratios (TBRs) of 99mTc-labeled N4-PSMA compound and [99mTc]Tc-PSMA-I&S in male LNCaP tumor-bearing CB17-SCID mice at 6 hours post-infection. Data are presented as mean ± standard deviation (n = 4-5). Mean values ​​were determined from TBRs calculated for individual animals. gl.: gland; submandibular. [Figure 5]Figure 1 shows the recommended workflow for clinical manufacturing of [99mTc]Tc-N4-PSMA-12. Patient-scale manufacturing is completed within 35 minutes after generator elution under non-optimized laboratory conditions. Optimization for clinical routine synthesis will further reduce manufacturing time. RT: room temperature; SPE: solid phase extraction. References 1. 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Claims

1. Formula (1): 【Chemistry 1】 (In the formula, R Ｔ is a PSMA binding group, L is a linking group; R Ｃ is a trivalent coupling group, R Ｓ is represented by the formula -C(O)-R Ｓ３ -SiR Ｓ１ R Ｓ２ is the silicon-containing moiety of OH, Ｓ１ and R Ｓ２ are independently selected from C3 to C10 alkyl, and R Ｓ３ is a group containing a 6-membered aromatic ring), R Ａ is an amino acid residue, and R ＣＨ teeth, (i) a branched, acyclic chelating moiety having four amino groups; and (ii) ９９ｍ Tc, ９４ｍ Tc, １８６ Re, and １８８ a chelating moiety in which a radioisotope selected from Re is chelated by a branched acyclic chelating moiety having four amino groups; (selected from or a salt thereof.

2. R in formula (1) Ｔ but has the following structure: 【Chemistry 2】 (Wherein, the wavy line represents R Ｔ (depicts the bond connecting the group to the remainder of the compound of formula (1)) 2. The compound or salt of claim 1, wherein the group is

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

4. R in formula (1) Ｓ has the following structure (S-3): 【Chemistry 4】 (In the formula, The wavy line is R Ｓ (depicts the bond connecting the group to the remainder of the compound of formula (1)) 4. A compound or salt according to any one of claims 1 to 3, wherein the group is

5. R in formula (1) Ａ but has the following structure: 【Transformation 5】 (In the formula, R Ａ１ is hydrogen, -(CH ２ ) ｋ -COOH, -CH ２ -Ar, and -Ar wherein k is 1, 2, or 3, preferably 2; and Ar is an optionally substituted phenyl group, including —OH and —NH ２ is a phenyl group which may have a substituent selected from The wavy line in the —C(O)— group shown in the formula represents R Ｃ The wavy line in the -NH- group shown in the formula represents the bond formed by R ＣＨ (The bond formed is shown below.) 5. A compound or salt according to any one of claims 1 to 4, wherein the group is

6. R in formula (1) ＣＨ but, (i) Formula: 【Transformation 6】 (In the formula, The wavy line is R ＣＨ depicts a bond connecting the group to the remainder of the compound of formula (1), and optionally one or more hydrogen atoms, such as one, two, or three, attached to the carbon atoms of the above formula may be replaced by a substituent. an N4 chelating group of (ii) ９９ｍ Tc, ９４ｍ Tc, １８６ Re, and １８８ a chelating moiety in which a radioisotope selected from Re is chelated by an N4 chelating group; 6. A compound or salt according to any one of claims 1 to 5, selected from:

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

8. 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 as well as a side chain having an additional functional group selected from a carboxylic acid group and an amino group.

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

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

11. 10. The compound or salt of claim 9 for use in identifying diseased tissue in vivo with overexpression of PSMA, preferably cancer tissue, more preferably prostate cancer tissue.

12. 12. The compound or salt of claim 11, wherein the diseased tissue is identified for removal of the diseased tissue in the context of radiation-guided surgery.

13. 10. The ex vivo or in vitro use of a compound or salt according to claim 9 for the identification of tissues or cells with PSMA overexpression, preferably cancer tissues or cells, more preferably prostate cancer tissues or cells.

14. 10. An ex vivo or in vitro method for identifying whether a tissue or cell overexpresses PSMA, comprising contacting the tissue or cell with a compound or salt of claim 9, wherein the tissue or cell is preferably a cancer tissue or cell, more preferably a prostate cancer tissue or cell.

15. 10. The compound or salt of claim 9 for use in treating or preventing a disease associated with overexpression of PSMA, preferably cancer, more preferably prostate cancer.

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