Functionalized bisaminothiol derivatives, complexes having these bisaminothiol derivatives, and use of said complexes for diagnosis and treatment

Abstract

The present invention relates to a compound of general formula I [wherein A is a chelator selected from the group consisting of A1, A2, A3 and A4; k is independently 0, 1 or 2 at each occurrence; m is independently 1, 2, 3, 4 or 5 at each occurrence; n is independently 0, 1, 2 or 3 at each occurrence; p is independently 1, 2 or 3 at each occurrence; q is independently 1, 2 or 3 at each occurrence; u is independently 0 or 1 at each occurrence; X and Y are substituted or unsubstituted amino acids; L is a bifunctional linker selected from the group consisting of L1, L2 and L3 (where v, x and y are independently 0, 1, 2 or 3, and z is 0, 1, 2, 3, 4 or 5), and R is H, methyl or ethyl]. TIFF2025517989000061.tif143170

Description

Technical Field

[0001] The present invention relates to functionalized bisaminothiol derivatives. Furthermore, the present invention relates to complexes of these functionalized bisaminothiol derivatives with metals, particularly radioactive metals. Furthermore, the present invention relates to the use of said complexes, in particular their use in the diagnosis and treatment of diseases involving prostate-specific membrane antigen (PSMA).

Background Art

[0002] In personalized medicine, radiolabeled agents used to target disease-specific biological structures for the diagnosis of the disease and subsequent treatment are called theranostics. This approach has been used to identify the location of metastatic disease and then eliminate it.

[0003] Compared to normal prostate epithelial cells, the expression of prostate-specific membrane antigen (PSMA) is increased in human prostate cancer and its metastatic malignancies. This highly specific expression of PSMA on the cell surface is a characteristic of localized and metastatic prostate cancer. Furthermore, PSMA expression has been observed in the neovasculature of multiple non-prostatic solid malignancies (Chang et al., Clin Cancer Res 1999, 5, 2674) (Non-Patent Document 1).

[0004] Therefore, PSMA is an attractive target for the diagnosis, staging, detection of recurrence, and treatment of prostate cancer (and other diseases in which PSMA is upregulated) using radiolabeled compounds. Such molecules typically include a pharmacophore for PSMA binding, a linker structure for optimizing pharmacological properties, and a chelator or family of ancillary molecules for incorporating an appropriate radionuclide. In recent years, mainly, 68 Ga-PSMA-11, 18 F-DCFPyL and 18 including F-PSMA-1007 18 F and 68Ga-labeled radiopharmaceuticals are being used for prostate cancer PET imaging.

[0005] 99m Tc has a longer half-life (t 1 / 2 = 6.01 h), low radiotoxicity, and 99 Mo / 99m Tc generators for widespread and decentralized availability, making it a common isotope in nuclear medicine for SPECT imaging. Due to similar coordination chemistry, technetium and rhenium are a theranostic pair, 99m when labeled with 186 Tc for SPECT imaging, 1 / 2 Re (t 188 = 89.25 h) or 1 / 2 Re (t

[0006] Initial 99m Tc-labeled PSMA ligands had slow distribution, high liver uptake, and slow clearance from the body. 99m The slow pharmacokinetic properties of

[0007] Known PSMA ligands with a HYNIC chelation moiety require a co-ligand to stabilize the technetium complex in a biological system and prevent oxidation by oxygen. The choice of co-ligand can affect the complex shape, the number of PSMA ligands coordinating to one Tc atom, and ultimately the biodistribution of the radiolabeled compound. The hydrazine moiety of HYNIC is a strong nucleophile, which can lead to unwanted by-products during radiolabeling or when administered to a biological system. The formation of a stable complex between rhenium and HYNIC as a chelator remains difficult (North et al., Inorg. Chem. 2017, 56, 9725 (Non-Patent Document 4); Philip J. Blower, Int. J. Nucl. Medi. Res. 2017, 39 (Non-Patent Document 5)).

[0008] As a result, the 99m Tc-labeled PSMA ligands for prior art SPECT imaging are inferior to current PSMA ligands labeled with PET radionuclides in terms of radiolabeling, stability, pharmacokinetics, and diagnostic performance. Therefore, it is desirable to develop a new 99m Tc-labeled PSMA ligand with optimized pharmacological properties while using a chelator that can also form a stable complex with other metals such as rhenium.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0010] The problem of the present invention is to eliminate the drawbacks of the prior art. In particular, a functionalized bisaminothiol derivative that enables the formation of stable complexes with metals, especially rhenium and technetium, should be provided. Furthermore, a complex that functions as a ligand for prostate-specific membrane antigen (PSMA) and can be used for the diagnosis and treatment of specific diseases involving PSMA should be provided.

Means for Solving the Problems

[0011] This problem is solved by the features of claims 1, 8, 10, 11, 13 and 14. Practical developments of the present invention are obtained from the features of the dependent claims.

[0012] According to the present invention, a compound of general formula I is provided

Chemical formula

Chemical formula

Chemical formula

[0013] Since the compound of general formula I contains group A, it is a bisaminothiol derivative. Hereinafter, group A is N 2 S 2 Also referred to as a chelator. The compound of general formula I can form a complex with a metal, especially a radioactive metal. The bisaminothiol moiety of the compound of general formula I enables complexation with a metal, especially a radioactive metal, thereby obtaining a complex. Due to the bisaminothiol moiety, the compound of general formula I is a ligand for complexation with a metal, especially a radioactive metal.

[0014] The compounds of general formula I are functionalized bisaminothiol derivatives because they are functionalized by different amino acid sequences as linkers and urea-based pharmacophore groups.

[0015] The complex can be used as a ligand that binds to prostate-specific membrane antigen (PSMA). That is, it has been found that the complex can be used as a PSMA inhibitor. Therefore, the complex can be used for the diagnosis and treatment of diseases involving PSMA. In particular, the complex can be used for the diagnosis and treatment of specific diseases in which PSMA is upregulated. Therefore, the complex can be used as a medicine in the diagnosis and treatment of prostate cancer.

[0016] According to the present invention, group X is one amino acid (p = 1) or a sequence of amino acids (p = 2 or 3). When p is 1, preferably, the amino acid is selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, and substituted or unsubstituted serine. Preferably, the amino acid is substituted or unsubstituted phenylalanine, or substituted or unsubstituted glutamic acid, and particularly preferably substituted or unsubstituted glutamic acid. When p is 2 or 3, preferably, each of the amino acids forming the amino acid sequence is independently selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, and substituted or unsubstituted serine. Preferably, each of the amino acids of the amino acid sequence is substituted or unsubstituted phenylalanine, or substituted or unsubstituted glutamic acid, and particularly preferably substituted or unsubstituted glutamic acid.

[0017] Group X may be bonded to group Y via a first peptide bond and to moiety BM via a second peptide bond. Moiety BM is of the general formula

Chemical formula

[0018] When group X has a sequence of two or three amino acids, the amino acids are each bonded to one another by peptide bonds. Group BM is a urea-based pharmacophore group. Groups X and Y form a linker between group BM on the one hand and moiety L-A on the other hand.

[0019] ​According to the present invention, the group Y is one amino acid (p = 1) or a sequence of amino acids (q = 2 or 3). When q is 1, preferably, the amino acid is selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, and substituted or unsubstituted serine. Preferably, the amino acid is substituted or unsubstituted phenylalanine, or substituted or unsubstituted glutamic acid, and particularly preferably substituted or unsubstituted glutamic acid. When q is 2 or 3, preferably, each of the amino acids forming the amino acid sequence is independently selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, and substituted or unsubstituted serine. Preferably, each of the amino acids of the amino acid sequence is substituted or unsubstituted phenylalanine, or substituted or unsubstituted glutamic acid, and particularly preferably substituted or unsubstituted glutamic acid.

[0020] The group Y may be bonded to the group X via a first peptide bond and to the moiety L via a second peptide bond. u When the group X has a sequence of two or three amino acids, the amino acids are bonded to each other by peptide bonds.

[0021] The groups X and Y may be the same or different. In one embodiment of the present invention, the term "substituted amino acid" refers to an amino acid having a phenyl ring, and the phenyl ring has one or two substituents independently selected from the group consisting of halogen and hydroxy. The term "halogen" refers to fluorine, chlorine, bromine or iodine unless otherwise specified. Preferably, the halogen is iodine. In a preferred embodiment, the phenyl ring has two substituents, one of the substituents is iodine, and the other substituent is hydroxy.

[0022] According to the present invention, L in the compound of formula I can be L1 or L2, wherein v, x and y are, independently of each other, 1, 2 or 3. In a more preferred embodiment of the present invention, the linker L can be L1 or L2, wherein v, x and y are, independently of each other, 1. In a more preferred embodiment of the present invention, the linker L is L1, wherein v is 1.

[0023] According to the present invention, A in the compound of formula I may be A1 or A2. In a more preferred embodiment of the present invention, A is A1.

[0024] In a more preferred embodiment of the present invention, k is 1, m is 3, n is 2, p is 1 or 2, q is 1 or 2, u is 1, preferably p and q are 1, and X and Y are independently substituted or unsubstituted phenylalanine or glutamic acid; L is L1 with v = 1, or L2 with x and y = 1, and A is A1 or A2, and a compound of general formula I can be provided.

[0025] In a first embodiment of the present invention, X is unsubstituted phenylalanine and Y is substituted or unsubstituted phenylalanine. Preferably, X is unsubstituted phenylalanine and Y is substituted phenylalanine, and more preferably, X is unsubstituted phenylalanine and Y is phenylalanine substituted with iodine and OH in the phenyl ring. In the first embodiment, L is preferably L1 or L2, and particularly preferably L1. Preferably, L is L1 with v = 1, or L2 with x and y = 1. In the first embodiment, A is preferably selected from the group consisting of A1 - A4. A is preferably A1 or A2, and particularly preferably A1. In the first embodiment, it is preferred that k is 1, m is 3, n is 2, p is 1, q is 1, and u is 1.

[0026] In a second embodiment of the present invention, X and Y are, independently of each other, phenylalanine or glutamic acid. Preferably, both X and Y are glutamic acid. In the second embodiment, L is preferably L1 or L2, and particularly preferably L1. Preferably, L is L1 with v = 1, or L2 with x and y = 1. In the first embodiment, A is preferably selected from the group consisting of A1 to A4. A is preferably A1 or A2, and particularly preferably A1. In the first embodiment, it is preferred that k is 1, m is 3, n is 2, p is 1, q is 1, and u is 1.

[0027] When part A is A4, it is preferred that k, m, n, p, q, v, x, y and z in A4 and X, Y and L have the same meaning as those in part BM.

[0028] According to the present invention, the following compounds 1, 2, 4, 5 and 6 are preferred.

[0029]

Chemical formula

[0030]

Chemical formula

[0031]

Chemical formula

[0032]

Chemical formula

[0033]

Chemical formula

[0034] Compounds of general formula I can exist in the form of tautomers or stereoisomers depending on their structure. Thus, compounds of general formula I include all enantiomers as well as all diastereomers. Accordingly, the present invention also encompasses tautomers, enantiomers or diastereomers of compounds of general formula I, and mixtures thereof. Stereoisomerically pure components can be isolated from mixtures of enantiomers and / or diastereomers as described above by known methods.

[0035] According to the present invention, there is further provided a complex comprising a compound of general formula I as a ligand and a metal. Such a complex can be used as a pharmaceutical, particularly as a pharmaceutical for the diagnosis and treatment of prostate diseases. The complexation of the metal is preferably carried out via the chelator moiety A. The complex is a coordination complex of the corresponding metal. The metal can exist as a metal ion or as a metal oxide. In the metal oxide, the metal can exist as an ion. In one embodiment, the complex can consist of a compound of general formula I as a ligand and a metal ion. In another embodiment, the complex can consist of a compound of general formula I as a ligand and a metal oxide. The metal forms the central particle of the complex of the present invention.

[0036] In a preferred embodiment of the present invention, the metal is selected from the group consisting of rhenium ions, technetium ions and copper ions. Preferably, the metal is a radioactive metal. Accordingly, a preferred complex is a metal complex containing a radionuclide and a compound of general formula I.

[0037] More preferably, the metal is 99m Tc, 99 Tc, 94m Tc, 186 Re, 188 Re, 64 Cu and 67 Cu, and is an isotope selected from the group consisting of. Even more preferably, the metal is 99m Tc, 186 Re and 188 Re, and is an isotope selected from the group consisting of. In the most preferred embodiment of the present invention, the isotope is 99m Tc.

[0038] In one embodiment of the present invention, the complex is a compound of formula ReO-5:

Chemical formula

[0039] In one embodiment of the present invention, the complex is a compound of formula TcO-5:

Chemical formula

Chemical formula

[0040] 99m The supply of them for the production and administration of Tc-containing complexes is achieved by reacting each ligand, i.e., each compound of general formula I, with sodium pertechnetate 99 Mo / 99m Tc in physiological saline as provided by a generator, such as 99m Tc], according to the methods described and adapted in the literature (IAEA. Labelling of small biomolecules using novel Technetium-99m Cores; International Atomic Energy Agency: Vienna, 2007). The 99m Tc(VII) present in sodium pertechnetate 99m Tc] is converted to 99m Tc(V)O present in the complex 99mTo enable the necessary reduction to Tc(V), a reducing agent, preferably stannous chloride, is used under optimized conditions. For example, its production is carried out under acidic conditions, preferably using dilute hydrochloric acid, under saturation by a stream of an inert gas, such as helium, and thus with the exclusion of oxygen. The labeling step is carried out under weakly acidic conditions, for example in the pH range of 5 to 6.5. The total volume of the reaction solution can range from 0.2 to 12 mL, and the amount of sodium pertechnetate ( 99m Tc) can range from 0.1 to 50 GBq. Depending on the volume of the generator eluate used, the amount of ligand used can vary (10 to 200 μg). The labeling step can be carried out at room temperature. In particular, the reaction parameters used may require elevated temperatures up to 100 °C, preferably up to 80 °C, to complete the reaction or to minimize the reaction time. Furthermore, heating for a specific period, for example heating at 80 °C for 20 minutes, results in the decomposition of the unreacted amount of the excess ligand and is thus advantageous for supporting the imaging properties of the complex. If the labeling is incomplete, the complex-containing solution can be purified by solid-phase extraction (SPE) method or semi-preparative HPLC using, for example, RP-C18 or Sephadex (G-25, superfine) as the stationary phase. Auxiliary materials that contribute to complete conversion and minimize the reaction time, such as salts of gluconic acid or heptagluconic acid, preferably calcium heptagluconate, can be used. Other auxiliary substances such as D-mannose, galactose or cyclodextrin can be used, among which D-mannose is preferred. Similarly, the use of stabilizers and antioxidants, such as dithiothreitol and ascorbic acid, is possible respectively.

[0041] The complexes according to the invention can bind to prostate-specific membrane antigen (PSMA). Thus, they are ligands for prostate-specific membrane antigen (PSMA). In the following, the complexes according to the invention are also referred to as ligands or PSMA ligands. When the complex has a radioactive metal, the complex is also called a radioactive ligand or a PSMA radioactive ligand.

[0042] The PSMA ligand according to the present invention comprises a urea-based pharmacophore group BM, various amino acid sequences in the linker -X-Y-, and various N 2 S 2 chelators. A synthetic strategy that does not require mercury-containing intermediates is used, enabling the production of the PSMA ligands of the present invention that are free of potentially toxic mercury impurities. Mercury salts are generally applied to cleave thiol protecting groups; see Peter G.M. Wuts, Greene’s Protective Groups in Organic Synthesis, Fifth Edition, Wiley, 2014.

[0043] Compounds of general formula I can be synthesized by various methods according to known synthetic routes, for example, by the use of Merrifield synthesis (see Robert Bruce Merrifield, Solid phase peptide synthesis, Journal of the American Chemical Society, Volume 85, issue 14 p. 2149-2154). In one method, a compound of general formula II is provided

Chemical formula

[0044] R 12 is Dde, and R 13When it is Fmoc, the first synthetic route can be used. In the first synthetic route, in the first step, Fmoc deprotection of the compound of general formula II is carried out, thereby obtaining the first reaction product of general formula III

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0045] In the fifth step, the fifth reaction product of general formula IX

Chemical formula

[0046] In the sixth step, the sixth reaction product of General Formula X [Chemical Formula] is synthesized. The moiety Y q is the moiety Y of General Formula I qCorresponding thereto, provided that the reactive side chain can be protected by a protecting group. For example, -COOH can be protected as -COOtBu. To synthesize the reaction product of general formula X, when q = 1, Fmoc deprotection of the reaction product of general formula IX is carried out, followed by amide coupling between the Fmoc-deprotected reaction product of general formula IX and a compound of general formula Fmoc-Y-OH. When q = 2, Fmoc deprotection of the reaction product of general formula IX is carried out, followed by a first amide coupling between the Fmoc-deprotected reaction product of general formula IX and a first compound of general formula Fmoc-Y-OH, thereby obtaining an intermediate. After Fmoc deprotection of the intermediate, a second amide coupling between the Fmoc-deprotected intermediate and a second compound of general formula Fmoc-Y-OH is carried out. When q = 3, Fmoc deprotection of the reaction product of general formula IX is carried out, followed by a first amide coupling between the Fmoc-deprotected reaction product of general formula IX and a first compound of general formula Fmoc-Y-OH, thereby obtaining a first intermediate. After Fmoc deprotection of the first intermediate, a second amide coupling between the Fmoc-deprotected first intermediate and a second compound of general formula Fmoc-Y-OH is carried out, thereby obtaining a second intermediate. After Fmoc deprotection of the second intermediate, a third amide coupling between the Fmoc-deprotected second intermediate and a third compound of general formula Fmoc-Y-OH is carried out.

[0047] When u = 1, in the seventh step, Fmoc deprotection of the reaction product of general formula X is carried out, followed by amide coupling between the obtained reaction product and a compound of general formula Fmoc-L-OH, thereby obtaining a reaction product of general formula XI

Chemical formula

[0048] In the 8th step, when u = 1, Fmoc deprotection of the reaction product of general formula XI or, when u = 0, of the reaction product of general formula X is carried out, followed by amide coupling between the resulting reaction product and a compound of general formula A-OH, whereby a reaction product of general formula XII [Chemical formula] is obtained. Moiety A corresponds to moiety A of general formula I, provided that the reactive side chains can be protected by protecting groups. For example, -COOH can be protected as -COOtBu and the PMB protecting group may be on the thiol group of A. In the next step, the reaction product of general formula XII is cleaved from the 2-CTC resin and deprotected by removing all the protecting groups, whereby a compound of general formula I is obtained. In formulas II - XII, the subscripts k, m, n, p, q, u, v, x, y and z have the meanings given in the context of general formula I.

[0049] R 12 is Fmoc and R 13 is Dde, a second synthetic route can be used. In the second synthetic route, in the first step, Fmoc deprotection of the compound of general formula II is carried out, whereby a first reaction product of general formula XIII [Chemical formula] is obtained. In the second step, a second reaction product of general formula XIV [Chemical formula] is synthesized. Moiety X p is moiety X of general formula I pCorresponding thereto, provided that the reactive side chain can be protected by a protecting group. For example, -COOH can be protected as -COOtBu. To synthesize the reaction product of general formula XIV, when p = 1, an amide coupling is carried out between the first reaction product of general formula XIII and the compound of general formula Fmoc-X-OH. When p = 2, a first amide coupling is carried out between the first reaction product of general formula XIII and the first compound of general formula Fmoc-X-OH, whereby an intermediate is obtained, and after the Fmoc deprotection of the intermediate, a second amide coupling is carried out between the Fmoc-deprotected intermediate and the second compound of general formula Fmoc-X-OH. When p = 3, a first amide coupling is carried out between the first reaction product of general formula XIII and the first compound of general formula Fmoc-X-OH, whereby a first intermediate is obtained, and after the Fmoc deprotection of the first intermediate, a second amide coupling is carried out between the Fmoc-deprotected first intermediate and the second compound of general formula Fmoc-X-OH, whereby a second intermediate is obtained, and after the Fmoc deprotection of the second intermediate, a third amide coupling is carried out between the Fmoc-deprotected second intermediate and the third compound of general formula Fmoc-X-OH.

[0050] In the third step, the third reaction product of general formula XV

Chemical formula

[0051] When u = 1, in the fourth step, Fmoc deprotection of the reaction product of general formula XV is carried out, followed by amide coupling between the obtained reaction product and a compound of general formula Fmoc-L-OH, thereby obtaining a reaction product of general formula XVI

Chemical formula

[0052] In the 5th step, when u = 1, Fmoc deprotection of the reaction product of general formula XVI or, when u = 0, Fmoc deprotection of the reaction product of general formula XV is carried out, followed by amide coupling between the resulting reaction product and a compound of general formula A-OH, whereby a reaction product of general formula XVII

Chemical formula

[0053] In the 6th step, Dde cleavage of the compound of general formula XVII is carried out, whereby a reaction product of general formula XVIII

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0054] The synthesis of the compounds of general formula I is described in more detail in the Examples section.

[0055] The N incorporated into the compound of general formula I 2 S 2 The chelator can be used to label these PSMA ligands with various metals by complex formation. The complex is useful as a diagnostic (agent) and therapeutic (agent) for diseases involving PSMA, such as prostate cancer. The complex is useful as a diagnostic (agent) and therapeutic (agent) for specific diseases in which PSMA is upregulated. An example of a disease in which PSMA is upregulated is prostate cancer.

[0056] Surprisingly, in the LNCaP xenograft model, particularly compound 5 was found to exhibit pharmacokinetic properties faster and more favorable organ distribution (high tumor uptake, low to moderate uptake in non-target organs such as the kidney) than those known from the prior art. High tumor-to-muscle and tumor-to-kidney ratios were achieved early, 1 hour after injection, which allows imaging at an early time point. Further increases in the tumor-to-muscle ratio and tumor-to-kidney ratio over time were observed. These findings suggest better visualization of tumors compared to compounds known from the prior art.

[0057] To demonstrate the influence of moieties X and Y of the compounds of the present invention, the inventors synthesized comparative compounds of general formula BM-L u -A (wherein BM, L, A and u have the meanings given in relation to general formula I). The compounds of general formula BM-L u -A are compounds of general formula I wherein p is 0 and q is 0. The comparative compound BM-L uAn example of -A is Comparative Compound 3.

[0058]

Chemical formula

[0059] a prostate cancer imaging agent approved by the US Food and Drug Administration (FDA), well studied, and widely used 68 compared to Ga-PSMA-11, which is a PET tracer 99m The complex of Tc and the compounds of general formula I surprisingly was found to have similar properties in the in vivo and in vitro experiments described herein. The compounds of the present invention showed similar affinity for PSMA, higher internalization in LNCaP tumor cells, and comparable tumor uptake values in the LNCaP xenograft model when comparing 1 hour after injection of each compound. Since technetium and rhenium are a seranostic pair, the inventors expect 186 the complex of Re and the compounds of general formula I, and 188 the complex of Re and the compounds of general formula I to have similar properties.

[0060] Therefore, the complex according to the present invention can be used as a medicament, particularly a medicament for diseases involving PSMA. Further, a pharmaceutically acceptable salt of the complex according to the present invention can be used as a medicament, particularly a medicament for diseases involving PSMA. In one embodiment, the complex according to the present invention can be used as a medicament in the diagnosis and treatment of diseases involving PSMA. Also, a pharmaceutically acceptable salt of the complex according to the present invention can be used as a medicament in the diagnosis and treatment of diseases involving PSMA. Diseases involving PSMA are, for example, diseases in which PSMA is upregulated. An example of a disease in which PSMA is upregulated is prostate cancer.

[0061] Therefore, according to the present invention, there is provided the use of the complex according to the present invention as a medicament. Further, there is provided the use of the complex according to the present invention as a medicament in the diagnosis and treatment of diseases involving PSMA. The medicament can be a radiopharmaceutical. Preferably, the medicament is a radiopharmaceutical for nuclear medicine imaging. For example, the medicament is a radiopharmaceutical for nuclear medicine imaging by single-photon emission computed tomography (SPECT) or positron emission tomography (PET). Also, preferably, the medicament is a radiopharmaceutical for radio-ligand therapy. In a preferred embodiment, 99m Tc, 186 Re, 188 Re and 67 a complex of the present invention containing a metal selected from the group consisting of Cu is used as a radiopharmaceutical for nuclear medicine imaging by single-photon emission computed tomography (SPECT). In another preferred embodiment, 94m Tc or 64 a complex of the present invention containing Cu is used as a radiopharmaceutical for nuclear medicine imaging by positron emission tomography (PET). In yet another preferred embodiment, 186 Re, 188 Re, 64 Cu and67 The complex of the present invention containing a metal selected from the group consisting of Cu is used as a radiopharmaceutical for radio - ligand therapy. Instead of the pharmaceutical containing the compound of general formula I according to the present invention, pharmaceutically acceptable salts of said compound can be used. Because of its long half - life of 211,000 years, 99 The complex of the present invention containing Tc can be used for elucidating the structure of the shape of the complex of the present invention.

[0062] According to the present invention, there is further provided a method for producing a complex containing a compound of general formula I as a ligand and a metal. The method includes the step of contacting the compound of general formula I with a metal. In a preferred embodiment, the compound of general formula I is contacted with the metal at a reaction temperature in the range of 20 - 100 °C. Preferably, the compound of general formula I according to the present invention is contacted with the metal at room temperature. Preferably, the compound of general formula I is contacted with the metal at ambient pressure. The molar ratio of the compound of general formula I to non - radioactive metal ions in solution can be 1:1. Preferably, the method according to the present invention is carried out using a protic solvent, such as water. The pH value can be selected according to the metal used.

[0063] Hereinafter, the present invention will be described in more detail with reference to examples, but it is not intended to limit the present invention with respect to the drawings.

Brief Description of the Drawings

[0064] Here Figure 1 shows the functional imaging of subcutaneous LNCaP tumor xenografts in mice using [99mTc]TcO - 5 [= [99mTc]TcO - ABX474], compared with a reference compound. 99m [99mTc]TcO - 5 [= 99m [99mTc]TcO - ABX474] Figure 2 shows a diagram showing the tumor - to - background kinetics of [99mTc]TcO - 5 [= [99mTc]TcO - ABX474] in LNCaP tumor - bearing mice, compared with a reference compound. 99m [99mTc]TcO - 5 [= 99m [99mTc]TcO - ABX474] Figure 3 shows in LNCaP tumor - bearing mice 99mFigure showing the tumor uptake of the [Tc] radiolabeled ligand. Figure 4 shows, in LNCaP tumor-bearing mice, 99m [Tc]TcO-5 [= 99m Figure showing the tumor-to-background kinetics of [Tc]TcO-ABX474 compared to a reference compound. Figure 5 shows the tumor-to-background ratio of the [Tc] radiolabeled ligand in LNCaP tumor-bearing mice. 99m Figure showing the tumor-to-background ratio of the [Tc] radiolabeled ligand.

Example

[0065] Example General synthetic methods for Compounds 1, 2, 4, 5 and 6

[0066] Compounds 1, 2, 4, 5 and 6 were synthesized by solid-phase peptide synthesis (SPPS) on 2-chlorotrityl resin.

[0067] Analysis of the synthesized molecules was performed using reverse-phase high-performance liquid chromatography (RP-HPLC; Ascentis Express C18, 150x4.6mm; Supelco, Germany) with a linear A-B gradient (from 5% B to 100% B in 10 minutes) at a flow rate of 1.5 mL / min. Purification was carried out using reverse-phase high-performance liquid chromatography (RP-HPLC; Gemini-NX C18, 250x50mm; Phenomenex, Germany) with a linear A-B gradient at a flow rate of 100 mL / min. Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in ACN.

[0068] The HPLC system (Dionex Ultimate 3000; Thermo-Fisher, Germany) was equipped with a UV detector. UV absorbance was measured at 200, 210 and 230 nm. Mass spectrometry was performed using an LC-MS System (Dionex 3000, Thermo-Fisher, Germany).

[0069] Examples 1 - 4 Synthesis of intermediates

[0070] To prepare Compounds 1 to 6, the following intermediates were prepared.

[0071] Example 1 Synthesis of Intermediate 101: Glu-CO-Im ((di-tert-butyl (1H-imidazole-1-carbonyl)-L-glutamate))

[0072] H-Glu(OtBu)-OtBu (29.59 g, 1 equivalent, 100 mmol) was dissolved in 400 ml of DCM. Triethylamine (25.3 g, 2.5 equivalents, 250 mmol) was slowly added. Carbonyldiimidazole (17.84 g, 1.1 equivalents, 110 mmol) was added in small portions. The reaction was stirred for 4 hours. The solution was washed with water, NaHCO 3 and brine. The organic phase was dried over Na 2 SO 4 and evaporated under reduced pressure to obtain the target compound 101 as an oil.

[0073] Example 2 Synthesis of Intermediate 103: N’-[2-(4-methoxy-benzylsulfanyl)-ethyl]-N’-{2-[4-methoxy-benzylsulfanyl)-ethylamino]-ethyl}-propane-1,3-diamine

Chemical formula

[0074] The synthesis of Intermediate 103 was carried out in the same manner as in WO2012 / 022812A1. Step 1: 2-(4-methoxy-benzylsulfanyl)-ethylamine (Compound 104)

[0075] Sodium (4.5 g, 196 mmol) was added to vigorously stirred methanol (150 ml, dry). Once the sodium had completely dissolved, 2-aminoethanethiol hydrochloride (10.8 g, 95.0 mmol) was added. Then, p-methoxybenzyl chloride (14.9 g, 95.5 mmol) was added via a dropping funnel. The mixture was heated to reflux at 70 °C for 30 minutes. Thereafter, the mixture was cooled to room temperature. The solid was removed by filtration and the filter cake was washed with methanol (3 times with 25 ml). The organic extracts were combined and the volatile substances were removed under reduced pressure. This residue was redissolved in DCM (75 ml), extracted with water (75 ml × 3), dried (MgS0 4 ) and filtered, and the solvent was removed to obtain Compound 104 as a colorless oil. Yield 18.5 g (99%).

[0076] Step 2: N-[(4-methoxy-benzylsulfanyl)-ethyl]-2-chloroacetamide (Compound 105)

[0077] Chloroacetyl chloride (4.24 ml, 53.2 mmol) in dry DCM (50 ml) was added dropwise over 90 minutes with stirring to a solution of 2-(4-methoxy-benzylsulfanyl)-ethylamine 104 (9.4 g, 47.5 mmol) and triethylamine (8.0 ml) in dry DCM (200 ml) cooled in an ice bath (0 °C). After the addition, the cooling bath was removed and stirring was continued for 60 minutes. The solution was extracted with water (2 × 250 ml), dried (MgSO 4 ) and filtered, and the solvent was evaporated under reduced pressure to obtain Compound 105 as a colored solid. Yield 12.93 g (99%).

[0078] Step 3: Methyl 3-[(4-methoxy-benzylsulfanyl)-ethylamino]-propanoate (Compound 106)

[0079] Methyl acrylate (4.46 ml, 49.2 mmol) in methanol (10 ml) was added to a stirred solution of 2-(4-methoxy-benzylsulfanyl)-ethylamine 104 (8.85 g, 48.3 mmol) in methanol (50 ml). The colorless solution was stirred at room temperature for 6 hours. The volatile substances were removed by rotary evaporation to obtain Compound 106 as a colorless viscous oil. Yield 1.35 g (97%).

[0080] Step 4: 3-[2-(4-methoxybenzylsulfanyl)-ethylamino]-propanamide (Compound 107)

[0081] Methyl 3-[(4-methoxy-benzylsulfanyl)-ethylamino]propanoate 106 (10.65 g, 37.6 mmol), methanol (120 ml) and ammonia solution (200 ml) were stirred at room temperature for 24 hours. The volatile substances were removed under reduced pressure to obtain Compound 107 as a nearly white solid. Yield 9.98 g (99%).

[0082] Step 5: 3-([2-(4-methoxy-benzylsulfanyl)-ethyl]-{[2-(4-methoxy-benzylsulfanyl)-ethylcarbamoyl]-methyl}-amino)-propionamide (Compound 108)

[0083] 3-[2-(4-Methoxybenzylsulfanyl)-ethylamino]propenamide 106 (10.33 g, 38.5 mmol), N-[(4-methoxy-benzylsulfanyl)-ethyl]-2-chloroacetamide 105 (10.54 g, 38.5 mmol), triethylamine (6.5 ml) and acetonitrile (80 ml) were heated at 70 °C overnight. Then, the mixture was cooled to room temperature. The solvent was removed under reduced pressure to obtain a brown residue, which was purified by silica eluting with DCM / methanol 20:1 to obtain Compound 108 as a colorless oil (yield 8.3 g, 43%).

[0084] Step 6: N’-[2-(4-methoxy-benzylsulfanyl)-ethyl]-N’-{2-[4-methoxy-benzylsulfanyl)-ethylamino]-ethyl}-propane-1,3-diamine (Compound 109)

[0085] 1.0 M borane in THF (102 ml, 102 mmol) was added via syringe to 3-([2-(4-methoxy-benzylsulfanyl)-ethyl]-{[2-(4-methoxybenzylsulfanyl)-ethylcarbamoyl]-methyl}-amino)-propionamide 108 (3.8 g, 7.5 mmol) under an argon atmosphere. The resulting colorless solution was heated to reflux at 70 °C overnight. After cooling to room temperature, water (40 ml) was added dropwise. The solvent was removed under reduced pressure to obtain a waxy solid, which was diluted with HCl (0.5 N, 400 ml). The mixture was heated to reflux at 100 °C for 3 hours. After cooling to room temperature, sodium hydroxide was added until a pH of 10 - 11 was obtained. This mixture was extracted with DCM (4 × 200 ml), the organic fractions were combined, dried (MgSO 4 ) and filtered. The solvent was evaporated to obtain a waxy solid, which was purified by silica eluting with DCM / methanol / NH 4 OH = 9:1:0.1 to give compound 109 as a colorless oil. Yield 1.29 mg (36%).

[0086] Example 3 Synthesis of Intermediate 4: N-Boc-N’-(5-carboethoxypentyl-N,N’-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine (Compound 110)

Chemical Structure

[0087] The synthesis of intermediate 110 was carried out in the same manner as in US5776428A.

[0088] Step 1: 2,2’-dithiobis(2-methylpropanal) (Compound 111)

[0089] Sulfur monochloride (27 g, 0.2 mol) was added to 2-methylpropanal (28.8 g, 0.4 mol) in carbon tetrachloride (30 ml). The reaction mixture was stirred at 50 °C for 16 hours. After cooling to room temperature, the volatile substances were evaporated in vacuo and the residue was purified by distillation in vacuo (bp: 98 - 102 °C at 0.5 Torr) to give 22.7 g (55%) of 2,2’-dithiobis(2-methylpropanal) 111.

[0090] Step 2: 3,3,10,10-tetramethyl-6,7-dihydro-1,2,5,8-dithiazocine (Compound 112)

[0091] To a stirred solution of 111 (5 g) of 2,2'-dithiobis(2-methylpropanal) in chloroform (20 mL) was added 1.8 g of ethylenediamine. The reaction mixture was stirred at room temperature for 2 h. After removal of the solvent, the residue was triturated with water until crystals began to form. The white crystals were collected by filtration and washed with ethanol to give 4.8 g (86%) of 3,3,10,10-tetramethyl-6,7-dihydro-1,2,5,8-dithiazocine 112.

[0092] Step 3: N,N’-bis(2-mercapto-2-methylpropyl)ethylenediamine (Compound 113)

[0093] To 4.1 g (18 mmol) of 3,3,10,10-tetramethyl-6,7-dihydro-1,2,5,8-dithiazocine 112 dissolved in 70 mL of dry THF was added 1.35 g of LiAlH 4 (0.36 mmol) with stirring (argon atmosphere). The reaction mixture was refluxed for 4 h and then hydrolyzed by careful addition of saturated NaK tartrate solution (20 mL), followed by addition of diethyl ether (100 mL). The sludge was separated by decantation or filtration (celite) and washed thoroughly with ether. The solvent was removed in vacuo to give 0.8 g (20%) of compound 113.

[0094] Step 4: N,N’-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine (Compound 114)

[0095] A solution of N,N'-bis(2-mercapto-2-methylpropyl)ethylenediamine 113 (1.1 g, 4.65 mmol) in methanol (50 mL) was cooled in an ice / water bath and then saturated with gaseous ammonia for 30 minutes. To this was added 4-methoxybenzyl chloride (1.9 g, 12.3 mmol). The reaction was warmed to room temperature overnight with stirring under argon. The methanol was evaporated under reduced pressure and the residue was then partitioned between diethyl ether (50 mL) and 0.5 M KOH (40 mL). The aqueous layer was further extracted with diethyl ether (2 x 25 mL). The combined organic layers were washed with NaCl solution and concentrated in vacuo to give a clear colorless oil. The oil was dissolved in diethyl ether (200 mL) and then acidified with 4.0 M HCl in dioxane. The white precipitate was collected by filtration and washed with diethyl ether. The HCl salt was partitioned between 1 M KOH (30 mL) and ethyl acetate (30 mL). The aqueous layer was extracted with ethyl acetate (2 x 30 mL), the combined organic layers were washed with NaCl, dried over Na 2 SO 4 and concentrated to give pure compound 114 (free base) as a pale yellow oil (0.99 g, 45% yield).

[0096] Step 5: N-(5-carboethoxypentyl-N,N’-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine (Compound 115)

[0097] To N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine 114 (921 mg, 1.93 mmol) in acetonitrile (15 mL) was added K 2 CO 3 (270 mg), followed by ethyl 5-bromovalerate (807 mg). The reaction was stirred at reflux overnight and then concentrated in vacuo. The residue was partitioned between ethyl acetate (50 mL) and 0.5 M KOH (50 mL). The aqueous layer was extracted with ethyl acetate (2 x 50 mL), the combined organic layers were washed with brine (50 mL), dried over Na 2 SO 4 and concentrated to give a yellow oil which was purified by silica eluting with DCM / methanol = 20:1 to give the desired compound 115 as a yellowish oil. Yield 635 mg (54%)

[0098] Step 6: N-Boc-N'-(5-carboethoxypentyl)-N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)-ethylenediamine (Compound 116)

[0099] To N-(5-carboethoxypentyl)-N,N'-bis-(2-(4-methoxybenzylthio)-2-methylpropyl)ethylenediamine 115 (635 mg, 1.05 mmol) in THF (40 mL) were added water (30 mL) and 1 M KOH (2.5 mL, 2.5 mmol). The homogeneous solution was refluxed overnight. Then, the solution was cooled to room temperature and the THF was removed under vacuum. The residue was diluted with 50 mL of water and the pH was adjusted to 2 - 3 by the addition of 1 M HCl. The solution was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine (50 mL) and dried over Na 2 SO 4 and concentrated in vacuo to give 575 mg of the crude intermediate.

[0100] The crude intermediate was dissolved in ACN (50 mL) and Boc 2 O (326 mg) and then triethylamine (0.280 mL) were added. The homogeneous solution was stirred overnight at room temperature under argon. Then, the solution was concentrated in vacuo and subsequently partitioned between ethyl acetate (25 mL) and 1 M KH 2 PO 4 (25 mL). The organic layer was washed with 5% citric acid (2 × 25 mL) and brine (25 mL), dried over Na 2 SO 4 and concentrated to give a yellow oil (890 mg). Purification by silica column chromatography eluting with DCM / methanol = 20:1 gave Compound 116 as a yellowish oil. Yield 455 mg (64%).

[0101] Example 4 Synthesis of Compound 5 [ABX 474]

[0102] Compound 5 of the present invention

Chemical formula

[0103]

Chem.

[0104] For the synthesis of compound 501, Fmoc-D-Lys(Dde)-OH was loaded onto 2-CTC resin (in Scheme EX-1, the resin is represented by a circle), and the Fmoc was deprotected with 20% piperidine in DMF. Then, to synthesize compounds 502 and 503, Fmoc-Glu(OtBu)-OH (2 equiv) was activated with HATU (2 equiv), HOAt (2 equiv), and DIPEA (5,6 equiv) in DMF and added to the resin. The reaction mixture was stirred at room temperature (r.t.) for 2 h. Fmoc deprotection was carried out using 20% piperidine in DMF. To synthesize compound 504, intermediate 110 was coupled to the peptide sequence at room temperature for 2 h using PyBOP (2 equiv) and DIPEA (2 equiv) in DMF. Dde deprotection was performed using 2% hydrazine monohydrate in DMF. Then, to synthesize compound 505, suberic acid mono-NHS ester (2 equiv) and DIPEA (2 equiv) were dissolved in DMF (1 mL) and reacted with the resin-bound peptide at room temperature for 2 h. Next, to synthesize compound 506, the free carboxylic acid was treated with TSTU (2 equiv) and DIPEA (2 equiv) in DMF (10 mL / resin 1 g) at room temperature for 1 h. After the formation of the NHS ester, the resin-bound peptide was treated with Fmoc-Lys-OtBu (2 equiv) and DIPEA (2 equiv) in DMF (2 mL) at room temperature for 2 h. The Fmoc was cleaved using 20% piperidine in DMF, and then the free amine was treated with Glu-CO-Im (intermediate 101, 3 equiv) and NMM (3 equiv) in DMF at room temperature for 2 h. After the final coupling, the resin was washed with DMF (3x5 mL), DCM (3x5 mL), IPA (3x5 mL), and Et 2 O (3x5 mL). For the synthesis of compound 5, cleavage from the resin and final deprotection were carried out by treatment with TFA / TIS / EDT / water (v / v / v / v; 92.5 / 2.5 / 2.5 / 2.5) at 0 °C followed by the dropwise addition of 10% TFMSA.

[0105] The crude peptide was precipitated from ice-cold diethyl ether and purified by RP-HPLC. After RP-HPLC purification, compound 4 [ABX 474] (TFA salt) was obtained as a white to off-white solid (isolation yield 38%). Calculated monoisotopic mass (C 51 H 89 N 9 O 18 S 2 ): 1179.58; found: m / z = 1180.5 [M+H] + , 590.79 [M+2H] 2+ .

[0106] Example 5 Synthesis of Compound 6 [ABX 490]

[0107] Compound 6 of the present invention [Chemical formula] The synthesis of [Chemical formula] is shown in Scheme EX-2

[0108] [Chemical formula] JPEG2025517989000042.jpg224170 In Scheme EX-2, letter a) indicates the use of 20% piperidine in DMF; letter b) indicates the use of Sub-NHS, DIPEA and DMF; letter c) indicates the use of TSTU, DIPEA and DMF, and a reaction time of 1 hour; letter d) indicates the use of Fmoc-Lys-OtBu, DIPEA and DMF; letter e) indicates the use of Glu-CO-Im, NMM and DMF; letter f) indicates the use of 2% N 2 H 2 .H 2 O; letter g) indicates the use of Fmoc-Glu(OtBu)-OH, PyBop, HOBt, DIPEA and DMF; letter h) indicates the use of succinic anhydride, DIPEA and DMF; letter i) indicates the use of intermediate 103, DIPEA and DMF; letter j) indicates TFA:TIS:H 2Shows the use of O: EDT (92.5:2.5:2.5:2.5); and letter k) shows the use of 10% TFMSA.

[0109] For the synthesis of compound 601, Dde-D-Lys(Fmoc)-OH was loaded onto 2-CTC resin (in Scheme EX-2, the resin is represented by a circle), and Fmoc was deprotected with 20% piperidine in DMF. Then, for the synthesis of compound 602, suberic acid mono-NHS ester (2 eq) and DIPEA (2 eq) were dissolved in DMF (1 mL) and reacted with the resin-bound peptide at room temperature for 2 h. Next, for the synthesis of compound 603, the free carboxylic acid was treated with TSTU (2 eq) and DIPEA (2 eq) dissolved in DMF (2 mL) and reacted at room temperature for 1 h. After the formation of the NHS ester, the resin-bound peptide was treated with Fmoc-Lys-OtBu (2 eq) and DIPEA (2 eq) in DMF (1 mL) at room temperature for 2 h. For the synthesis of compound 604, Fmoc was cleaved using 20% piperidine in DMF, and then the free amine was treated with Glu-CO-Im101 (3 eq) and NMM (3 eq) in DMF at room temperature for 2 h. Dde deprotection was carried out using 2% hydrazine monohydrate in DMF. Then, for the synthesis of compound 605, Fmoc-Glu(OtBu)-OH (2 eq) was activated with PyBOP (2 eq), HOBt (2 eq) and DIPEA (2 eq) in DMF and added to the resin. The reaction mixture was stirred at room temperature for 2 h. For the synthesis of compound 606, Fmoc was deprotected with 20% piperidine in DMF. Succinic anhydride (2 eq) and DIPEA (2 eq) dissolved in DMF (2 mL) were added to the resin-bound peptide and stirred at room temperature for 2 h. Then, the free carboxylic acid was activated with TSTU (2 eq) and DIPEA (2 eq) dissolved in DMF (2 mL) and reacted at room temperature for 1 h. After the formation of the NHS ester, the resin-bound peptide was treated with intermediate 103 (1 eq) and DIPEA (2 eq) in DMF (2 mL) for 2 h to synthesize compound 607. After completion of the reaction, the resin was washed with DMF (3×5 mL), DCM (3×5 mL), IPA (3×5 mL), Et 2It was washed with O(3×5 mL). For the synthesis of Compound 6, cleavage from the resin and final deprotection were carried out by treatment with TFA / TIS / EDT / water (v / v / v / v; 92.5 / 2.5 / 2.5 / 2.5) at 0 °C and subsequent dropwise addition of 10% TFMSA.

[0110] The crude peptide was precipitated from ice-cold diethyl ether and purified by preparative RP-HPLC. After RP-HPLC purification, Compound 6 [ABX 490 (TFA salt)] was obtained as a white to off-white solid (yield 12 percent). Calculated monoisotopic mass (C 49 H 84 N 10 O 19 S 2 ): 1180.54; Found: m / z = 1181.38 [M+H] + , 591.27 [M+2H] 2+ .

[0111] Example 6 Synthesis of Compound 1 [ABX 408]

[0112] Compound 1 of the present invention

Chemical formula

[0113] Example 7 Synthesis of Compound 2 [ABX 451]

[0114] Compound 2 of the present invention [Chemical formula] was synthesized according to the procedure described in Example 4, except that Fmoc-Phe-OH was used instead of Fmoc-Glu(OtBu)-OH. The crude peptide was purified by preparative RP-HPLC. Calculated monoisotopic mass (C 59 H 93 N 9 O 14 S 2 ): 1215.63; Found: m / z = 1216.70 [M+H] + .

[0115] Comparative Example 1 Synthesis of Compound 3 [ABX 455]

[0116] Comparative Compound 3 [Chemical formula] was synthesized according to the procedure described in Example 4, except for the omission of Fmoc-Glu(OtBu)-OH. The crude peptide was purified by preparative RP-HPLC. Calculated monoisotopic mass (C 41 H 75 N 7 O 12 S 2 ): 921.49; Found: m / z = 922.45 [M+H] + .

[0117] Example 8 Synthesis of Compound 4 [ABX 456]

[0118] Compound 4 of the present invention [Chemical formula] was synthesized according to the procedure described in Example 4, except that Fmoc-D-Phe-OH and Fmoc-D-Tyr(3I)-OH were used instead of Fmoc-Glu(OtBu)-OH. The crude peptide was purified by preparative RP-HPLC. Calculated monoisotopic mass (C 59 H92 N 9 O 15 S 2 ): 1357.52; Measured value: m / z = 1356.70 [M-H] + 。

[0119] Example 9 nat Synthesis of Re Complex

[0120] Complexes with the naturally abundant Re isotope ( nat Re) were synthesized using Compounds 1, 3, and 5. These synthetic complexes can be used as PSMA ligands.

[0121] 0.06 mmol of the corresponding Compound 1, 3, or 5 was dissolved in 2 ml of methanol. Subsequently, 1 ml of 1N NaOAc solution and 0.06 mmol of oxotrichloro[(dimethylsulfide)-triphenylphosphine oxide]-rhenium(V) were added. The reaction mixture was stirred at room temperature overnight. The crude product was precipitated with ice-cold diethyl ether and purified by preparative RP-HPLC. Table Ex-1 shows the results of LC-MS analysis of the synthesized rhenium complexes.

[0122]

Table 1

[0123] The nat Re complexes of Compounds 1, 3, and 5 can be used as standards for the radiochemical synthesis of 186 Re and 188 Re-labeled PSMA ligands. Similarly, the nat Re complexes of Compounds 2, 4, and 6 can be used as standards for the radiochemical synthesis of 186 Re and 188 Re-labeled PSMA ligands.

[0124] Example 10 99m Radioactive Labeling of Compounds 1 - 6 with Tc

[0125] Using Compounds 1 to 6, 99m complexes containing Tc were synthesized. These synthesized complexes can be used as PSMA radioligands.

[0126] The corresponding Compound 1, 2, 3, 4, 5 or 6 (50 μg), mannitol (1 mg) and calcium heptagluconate (10 μg) were stirred in 1 - 1.5 mL of sodium pertechnetate / saline aqueous solution (from a commercially available 99 Mo / 99m Tc generator, 0.2 - 4 GBq) at room temperature for 10 minutes. After adding stannous chloride (1 μg in 100 μL of 0.01 N HCl; a solution saturated with helium), the solution was held at room temperature for 20 minutes and finally at 80 °C for a further 20 minutes. After cooling, the product was ready for further use. The product is a 99m complex of Tc with the corresponding Compound 1, 2, 3, 4, 5 or 6. These complexes were provided as solutions of the respective complexes.

[0127] Quality control of the product was carried out by radio - RP - HPLC using a Poroshell 120 EC - C18 column (3.5 μm, 100×3 mm; Agilent Technologies Deutschland, Waldbronn, Germany) and a solvent system consisting of water (0.1% TFA) and ACN (0.1% TFA). The analysis was performed using two different gradient elution methods: a) 0 - 0.5 min 0%, 0.5 - 3 min 0 - 100%, 3 - 5 min 100%, 5 - 8 min 0% ACN (0.1% TFA); flow rate: 0 - 0.5 min 0.4 mL / min, 0.5 - 8 min 0.7 mL / min, and b) 0 - 1.5 min 0%, 1.5 - 10 min 0 - 100%, 10 - 12 min 100%, 12 - 15 min 0% ACN (0.1% TFA); flow rate: 0.7 mL / min, and an isocratic elution method using a specific ratio of ACN (0.1% TFA) suitable for each radioligand. Furthermore, the possible 99m Tc / 99For the determination of Tc colloid formation, radio-thin layer chromatography (radio-TLC) was carried out on a silica gel TLC sheet (POLYGRAM SIL G UV254, Macherey-Nagel, Dueren, Germany) using methanol / ammonium acetate (2M) 1:1. Table 2 shows the radiochemical purity and retention time t R of the obtained complexes.

[0128]

Table 2

[0129] Example 11 Determination of logD Value

[0130] For various media, LogD values were determined by the shake-flask method (Andres, A.; Roses, M.; Rafols, C.; Bosch, E.; Espinosa, S.; Segarra, V.; Huerta, J. M. Setup and validation of shake-flask procedures for the determination of partition coefficients (logD) from low drug amounts. Eur. J. Pharm. Sci. 2015, 76, 181-191). In the preparation, octanol was saturated with each of the buffer solutions used and vice versa. To 3 mL of octanol, together with 3 mL of buffer solution, 200 - 300 μL of 99m Tc-labeled radioligand (15 - 25 MBq, triple samples) was added and shaken vigorously for 30 minutes. After centrifugation at 9,000 rpm for 15 minutes, samples were taken from both separated phases and measured by a gamma counter. The LogD value was calculated as the ratio of the radioactivities determined in the octanol and aqueous phases. Table EX-3 shows the determined LogD values of the 99m Tc complexes in Na phosphate, TRIS buffer and PBS.

[0131]

Table 3

[0132] Example 12 Stability Test:

[0133] 99m The stability of the complexes of Tc with Compounds 1 - 6 was measured in various media.

[0134] Briefly, freshly generated 99m The Tc - labeled radioligand solution (10 - 20 MBq, 20 - 100 μL) was added to each medium (200 - 400 μL), vortexed rapidly, and gently shaken at each of the temperatures described below. At specific time points, samples were taken, diluted, and measured by radio RP - HPLC. Here, the investigation of plasma samples included protein precipitation using a 4 - fold volume of an ice - cold mixture of methanol / water (4 / 1, v / v), vigorous shaking (5 minutes), and centrifugation (14,000 rpm, 10 minutes) prior to the examination of the supernatant. Also, after measuring the activities of the supernatant and residue by a gamma counter, the recovery of the extracted activity was calculated.

[0135]

Table 4

[0136]

Table 5

[0137]

Table 6

[0138]

Table 7

[0139] Example 11 In vitro Assay

[0140] Example 11 describes an in vitro assay performed to determine the properties of compounds 1, 2, 3, 4, 5, and 6. For this reason, a complex of compound 1, 2, 3, 4, 5, or 6 described in Example 10 with 99m Tc, and a complex of compound 1, 2, 3, 4, 5, or 6 described in Example 9 with nat Re were prepared.

[0141] a) Cell Culture

[0142] Competition, saturation, and internalization assays were evaluated using the high PSMA-expressing human prostate cancer cell line LNCaP (ATCC (登録商標) CRL-1740). Cells were grown as a monolayer at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air in RPMI medium containing 10% FCS (Merck KGaA, Germany). Confluent cells were washed twice with phosphate-buffered saline (PBS), detached with trypsin / EDTA (0.05% / 0.02%), and then the cells were suspended in medium and counted (Casy TT, Omni Life Science, Germany).

[0143] b) 68 Ga Labeling of PSMA-11

[0144] 68 The Ga generator was purchased from iThemba LABS (Republic of South Africa). PSMA-11 (2 - 4 μg = 2.11 - 4.21 nmol) was dissolved in a mixture of ammonium acetate (2 M) and HCl at pH 4.5 68It was labeled with Ga (100 - 200 MBq). The reaction mixture was incubated at 90 °C for 10 minutes. Quality control of the radiolabeled PSMA-11 was performed using high-performance liquid chromatography (HPLC) with a C-18 reverse-phase column (semi-preparative Zorbax 300SB-C18, 9.4 x 250 mm 5 μm; Agilent Technologies, USA). For 68 Ga]Ga-PSMA-11 with a molar activity of 30 - 60 GBq / μmol, the radiochemical yield was >97%.

[0145] c) Determination of Competitive and Direct Binding Affinity (Competition and Saturation)

[0146] For competition, two days before the assay, a monolayer of LNCaP cells was seeded at 1×10 5 cells / well in a 24-well plate. On the day of competition, after aspirating the medium, 100 μL of PBS and 100 μL of test compounds at different concentrations in PBS (10 -12 ~10 -6 M) for competition were pipetted into the wells, and simultaneously 400 μL of medium containing 1 nM of 68 Ga]Ga-PSMA-11 was added.

[0147] For saturation, two days before the assay, a monolayer of LNCaP cells was seeded at 5×10 4 cells / well in a 48-well plate. On the day of saturation, after aspirating the medium, for non-specific binding samples containing 100 μM 2-PMPA, 160 μL of medium per well (for 99m Tc-labeled radioligand (1 mg / mL) containing D-mannitol) was pipetted in. After a 5-minute pre-incubation, 40 μL of the active substance ( 68 Ga]Ga-PSMA-11 or 99m Tc-labeled radioligand solution) was added (total volume / well: 200 μL). The eight concentrations ranged from 0.3 - 40 nM.

[0148] For both the competitive and saturated samples, after incubation at 37 °C for 1 hour, the supernatant was aspirated and the cells were washed twice with cold PBS. The cell lawn was lysed by shaking with 500 μL of NaOH / SDS (0.1 M / 1%) for 3 - 5 minutes. After transferring the lysate to a measuring tube, the activity of the sample was measured with a gamma counter (2480 Automatic Gamma Counter Wizard 2, Perkin Elmer, USA).

[0149] Using a non - linear curve fitting program (GraphPad Prism 9), the half maximal inhibitory concentrations (IC50) and dissociation constants (K d ) were calculated. Using the K 68 value of d Ga]Ga - PSMA - 11 in LNCaP and the known concentrations of 68 Ga]Ga - PSMA - 11 in the competitive assay, the inhibition constant (K i ) was also determined using the curve fitting program. nat The K i values of the Re complex and 99m the K d values of the

[0150]

Table 8

[0151] d) Determination of Internalization

[0152] For internalization, 2 days before the assay, LNCaP and PC3 cells were seeded at 1×10 5 cells / well in a 24 - well plate. On the day of internalization, after aspirating the medium, 100 μL of PBS and, for non - specific binding samples, 100 μL of 2 - PMPA (100 μM) were pipetted into the wells, followed by 400 μL of medium containing the active substance. 99mThe concentration of the Tc-labeled radioactive ligand was 25 nM. After incubating at 37 °C and the adjacent well plate at 4 °C for 1 hour, the supernatant was removed and the cells were washed with cold PBS. The surface binding activity was stripped with a cold acid wash buffer (0.2 M glycine, pH 2.8) at 4 °C for 5 minutes. The acid wash buffer was transferred from the plate well to the measurement tube as was the PBS buffer (bound on the surface) after one wash. The cytoplasmic activity was determined after treatment with cell lysis buffer (0.1 M NaOH / 1% SDS). The cell surface and cytoplasmic activities were measured separately with a gamma counter. The determination of the protein content rate from the cell lysate was performed at an absorbance of 280 nm using a spectrophotometer (NanoDrop, Thermo Fisher Scientific, USA). The results are shown in Table EX-9.

[0153]

Table 9

[0154] Compared with Comparative Compound 3, the addition of substituted or unsubstituted amino acids X and Y results in an increase in the %AD / internalized per 1 mg of protein.

[0155] Example 12 SPECT / CT and PET / CT Imaging

[0156] All animal experiments were carried out in accordance with the guidelines of the German Regulations for Animal Welfare and approved by the Ethical Committee for Animal Experiments of the region.

[0157] Male nude mice, 8 - 12 weeks old (Rj:NMRI-Foxn1 nu / nu, a subcutaneous injection of human LNCaP cells into the right shoulder of Janvier Labs, Le Genest-Saint-Isle, France) was used to create a prostate cancer xenograft model. When the tumor reached a diameter exceeding 6 mm, an imaging test was performed. General anesthesia was induced and maintained by inhalation of 10% (v / v) desflurane in 30 / 10% (v / v) oxygen / air. During anesthesia, the animals were continuously warmed at 37 °C.

[0158] Single-photon emission computed tomography (SPECT) for small animals was performed using a nanoSPECT / CT scanner (Mediso Medical Imaging Systems) equipped with an APT63 aperture consisting of four M3 multi-pinhole collimators. Each animal was given a single intravenous injection via a tail vein catheter of 30 MBq of 99m Tc-labeled compound supplied in 0.2 mL of Dulbecco's phosphate-buffered saline. Photon emissions were recorded using frame times of 60 seconds (1-hour scan: 40 - 70 minutes), 90 seconds (4-hour scan, 3.5 - 4.5 hours), and 320 seconds (20-hour scan, 18.5 - 21.5 hours) and were simultaneously binned within a 20% energy window of the 140.5 keV photopeak. In each SPECT scan, the corresponding CT image was recorded and used for anatomical reference and attenuation correction. The SPECT images were reconstructed using a Tera-Tomo (商標) Three-dimensional (3D) algorithm with a voxel size of 0.4 mm in the normal range, and corrections for scatter attenuation and decay were applied.

[0159] Positron emission tomography (PET) for small animals was performed using a nanoPET / CT scanner (Mediso Medical Imaging Systems). 10 MBq of a radiolabeled reference compound supplied in Dulbecco's phosphate-buffered saline 68[Ga]Ga-PSMA-11 was given as a single intravenous injection via a tail vein catheter. Emission of 511 keV annihilation photons was recorded continuously in 1:5 coincidence mode for 60 min after radiotracer injection. For each PET scan, a corresponding CT image was recorded and used for anatomical referencing and attenuation correction. Three-dimensional list-mode data were binned using an energy window of 400–600 keV. PET images from 40–60 min time frames were imaged with Tera-Tomo at a voxel size of 0.4 mm. (商標) Reconstruction was performed using a three-dimensional (3D) algorithm and corrections for random events, scattering, attenuation and decay were applied.

[0160] All images were post-processed, analyzed using ROVER (ABX) and displayed as maximum intensity projections with the indicated scaling. Three-dimensional VOIs were created by applying fixed thresholds for the delineation of tumor (30%), muscle (0%) and kidney (39%). Standardized uptake values ​​(SUV = [MBq activity detected / mL tissue] / [MBq activity injected / g body weight], mL / g) were determined and reported as maximum SUV (VOI maximum value). Time-activity curves were generated and further analyzed using Prism (GraphPad Software, San Diego CA, USA).

[0161] In LNCaP tumor-bearing mice, 99m The tumor uptake of [Tc]-labeled radioligands is shown in Figure 3 (SUV = standardized uptake value; error bars represent SEM of measurements). In contrast to internalization in cell culture (Example 11), no significant increase in tumor uptake was found for X and / or Y represented by phenylalanine or substituted phenylalanines. Surprisingly, a marked or significant increase in tumor uptake was found for X and Y represented by glutamic acid (highest uptake for A = A1, second highest uptake for A = A2).

[0162] FIG. 5 shows the results of the [ 99mThe tumor-to-muscle ratio and tumor-to-liver ratio of the [99mTc]-labeled radioligand are shown (SUV = standardized uptake value). In particular, X and Y represented by glutamic acid ( 99m [99mTc]TcO-5 [= 99m [99mTc]TcO-ABX474] and 99m [99mTc]TcO-6 [= 99m [99mTc]TcO-ABX490]) showed a marked or significant increase in the tumor-to-muscle ratio and tumor-to-liver ratio. This indicates that, in particular, 99m [99mTc]TcO-5 [= 99m [99mTc]TcO-ABX474] provides the most favorable tumor visualization of the [99mTc]-labeled radioligands of the present invention.

[0163] Figure 1 shows the functional imaging of subcutaneous LNCaP tumor xenografts in mice using 99m [99mTc]TcO-5 [= 99m [99mTc]TcO-ABX474], compared to a reference compound (maximum intensity projection; (SUV) standardized uptake value).

[0164] Figure 2 shows the tumor-to-background kinetics of 99m [99mTc]TcO-5 [= 99m [99mTc]TcO-ABX474] in LNCaP tumor-bearing mice, compared to a reference compound ((SUV) standardized uptake value, (RT) radioactive tracer).

[0165] 99m [99mTc]TcO-5 [= 99m [99mTc]TcO-ABX474] showed similar uptake in tumors and lower uptake in the kidneys compared to the reference compound, with high contrast compared to 99m [99mTc]TcO-PSMA-I&S and high image resolution compared to 68 [68Ga]Ga-PSMA-11 (see Figure 1). As a result, 99m [99mTc]TcO-5 [= 99m [99mTc]TcO-ABX474] is a reference compound 99m [99mTc]TcO-PSMA-I&S and​68 Compared with Ga-PSMA-11, it showed higher tumor-to-muscle and tumor-to-kidney ratios within the first 4 hours after injection (see Figure 2). These results 99m Tc]TcO-5 [= 99m Tc]TcO-ABX474] has been shown to result in improved tumor visualization compared to the reference compound. Blockade of radioactive tracer uptake in tumors by co-administration of 1.5 mg of 2-PMPA confirmed PSMA-specific binding (see Figure 2).

[0166] In Figure 4, 99m Tc]TcO-5 [= 99m Tc]TcO-ABX474] also showed higher tumor-to-liver ratios within the first 1 hour after injection compared to the reference compounds 99m Tc]TcO-PSMA-I&S and 68 Ga]Ga-PSMA-11.

[0167] List of Abbreviations Ac Acetate ACN Acetonitrile AD Applied dose Boc tert-Butoxycarbonyl Boc 2 O Di-tert-butyl dicarbonate Bp Boiling point CT Computed tomography 2-CTC 2-Chlorotrityl chloride DCM Dichloromethane Dde N-(1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl) DIPEA N,N-Diisopropylethylamine DMF Dimethylformamide DPBS Dulbecco's phosphate-buffered saline EDT 1,2-Ethanedithiol EDTA Ethylenediaminetetraacetic acid Et 2 O Diethyl ether FCS Fetal Calf Serum Fmoc Fluorenylmethyloxycarbonyl HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HOAt 1-Hydroxy-7-azabenzotriazole HYNIC Hydrazinonicotinic acid IPA 2-Propanol LNCaP Lymph Node Carcinoma of the Prostate (Human Prostate Cancer Cell Line) logD Partition Coefficient NaOAc Sodium Acetate NMM 4-Methylmorpholine NHS N-Hydroxysuccinimide PBS Phosphate Buffered Saline PC3 Human Prostate Cancer Cell Line PET Positron Emission Tomography 2-PMPA 2-(Phosphonomethyl)pentanedioic acid PSMA Prostate-Specific Membrane Antigen PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RLT Radioactive Ligand Therapy r.t. Room Temperature RP-HPLC Reverse Phase High Performance Liquid Chromatography RPMI RPMI is a growth medium for cell culture RT Radioactive Tracer SDS Sodium Dodecyl Sulfate SEM Standard Error of the Mean SPECT Single Photon Emission Computed Tomography SPPS Solid Phase Peptide Synthesis Sub Suberic Acid SUV Standardized Uptake Value tBu tert-Butyl TFA Trifluoroacetic Acid TFMSA Trifluoromethanesulfonic Acid THF Tetrahydrofuran TIS Triisopropylsilane TRIS Tris(hydroxymethyl)aminomethane TSTU O-(N-Succinimidyl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate VOI Volume of interest

Claims

1. A compound of general formula I 【Chemical 1】 [wherein A is a chelator selected from the group consisting of the following, [Chemical Formula 2] k is, independently at each occurrence, 0, 1 or 2; m is, independently at each occurrence, 1, 2, 3, 4 or 5; n is, independently at each occurrence, 0, 1, 2 or 3; p is, independently at each occurrence, 1, 2 or 3; q is, independently at each occurrence, 1, 2 or 3; u is, independently at each occurrence, 0 or 1; X and Y are substituted or unsubstituted amino acids; L is a bifunctional linker selected from the group consisting of the following [Chemical Formula 3] (wherein v, x and y are, independently of one another, 0, 1, 2 or 3, and z is 0, 1, 2, 3, 4 or 5); and R is H, methyl or ethyl].

2. The compound of general formula I according to claim 1, wherein the amino acid is substituted or unsubstituted glutamic acid, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine and substituted or unsubstituted serine.

3. The compound of general formula I according to claim 1 or 2, wherein the amino acid is substituted or unsubstituted phenylalanine, or substituted or unsubstituted glutamic acid.

4. The compound of general formula I according to any one of claims 1 to 3, wherein L is L1 or L2.

5. The compound of general formula I according to any one of claims 1 to 4, wherein A is A1 or A2.

6. The compound of general formula I according to any one of claims 1 to 5, wherein k is 1, m is 3, n is 2, p is 1 or 2, q is 1 or 2, u is 1, X and Y are independently substituted or unsubstituted phenylalanine or glutamic acid; L is L1 where v is 1, or L2 where x and y are 1, and A is A1 or A2.

7. The compound of general formula I is selected from the group consisting of the following: [Chemical Formula 4] 【Chem.】 The compound of general formula I according to any one of claims 1 to 6.

8. A complex of a compound according to any one of claims 1 to 7 as a ligand and a metal.

9. wherein the metal is ９９ｍ Tc, ９９ Tc, ９４ｍ Tc, １８６ Re, １８８ Re, ６４ Cu and ６７ an isotope selected from the group consisting of Cu, the complex according to claim 8.

10. The complex according to claim 8 or 9 for use as a medicament.

11. The complex according to claim 8 or 9 for use as a medicament for the diagnosis and treatment of diseases involving PSMA.

12. The complex according to claim 10 or 11, wherein the medicament is a radiopharmaceutical for nuclear medicine imaging or radioligand therapy.

13. A medicament comprising the complex according to claim 8 or 9 or a pharmaceutically acceptable salt thereof.

14. A method for producing the complex according to claim 8 or 9, wherein the compound according to any one of claims 1 to 7 is brought into contact with the metal.

15. The method according to claim 14, wherein the compound is brought into contact with the metal at a reaction temperature in the range of 20 to 100 °C at ambient pressure.

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