PSMA-binding dual-mode radiotracers and therapy

Abstract

To provide compositions for use in radio-diagnostics and radio-therapeutics which contain a silicone fluoride acceptor and which are, at the same time, characterized by favourable in-vivo properties.SOLUTION: The present invention relates to a compound according to formula (V) or a pharmaceutically acceptable salt thereof, optionally containing a chelated radioactive cation, wherein F is optionally 18F.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention provides a compound according to formula (V):

[0002] [ka]

[0003] or a pharmaceutically acceptable salt thereof, containing a chelated radioactive cation, or wherein F is optionally 18 F. The present invention also relates to a method for synthesizing a compound that prevents racemization during synthesis.

[0004] Many documents are cited herein, including patent applications and manufacturer's manuals. The disclosures of these documents are not considered relevant to the patentability of this invention and are incorporated herein by reference in their entirety. More particularly, all documents referenced are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Background technology]

[0005] Prostate cancer Prostate cancer (PCa) remains the most common malignant disease in men, with a high incidence and poor survival rate. Due to its overexpression in prostate cancer, prostate-specific membrane antigen (PSMA), or glutamate carboxypeptidase II (GCP II), has proven its suitability as an excellent target for the development of highly sensitive radiolabeled drugs for internal radiotherapy and imaging of PCa. Prostate-specific membrane antigen (PSMA) is an extracellular hydrolase whose catalytic center contains two zinc(II) ions with bridging hydroxide ligands. It is highly upregulated in metastatic and hormone-refractory prostate cancer, but its physiological expression has also been reported in the kidney, salivary gland, small intestine, brain, and, to a lesser extent, healthy prostate tissue. In the intestine, PSMA facilitates the absorption of folate by converting pteroylpoly-γ-glutamate to pteroylglutamate (folate). In the brain, it hydrolyzes N-acetyl-L-aspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate and glutamate.

[0006] Prostate-specific membrane antigen (PSMA) Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is highly overexpressed in prostate cancer epithelial cells. Despite its name, PSMA is also expressed to varying degrees. It is expressed in the neovasculature of a wide range of non-prostate cancers, to a large extent. Among the non-prostate cancers most commonly found to demonstrate PSMA expression are breast, lung, colorectal, and renal cell carcinoma.

[0007] The generally required structure of a PSMA-targeting molecule includes a linking unit containing a zinc-binding group (e.g., urea, phosphinate, or phosphoramidate) connected to the P1' glutamate moiety, ensuring high affinity and specificity for PSMA, which is usually further connected to an effector functional group. The effector moiety is more flexible and, to some extent, resistant to structural modifications. The entrance tunnel houses two other distinctive structural features that are important for ligand binding. The first is a mechanistic explanation for the arginine patch, a positively charged region in the wall of the entrance funnel, and the preference for a negatively charged functional group at the P1 position of PSMA. This is likely the reason for the preferred incorporation of negatively charged residues within the ligand scaffold. To our knowledge, no thorough analysis of the effect of positive charges on PSMA ligands has been performed to date. After binding, a concerted rearrangement of the arginine side chain can open a second key structure, the S1 hydrophobic accessory pocket, which has been shown to accommodate the iodo-benzyl group of several urea-based inhibitors, thus contributing to their high affinity for PSMA.

[0008] Zhang et al. developed a remote binding site for PSMA that can be used in a bidentate binding mode (Zhang et al., Journal of the American Chemical Society 132, 12711-12716 (2010)). The so-called arene-binding site is a simple structural motif formed by the side chains of Arg463, Arg511, and Trp541, and is part of the GCPII entrance lid. Binding of the arene-binding site by a distal inhibitor moiety can significantly increase inhibitor affinity for PSMA through an avidity effect. PSMA I&T was developed with the intention of interacting with PSMA in this way, even though crystal structure analysis of the binding mode is not available. The necessary feature according to Zhang et al. is a linker unit (suberic acid in the case of PSMA I&T) that facilitates an open conformation of the GCPII entrance lid, thereby enabling access to the arene-binding site. The structural composition of the linker has further been shown to have a significant impact on tumor targeting and bioactivity, as well as on imaging contrast and pharmacokinetics (Liu et al., Bioorganic & Medicinal Chemistry Letters 21, 7013-7016 (2011)), properties that are crucial for both high imaging quality and efficient targeted internal radiotherapy.

[0009] Two categories of PSMA-targeted inhibitors are currently used in clinical settings: on the one hand, tracers with chelating units for radionuclide conjugation, such as PSMA I&T or related compounds, and on the other hand, small molecules containing a targeting unit and an effector molecule.

[0010] The most commonly used agents for selective PSMA imaging are PSMA HBED-CC, PSMA-617, and PSMA I&T, which are 68 Ga(88.9% β + , E β+, max = 1.89 MeV, t 1 / 2 = 68 min). Among these, 68Ga-PSMA-HBED-CC( 68 Ga-PSMA-11) is currently considered the gold standard for PET imaging of PCa.

[0011] 18 F labeling In recent years, several groups have developed novel immunotherapeutic agents for PCa diagnosis. 18 The focus was on developing F-labeled urea-based inhibitors. 68 Ge / 68 Ga radionuclide generator -( 68 Ge;t 1 / 2 =270.8d) can be obtained from radioactive metals 68 Unlike Ga and radioactive isotopes 18 F-Fluoride (96.7% β + , E β+, max =634k eV) requires an on-site cyclotron for its production. Despite this limitation, 18 F is its half-life (t 1 / 2 = 109.8 min) is long and its positron energy The low cost offers significant advantages in terms of routine handling and image quality. Furthermore, there is potential for high volume production in cyclotrons, which should be beneficial for higher patient throughput and reduced manufacturing costs. 18 F-labeled urea-based PSMA inhibitors 18 F-DCFPyl has shown promising results in the detection of primary and metastatic PCa (Rowe et al., Molecular Imaging and Biology, 1-9 (2016)) and in comparative studies 68 Based on the structure of PSMA-617, we demonstrated its superiority over Ga-PSMA-HBED-CC (Dietlein et al., Molecular Imaging and Biology 17, 575-584 (2015)). 18A labeled analog, PSMA-1007, was recently developed, which showed comparable tumor-to-organ ratios (Cardinale et al., Journal of nuclear medicine: official publication, Society of Nuclear Medicine 58:425-431 (2017); Giesel et al., European journal of nuclear medicine and molecular imaging 43:1929-1930 (2016)). 68 A comparative study with Ga-PSMA-HBED-CC demonstrated similar diagnostic accuracy of both tracers and allowed for better assessment of the prostate. 18 Decreased urinary clearance of F-PSMA-1007 was demonstrated (Giesel et al., European Journal of Nuclear Medicine and Molecular Imaging, Vol. 44, pp. 678-688 (2017)).

[0012] 18An attractive approach for introducing F labels is the use of fluorinated silicon acceptors (SIFAs). Fluorinated silicon acceptors are described, for example, in Lindner et al., Bioconjugate Chemistry, vol. 25, pp. 738-749 (2014). To maintain the fluorinated silicon bond, the use of fluorinated silicon acceptors creates the need for sterically bulky groups around the silicon atom. This, in turn, makes the fluorinated silicon acceptor highly hydrophobic. For binding to target molecules, particularly PSMA, the hydrophobic moiety provided by fluorinated silicon acceptors can be utilized to establish interactions with radiodiagnostic or radiotherapeutic compounds with hydrophobic pockets, as described in Zhang et al., Journal of the American Chemical Society, vol. 132, pp. 12711-12716 (2010). Furthermore, the higher extent of lipophilicity introduced into the molecule prior to conjugation poses serious problems for the development of radiopharmaceuticals suitable for in vivo biodistribution, i.e., with low non-specific binding in non-target tissues.

[0013] Failure to solve the hydrophobic problem Despite many attempts, the hydrophobicity problem posed by fluorinated silicon acceptors has not been satisfactorily resolved in the prior art.

[0014] To further illustrate, Schirrmacher E. et al. (Bioconjugate Chem. 2007, Vol. 18, pp. 2085-2089) have described a highly effective labeling synthon, p-(di-tert-butylfluorosilyl)benzaldehyde ([ 18 F]SIFA-A) to differentiate 18 We synthesized F-labeled peptides, which are an example of a silicon fluoride acceptor. The SIFA technique is an unexpectedly efficient isotope 19 F- 18 F exchange in near quantitative yields at high specific activities between 225 and 680 GBq / μmol (6081-18378 Ci / mmol) without the need for HPLC purification.18 F-synthon was generated. 18 [F]SIFA-benzaldehyde was synthesized in high radiochemical yields using N It was finally used to label the terminal amino-oxy (N-AO) derivatized peptides AO-Tyr3-octreotate (AO-TATE), cyclo(fK(AO-N)RGD), and N-AO-PEG2-[D-Tyr-Gln-Trp-Ala-Val-Ala-His-Thi-Nle-NH2] (AO-BZH3, a bombesin derivative). Nevertheless, the labeled peptides are highly lipophilic (as can be captured from HPLC retention times using the conditions described herein) and therefore not suitable for further evaluation in animal models or humans.

[0015] Wangler C. et al. (Bioconjugate Chem., 2009, Vol. 20(2), pp. 317-321) describe the first SIFA-based Kit-like radiofluorination of a protein (rat serum albumin, RSA). As a labeling agent, 4-(di-tert-butyl[ 18 F]fluorosilyl)benzenethiol (Si[ 18 [F]FA-SH) was generated by simple isotope exchange with a radiochemical yield (RCY) of 40–60% and coupled directly to maleimide-derivatized serum albumin within 20–30 min with an overall RCY of 12%. This technically simple labeling procedure does not require any detailed purification steps and is a clear example of the successful application of Si-18F chemistry for in vivo imaging using PET. Time-activity curves and μPET images in mice showed that most of the activity was localized in the liver, thus demonstrating that the labeling agent is highly lipophilic, directing the in vivo probe to hepatobiliary excretion and extensive hepatic metabolism.

[0016] Wangler C. et al. (see Bioconjug Chem. 2010 Dec. 15; Vol. 21(12):2289-96) subsequently sought to overcome a major weakness of SIFA technology, the high lipophilicity of the resulting radiopharmaceuticals, by synthesizing and evaluating new SIFA-octreotate analogs (SIFA-Tyr3-octreotate, SIFA-Asn(AcNH-β-Glc)-Tyr3-octreotate, and SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate). In these compounds, hydrophilic linkers and pharmacokinetic modifiers were introduced between the peptide and the SIFA-moiety, i.e., carbohydrate and carbohydrate-added PEG linkers. As a measure of the lipophilicity of the conjugate, log P(ow) was determined, and the SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate analogs were evaluated. 3 -octreotate 0.96 and SIFA-Asn(AcNH-β-Glc)-Tyr 3 The RI for -octreotate was found to be 1.23. These results indicate that the high lipophilicity of the SIFA moiety can only be slightly compensated for by the addition of a hydrophilic moiety. The first imaging study demonstrated excessive hepatic clearance / uptake and therefore was never transferred to the first human study.

[0017] Bernard-Gauthier et al. (Biomed Res Int. 2014; 2014:454503) review the significant diversity of different SIFA species reported in the literature, ranging from small prosthetic groups and other compounds of low molecular weight to labeled peptides and, more recently, antibody molecules. Based on these data, the issue of lipophilicity of SIFA-based prosthetic groups has not yet been resolved; i.e., no methodology has been described to reduce the overall lipophilicity of SIFA-conjugated peptides to log D values ​​below approximately -2.0.

[0018] In Lindner S. et al. (Bioconjug Chem. 2014 April 16; Vol. 25(4):738-49), pegylated bombesin (PESIN) derivatives as specific GRP receptor ligands and RGD (single-letter code for arginine-glycine-aspartic acid) peptides as specific αvβ3 binders were synthesized and tagged with silicon-fluorine-acceptor (SIFA) moieties. To compensate for the high lipophilicity of the SIFA moiety, To achieve this, various hydrophilic structural modifications were introduced to reduce the log D values: SIFA-Asn(AcNH-β-Glc)-PESIN, SIFA-Ser(β-Lac)-PESIN, SIFA-Cya-PESIN, SIFA-LysMe3-PESIN, SIFA-γ-carboxy-d-Glu-PESIN, SIFA-Cya2-PESIN, SIFA-LysMe3-γ-carboxy-d-Glu-PESIN, SIFA-(γ-carboxy-d-Glu)2-PESIN, SIFA-RGD, SIFA-γ-carboxy-d-Glu-RGD, SIFA-(γ-carboxy-d-Glu)2-RGD, and SIFA-LysMe3-γ-carboxy-d-Glu-RGD. All of these peptides, which had already been modified and derivatized with the goal of reducing lipophilicity, showed log D values ​​between +2 and -1.22.

[0019] Niedermoser S. et al. (J Nucl Med. 2015 July; 56(7):1100-5) reported a newly developed 18 F-SIFA- and 18 F-SIFAlin- (SIFA = silicon fluoride acceptor) modified TATE derivatives are the current clinical gold standard for high-quality imaging of somatostatin receptor-bearing tumors. 68 For this purpose, 18 F-SIFA-TATE and two fairly complex analogues, 18 F-SIFA-Glc-PEG1-TATE, 18 F-SIFAlin-Glc-Asp2-PEG1-TATE was developed. None of these drugs showed logD<-1.5. [Prior art documents] [Non-patent literature]

[0020] [Non-Patent Document 1] Zhang et al., Journal of the American Chemical Society 132, 12711–12716 (2010) [Non-patent document 2] Liu et al., Bioorganic & medicinal chemistry letters, vol. 21, pp. 7013-7016 (2011) [Non-patent document 3] Rowe et al., Molecular Imaging and Biology, 1–9 (2016) [Non-patent document 4] Dietlein et al., Molecular Imaging and Biology 17, 575-584 (2015) [Non-Patent Document 5] Cardinale et al., Journal of nuclear medicine: official publication, Society of Nuclear Medicine, Vol. 58, pp. 425-431 (2017) [Non-patent document 6] Giesel et al., European Journal of Nuclear Medicine and Molecular Imaging, Vol. 43, 1929-1930 (2016) [Non-Patent Document 7] Giesel et al., European Journal of Nuclear Medicine and Molecular Imaging, Vol. 44, pp. 678-688 (2017) [Non-patent document 8] Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014) [Non-Patent Document 9] Schirrmacher E. et al. (Bioconjugate Chem. 2007, Vol. 18, 2085-2089) [Non-Patent Document 10] Wangler C. et al. (Bioconjugate Chem., 2009, Vol. 20(2), pp. 317-321) [Non-Patent Document 11] Wangler C. et al. (Bioconjug Chem. 2010, December 15; Vol. 21(12): 2289-96) [Non-Patent Document 12] Bernard-Gauthier et al. (Biomed Res Int. 2014;2014:454503) [Non-Patent Document 13] Niedermoser S. et al. (J Nucl Med. 2015 July; 56(7):1100-5) Summary of the Invention [Problem to be solved by the invention]

[0021] In view of the above, the technical problem underlying the present invention can be stated in providing radiodiagnosis and radiotherapy agents which contain fluorosilicone acceptors and which at the same time are characterized by favorable in-vivo properties.

[0022] As will become apparent below, the present invention has established proof of principle using specific conjugates that bind with high affinity to prostate-specific antigen (PSMA) as a target. Thus, a further technical problem underlying the present invention can be presented in providing improved radiotherapy and radiodiagnosis for the medical indication of cancer, preferably prostate cancer.

[0023] PCT / EP2018 / 070533 discloses a genus of PSMA-binding compounds. Disclosed herein is an advantageous subset of compounds from the earlier application. The application herein is a selection of advantageous features not recognized by the inventors at the time of filing PCT / EP2018 / 070533. [Means for solving the problem]

[0024] These technical problems are solved by the subject matter of the claims. Thus, in some embodiments, the present invention provides a compound according to formula (V):

[0025] [ka]

[0026] or a pharmaceutically acceptable salt thereof, containing a chelated radioactive cation, or wherein F is optionally 18 F. The disclosed compounds may be in the form of a salt. The present invention also provides compounds of formula (Va):

[0027] [ka]

[0028] or a pharmaceutically acceptable salt thereof, Each X is independently OH or O - and; M is either a chelated radioactive cation or is absent; F is, in some cases, 18 F].

[0029] In addition, the use of chelating agents and 18 By a combination of isotope exchange at SIFA with F-fluoride, by on-site cyclotron or by transport from the cyclotron center. 18 F-fluoride is obtained at the center, 18 F][ nat Ion] tracer, while providing a "pair" diagnostic tracer that can be used either as a 18In centers where F-fluoride is not available but radioisotope generators, e.g., Ge-68 / Ga-68 generators, corresponding versions, e.g., nat F][ 68 A [Ga] tracer can be used.

[0030] Importantly, in both cases, chemically identical radiopharmaceuticals are injected, and therefore differences in in vivo behavior are not expected. On the other hand, chemical differences currently exist between different patient cohorts at a given site. 18 Clinical data on F-labeled compounds will be provided by other groups at other sites. 68 While clinical data for Ga-analogues cannot be directly compared, radiopharmaceuticals and / or diagnostics according to the present invention can be directly compared, thus making it possible to link such data (e.g., data obtained from a center working in Europe with F-18 and another center working in India with Ga-68).

[0031] Furthermore, when appropriately selected, chelates can also be used to label with therapeutic isotopes, such as beta-emitting isotopes, Lu-177, Y-90, etc., or alpha-emitting isotopes, such as Ac-225, and thus can be used for diagnostic purposes ([ 18 F][ nat Lu] tracers) and therapeutic radiopharmaceuticals ([ nat F][ 177 It is possible to extend the concept of "paired" tracers to bridge [Lu].

[0032] A further advantage of the compounds, particularly the PSMA-targeted compounds of the present invention, is their surprisingly low accumulation in the kidneys of mice when compared to other PSMA-targeted radiopharmaceuticals, such as PSMA I&T. Without wishing to be bound by any particular theory, it is believed that it is the combination of the structural element SIFA with the chelator that unexpectedly reduces accumulation in the kidneys.

[0033] In terms of lipophilicity / hydrophilicity, the logP value (sometimes also referred to as the logD value) is an established measure in the art. The term "lipophilic" refers to the strength of dissolving in a lipid solution, or being absorbed in it, or being adsorbed on a lipid-like surface or matrix. This refers to a preference for lipids (literally), or for organic or non-polar lipids, or for liquids, solutions, or surfaces that contain a small dipole moment compared to water. The term "hydrophobic" is used in this specification with an equivalent meaning. The adjectives lipophilic and hydrophobic are used in the corresponding sense to the nouns described above.

[0034] The mass flux of a molecule at the interface of two immiscible or nearly immiscible solvents is governed by its lipophilicity. The more lipophilic the molecule, the more soluble it is in the lipophilic organic phase. The partition coefficient of a molecule observed between water and n-octanol is used as the standard unit of lipophilicity. The partition coefficient P of a species A is given by P = [A] n-octanol / [A] water The partition coefficient is defined as the ratio of the total lipophilicity of the ionizable species to the total lipophilicity of the ionizable species. The commonly reported value is the logP value, which is the logarithm of the partition coefficient. When a molecule is ionizable, several different microspecies (ionized and non-ionized forms of the molecule) are in principle present in both phases. The content, which describes the total lipophilicity of the ionizable species, is the ratio D = [sum of the concentrations of all microspecies] n-octanol / [sum of concentrations of all microspecies] water The partition coefficient, D, is defined as: Analogous to logP, logD, the logarithm of the partition coefficient, is frequently reported. Often, a buffer system, such as phosphate buffered saline, is used as a substitute for water in the above-described determination of logP.

[0035] If the lipophilic character of a substituent on a first molecule is to be evaluated and / or quantitatively determined, this can be done by evaluating a second molecule corresponding to that substituent, said second molecule being obtained, for example, by cleaving the bond connecting said substituent to the rest of the first molecule and connecting the resulting free valence(s) to hydrogen(s).

[0036] Alternatively, the contribution of a substituent to the log P of a molecule can be determined: the contribution of a substituent X to the log P of a molecule RX, π X X is π X X =logP R-X -logP R-H where RH is the unsubstituted parent compound.

[0037] Values ​​of P and D greater than 1 and logP, logD, and π greater than 0 X X The values ​​indicate lipophilic / hydrophobic characteristics, with P and D values ​​less than 1 and logP, logD and π less than 0. X X The value indicates the hydrophilic character of the respective molecule or substituent.

[0038] The above-described parameters characterizing the lipophilicity of a lipophilic group or of the entire molecule according to the present invention can be determined by experimental means and / or predicted by calculation methods known in the art (see, for example, Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, John Wiley & Sons, Chichester. (1997)).

[0039] In a preferred embodiment, the logP value of the compounds of the present invention is between −5 and −1.5, and it is particularly preferred that the logP value is between −3.5 and −2.0. In a preferred embodiment, the chelating group comprises a chelated cation that is radioactive, more preferably a chelated radioactive metal isotope.

[0040] Preferred examples of cations that can be chelated by the chelating group are: 43 Sc, 4 4 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co,52 Fe, 56 Ni, 57 Ni, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga 68 Ga, 89 Zr, 90 Y, 89 Y, <Tc、 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 165 Dy, 169 Er, 169 Yb, 175 Yb, 172 Tm, 177 Lu, 186 Re, 188 Re, 191 Pt, 197 Hg, 198 Au, 199 Au, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th cation, 18 F or cationic molecules containing cations, e.g. 18 F-[AlF] 2+ etc.; more preferably, 44 Sc,47 Sc, 64 Cu, 67 Cu, 68 Ga, 90 Y, 111 In, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th cation or 18 It is a cationic molecule containing F. The cation can be selected from Lu-177, Y-90, or Ac-225.

[0041] In a further aspect, the present invention provides a pharmaceutical composition comprising or consisting of one or more compounds of the invention as disclosed hereinabove. In a further aspect, the present invention provides a diagnostic composition comprising or consisting of one or more compounds of the invention as disclosed hereinabove.

[0042] In a further aspect, the present invention provides therapeutic compositions comprising or consisting of one or more compounds of the invention as disclosed hereinabove. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, and / or diluent. Examples of suitable pharmaceutical carriers, excipients, and / or diluents are well known in the art and include phosphate-buffered saline, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated using well-known, conventional methods. These pharmaceutical compositions can be administered to a subject at an appropriate dose. The appropriate composition can be administered in different ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, intradermally, intranasally, or intrabronchially. It is particularly preferred that the administration be by injection and / or delivery to a site in the pancreas, a cerebral artery, or directly into brain tissue. The composition can also be administered directly to the target site, for example, by biolistic delivery to an external or internal target site, such as the pancreas or brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concomitantly. The pharmaceutically active substance can be present in an amount between 0.1 ng and 10 mg / kg of body weight per dose; however, doses below or above this exemplary range are envisioned, particularly considering the factors mentioned above.

[0043] In a further aspect, the present invention provides one or more compounds of the invention as disclosed hereinabove for use in medicine. A preferred use in medicine is in nuclear medicine, such as nuclear diagnostic imaging, also designated nuclear molecular imaging, and / or targeted radiotherapy of diseases with overexpression, preferably PSMA, in affected tissues.

[0044] In a further aspect, the present invention provides a compound of the present invention as defined hereinabove for use in a method for diagnosing and / or staging cancer, preferably prostate cancer.Prostate cancer is not the only cancer that expresses PSMA.Non-prostate cancers known to demonstrate PSMA expression include breast cancer, lung cancer, colorectal cancer, and renal cell carcinoma. Thus, any compound described herein having a PSMA-binding moiety can be used in the diagnosis, imaging, or treatment of cancers that have PSMA expression.

[0045] Preferred applications include the detection or staging of cancer, including but not limited to high-grade glioma, lung cancer, and particularly prostate cancer and metastatic prostate cancer, the detection of metastatic disease in patients with intermediate-risk to high-risk primary prostate cancer, and the detection of metastatic sites, as well as low serum PSA levels in patients with biochemically recurrent prostate cancer. Another preferred indication is the imaging and visualization of neoangiogenesis.

[0046] With respect to medical indications for which therapy, particularly radiation therapy, is desired, cancer is a preferred indication, with prostate cancer being a particularly preferred indication. In a further aspect, the present invention provides a compound of the invention, as defined herein above, for use in a method of diagnosing and / or staging cancer, preferably prostate cancer.

[0047] The present disclosure further relates to the following: Compounds according to formula (V):

[0048] [ka]

[0049] or a pharmaceutically acceptable salt thereof, containing a chelated radioactive cation, or wherein F is optionally 18 F, the compound. The compound can include a chelating cation selected from the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Er, and Th. The chelating cation can be radioactive. The chelating radioactive cation can be any radioactive isotope(s) of gallium, erbium, copper, scandium, lutetium, or yttrium.

[0050] Compounds of formula (Va):

[0051] [ka]

[0052] or a pharmaceutically acceptable salt thereof, Each X is independently OH or O - and; M is a chelated radioactive cation or is absent; F is, in some cases, 18 F].

[0053] In the compound of formula (Va), X can be OH. - When M is present, one or more of the X groups can be chelated to M. In the compound of formula (Va), M can be a chelated radioactive cation. M can be absent. M can be a radioactive cation chelated to one or more X groups. M can be a radioactive cation chelated to one or more N atoms. M can be a radioactive cation chelated to one or more N atoms or one or more X groups. M can be a radioactive cation chelated to one or more N atoms and one or more X groups.

[0054] In the compounds of formula (V) or (Va), F is 18 F can be 19 It can be F. In the compounds of formula (V) or (Va), the chelated radioactive cation is 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co, 52 Fe, 56 Ni, 57 Ni, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga 68 Ga, 89 Zr, 90 Y, 89 Y, <Tc、 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In, 111 In, 113m In, 114m In, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 165 Dy, 169 Er, 169 Yb, 175 Yb, 172 Tm, 177 Lu, 186 Re, 188 Re, 191 Pt, 197 Hg, 198 Au, 199 Au, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra,225 Ac, 227 Th cation, 18 F or cationic molecules containing cations, e.g. 18 F-[AlF] 2+ etc.; more preferably, 44 Sc, 47 Sc, 64 Cu, 67 Cu, 68 Ga, 90 Y, 111 In, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th cation or 18 The chelated radioactive cation can be selected from cationic molecules containing F. The chelated radioactive cation can be selected from cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Er, and Th. The chelated radioactive cation can be Ga. The chelated radioactive cation can be Lu-177, Y-90, or Ac-225.

[0055] In the compound of formula (Va), M is 43 Sc, 44 Sc, 47 Sc, 51 Cr, 52m Mn, 58 Co, 52 Fe, 56 Ni, 57 Ni, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga 68 Ga, 89 Zr, 90 Y, 89 Y, <Tc、 99m Tc, 97 Ru, 105 Rh, 109 Pd, 111 Ag, 110m In,111 In, 113m In, 114m In, 117m Sn, 121 Sn, 127 Te, 142 Pr, 143 Pr, 149 Pm, 151 Pm, 149 Tb, 152 Tb, 155 Tb, 161 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 165 Dy, 169 Er, 169 Yb, 175 Yb, 172 Tm, 177 Lu, 186 Re, 188 Re, 191 Pt, 197 Hg, 198 Au, 199 Au, 212 Pb, 203 Pb, 211 At, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th cation, 18 F or cationic molecules containing cations, e.g. 18 F-[AlF] 2+ etc.; more preferably, 44 Sc, 47 Sc, 64 Cu, 67 Cu, 68 Ga, 90 Y, 111 In, 161 Tb, 166 Ho, 177 Lu, 188 Re, 212 Pb, 212 Bi, 213 Bi, 225 Ac, and 227 Th cation or 18M can be selected from cationic molecules containing F. M can be selected from cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Er, and Th. M can be Ga. M can be Lu-177, Y-90, or Ac-225.

[0056] It is advantageous to synthesize a compound with as few isomers as possible. These isomers can be separated and only a single isomer can be used in vivo; therefore, the incorrect isomer is simply discarded and not used. Therefore, conditions that minimize racemization or inversion of any of the defined asymmetric centers are ideally avoided.

[0057] Again, it is stated that a) a compound of formula (I):

[0058] [ka]

[0059] with a compound of formula (II):

[0060] [ka]

[0061] to produce a compound of formula (III):

[0062] [ka]

[0063] wherein PG1 is tBu, PG2 is Fmoc, and PG3 is Dde; the reaction conditions involve the use of a base, the base being 2,4,6-collidine or 2,6-dimethylpyridine; b) a compound of formula (IV):

[0064] [ka]

[0065] reacting a compound of formula (III) under conditions suitable to form c) Compound (V):

[0066] [ka]

[0067] reacting a compound of formula (IV) under conditions suitable to form A method for producing a compound comprising: The method according to the present invention includes a method in which compound (II) is preactivated by reacting it with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU), 1-hydroxy-7-azabenzotriazole (HOAt), and 2,4,6-collidine prior to reaction with compound (I). The preactivation is carried out within 5 minutes.

[0068] The use of 2,4,6-collidine or 2,6-dimethylpyridine as the base, along with a short activation time, helps minimize racemization of the activated chiral compound (II) when compared to other nitrogenous bases, such as DIPEA. The more sterically hindered base does not abstract the acidic proton at the asymmetric center of the acid, thus reducing racemization prior to coupling to the amine.

[0069] Disclosed herein is a compound of formula (VI):

[0070] [ka]

[0071] or a pharmaceutically acceptable salt thereof. Where a chelated metal is shown, the chelated acid group is represented merely by COO - The equivalent fourth acid may also be partially chelated and therefore need not be literally COOH.

[0072] Disclosed herein is a compound of formula (VII)

[0073] [ka]

[0074] or a pharmaceutically acceptable salt thereof. Disclosed herein are pharmaceutical or diagnostic compositions comprising a compound of formula (V) or (VI). The conjugate, compound, or composition can be used as a cancer diagnostic or imaging agent.

[0075] Disclosed is a method of imaging and / or diagnosing cancer comprising administering to a patient in need thereof a conjugate, compound or composition according to formula (V) or (VI).

[0076] Disclosed is a conjugate, compound or composition according to formula (V) or (VI) for use in the treatment of cancer. Disclosed are conjugates, compounds or compositions according to formula (V) or (VI) for use in the diagnosis, imaging or prevention of angiogenesis / vasculogenesis.

[0077] Disclosed is a conjugate, compound or composition according to formula (V) or (VI) for use as a cancer diagnostic or imaging agent or for use in the treatment of cancer, wherein the cancer is prostate, breast, lung, colorectal or renal cell carcinoma.

[0078] The diagram is illustrated as follows: [Brief explanation of the drawings]

[0079] [Figure 1] FIG. 1 shows exemplary correlations of determinations of nine reference materials in OriginPro 2016G. [Figure 2a] Quality control of [19F][natGa]-rhPSMA7-rac ([19F][natGa]D / L-Dap-R / S-DOTAGA-rhPSMA-7-rac), (batch 10, precursor for the production of [18F][natGa]D / L-Dap-R / S-DOTAGA-rhPSMA-7) at the Department of Nuclear Medicine, TUM. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B in 0-40 min, 95-95% B in 40-45 min, 35-35% B in 45-50 min; flow rate: 1 mL / min; column: Nucleosil 100-5 C18, 125 x 4.6 mm; sample: 10 μL of 1 mM (DMSO). [Figure 2b] Peak assignment: D-Dap-R-DOTAGA-rhPSMA-7.1;rhPSM7-rac from Figure 2a co-injected with enantiopure D-Dap-R-DOTAGA-rhPSMA-7.1. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B (0-40 min), 95-95% B (40-45 min), 35-35% B (45-50 min); Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 2c] HPLC profile of D-Dap-R-DOTAGA-rhPSMA-7.1. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B in 0-40 min, 95-95% B in 40-45 min, 35-35% B in 45-50 min; Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 3a]Peak assignment: L-Dap-R-DOTAGA-rhPSMA-7.2;rhPSM7-rac from Figure 2a co-injected with enantiopure L-Dap-R-DOTAGA-rhPSMA-7.2. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B (0-40 min), 95-95% B (40-45 min), 35-35% B (45-50 min); Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 3b] HPLC profile of L-Dap-R-DOTAGA-rhPSMA-7.2. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B in 0-40 min, 95-95% B in 40-45 min, 35-35% B in 45-50 min; Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 4a] Peak assignment: D-Dap-S-DOTAGA-rhPSMA-7.3;rhPSM7-rac from Figure 2a co-injected with enantiopure D-Dap-S-DOTAGA-rhPSMA-7.3. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B (0-40 min), 95-95% B (40-45 min), 35-35% B (45-50 min); Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 4b] HPLC profile of D-Dap-D-DOTAGA-rhPSMA-7.3. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B in 0-40 min, 95-95% B in 40-45 min, 35-35% B in 45-50 min; Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 5a]Peak assignment: L-Dap-S-DOTAGA-rhPSMA-7.4; rhPSM7-rac obtained from Figure 2a co-injected with enantiopure D-Dap-S-DOTAGA-rhPSMA-7.3. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B (0-40 min), 95-95% B (40-45 min), 35-35% B (45-50 min); Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 5b] HPLC profile of L-Dap-D-DOTAGA-rhPSMA-7.4. HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA. Gradient: 25-35% B in 0-40 min, 95-95% B in 40-45 min, 35-35% B in 45-50 min; Flow rate: 1 mL / min. Column: Nucleosil 100-5 C18, 125 x 4.6 mm. Sample: 10 μL of 1 mM DMSO. [Figure 6a] Figure 1 shows the binding affinity (IC50 [nM]) of rhPSMA7.1 and 7.2 to PSMA. The affinity was determined using LNCaP cells (150,000 cells / well) and (4-[I]iodobenzoyl) KuE ([I]IB-KuE; c = 0.2 nM) as the radioligand (1 h, 4°C, HBSS + 1% BSA). Data are presented as mean ± SD (n = 3 in three different experiments). [Figure 6b-1] Figure 6 shows the determination of the binding affinity [nM] of rhPSMA7 isomers to PSMA. Each of the four columns shows an individual affinity measurement for rhPSAM7.1 (left) through rhPSMA7.4 (right). Conditions are as described in the legend to Figure 6a. [Figure 6b-2] Figure 6b continued. [Figure 6b-3] Figure 6b continued. [Figure 7]Figure 6 shows a depiction of the individual IC50 [nM] measurements shown in Figures 6a and 6b. Value number 5 for rhPSMA7.1 has been omitted for the conditions described in the legend to Figure 6a. [Figure 8] Figure 1 shows depiction of individual internalization measurements [% of [I]IB-KuE]. Internalized activity (c = 0.5 nM) at 1 h as % of the reference ligand ([I]I-BA) KuE (c = 0.2 nM) determined in LNCaP cells (37 °C, DMEM F12 + 5% BSA, 125,000 cells / well). Data are corrected for nonspecific binding (10 μmol PMPA) and are presented as mean ± SD (n = 3). [Figure 9] FIG. 1 depicts the individual measurements of logP's of rhPSMA isomers. [Figure 10] Figure 1 shows the biodistribution (%ID / g) of 18F-labeled rhPSMA tracer in LNCaP tumor-bearing SCID mice at 1 h pi. Data are expressed as mean ± SD (n = 4 for rhPSMA7.1, n = 5 for 7.2, n = 4 for 7.3, n = 5 for 7.4, and n = 3 for 7-rac). [Figure 11] Figure 1 shows the biodistribution [%ID / g] of 18F-rhPSMAs co-injected with PMPA (8 mg / kg) at 1 h pi in LNCaP tumor-bearing SCID mice. Data are expressed as mean ± SD (n=3). [Figure 12] Figure 1 shows possible species generated by metabolic cleavage of the amide bond: iL: cleavage forming species with increased lipophilicity; DF: defluorinated; nd: not radioactive and therefore not detectable. [Figure 13] Left: Graphical analysis of overlapping peaks 1 (rhPSMA7.2) and 2 (rhPSMA7.3); Right: Deconvolution and integration of peak profiles with Systat PeakFit software); Top: Fitted experimental data; Bottom: Deconvoluted single peak. [Figure 14a]Figure 1 shows quantification of relative change (% change of injected racemic mixture) to assess the reproducibility of conventional integration (by HPLC program) and deconvolution (by "PeakFit") of peak 4 (rhPSMA7.1). Both methods demonstrate similar performance for this peak. [Figure 14b] Figure 1 shows quantification of relative change (% change of injected racemic mixture) to assess the reproducibility of conventional integration (by HPLC program) and deconvolution (by "PeakFit") of peak 3 (rhPSMA7.4). Both methods demonstrate similar performance for this peak. [Figure 15] Figure 16 shows the percentage change of each rhPSAM7.1-7.4 isomer in blood, liver, kidney, tumor, and urine relative to its proportion in the injection solution ([F][natGa]rhPSMA7-rac). Data are expressed as mean ± SD (n=4; also see Figure 16). [Figure 16] Figure 1 shows the percentage change of each rhPSAM7.1-7.4 isomer for each sample and experiment in blood, liver, kidney, tumor, and urine relative to its proportion in the injection solution ([F][natGa]rhPSMA7-rac). Analysis was performed with Systat PeakFit. [Figure 17] Left: TLC scanner profile of a TLC plate containing liver samples (30.07.2018, total cts: 142 cts). Datasets with cts < 200 were removed due to their poor statistics and limited validity. Right: Photoimage of a TLC plate containing liver samples showing the long tail of the mobile tracer. [Figure 18] Left: TLC scanner profile of a TLC plate containing quality control samples (01.08.2018, total cts: 384). Right: Exemplary photographic images of TLC plates with urine, kidney, liver, tumor, blood and QK samples. [Figure 19]Radio-TLC of [F-18]rhPSMA7-rac (30.07.2018) as part of quality control in the nuclear medicine department before clinical application of the tracer. It is noted that tailing of the tracer is further observed in the formulation buffer (thus in the absence of protein). [Figure 20] FIG. 1 shows quantification of free [F-18]fluoride and "unchanged" [F-18]rhPSMA7-rac by radio-TLC of urine samples (30.07.2018). [Figure 21-1] Left: Radio-HPLC analysis of pooled urine from four normal mice injected with each [F-18]rhPSAM-7.x tracer. Right: Radio-HPLC analysis of "cold" urine spiked with each [F-18]rhPSAM-7.x tracer over 1 h (7.1, 7.2), 0.5 h (7.3), and 2 h (7.4). HPLC conditions: Solvent A: H2O + 0.1% TFA; Solvent B: MeCN + 0.1% TFA; Gradient: Isocratic 5% B 0-3 min, 25-35% B 3-43 min, 95-95% B 43-48 min; Flow rate: 1 mL / min; Column: Nucleosil 100-5 C18, 125 x 4.6 mm. [Figure 21-2] Figure 21 continued. [Figure 22]Separation of radioactive species in urine by cartridge immobilization and TLC. Top: Radio-HPLC analysis of mouse urine 30 min pi for [F-18]rhPSMA7.3, showing a small fraction at 1.6 min and unchanged tracer at approximately 34.5 min. Bottom (left): Mouse urine 30 min pi for [F-18]rhPSMA7.3 was diluted and subjected to STRATA-X cartridge immobilization. The cartridge was washed and eluted with MeCN / water (60 / 40 v / v + 1% TFA); only unchanged tracer was detected. Bottom (right): The breakthrough obtained from cartridge immobilization (unretained components) and the final fraction eluted from the cartridge with MeCN / water were analyzed by TLC (bottom, right). Only 96.1% [F-18]rhPSMA7.3 and 3.9% [F-18]fluoride were found in the eluate of the cartridge, and the opposite ratio was found at breakthrough of the cartridge (only 3.4% [F-18]rhPSMA7.3 and 96.6% [F-18]fluoride). [Figure 23] Fresh, non-radioactive mouse [F-18]rhPSMA7.3 was added to the urine, followed by 0.5 μmol of cold F-19-fluoride; incubation was continued for 2 h. Radioactivity was completely converted (98.5%) to a highly hydrophilic fraction representing [F-18]fluoride (peak at 1.6 min). Note: The peak at 1.6 min was subsequently immobilized on a QMA cartridge and eluted with NaCl (1 M) (= fluoride). [Figure 24] Figure 1 shows the clinical biodistribution and uptake in tumor lesions of 18F-rhPSMA-7 (left) and 18F-rhPSMA-7.3 (right) as demonstrated by SUVmax. Data are presented as mean ± SD. [Figure 25] 1 shows the clinical biodistribution and uptake in tumor lesions of 18F-rhPSMA-7 (left) and 18F-rhPSMA-7.3 (right) as demonstrated by SUV average. Data are presented as mean ± SD. [Figure 26]Figure 1 shows the clinical biodistribution and uptake in tumor lesions of 18F-rhPSMA-7 (left) and 18F-rhPSMA-7.3 (right), as demonstrated by the SUVmax-to-background ratio. Data are presented as mean ± SD. [Figure 27] Figure 1 shows the clinical biodistribution and uptake in tumor lesions of 18F-rhPSMA-7 (left) and 18F-rhPSMA-7.3 (right), as demonstrated by the SUV mean-to-background ratio. Data are presented as mean ± SD. [Figure 28] FIG. 1 shows two clinical cases of 18F-rhPSMA-7.3 PET-imaging. DETAILED DESCRIPTION OF THE INVENTION

[0080] These examples illustrate the present invention. [Example]

[0081] Example 1: Materials and Methods Fmoc-(9-fluorenylmethoxycarbonyl) and all other protected amino acid analogs were purchased from Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). Trityl chloride polystyrene (TCP) resin was obtained from PepChem (Tübingen, Germany). The chelating agents DOTAGA-anhydride, (R)-DOTA-GA(tBu)4, and (S)-DOTA-GA(tBu)4 were supplied by Chematech (Dijon, France). All necessary solvents and other organic reagents were purchased from Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany), or VWR (Darmstadt, Germany). Solid-phase peptide synthesis was performed manually using an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). Analytical and preparative reversed-phase high-pressure chromatography (RP-HPLC) were performed using a Shimadzu gradient system (Shimadzu Deutschland GmbH, Neufahrn, Germany) equipped with an SPD-20A UV / Vis detector (220 nm, 254 nm), respectively. A Nucleosil 100 C18 (125 × 4.6 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany) was used for analytical measurements at a flow rate of 1 mL / min. The specific gradient and corresponding retention time t Rare cited in the text. Preparative HPLC purification was performed using a Multospher 100 RP 18 (250 × 10 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany) at a constant flow rate of 5 mL / min. Analytical and preparative radioactive RP-HPLC were performed using a Nucleosil 100 C18 (5 μm, 125 × 4.0 mm) column (CS GmbH, Langerwehe, Germany). The eluents for all HPLC runs were water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. Electrospray ionization-mass spectra for characterization of the materials were obtained using the expression L NMR spectra were acquired on a CMS mass spectrometer (Advion Ltd., Harlow, UK). NMR spectra were recorded on a Bruker AVHD-300 or AVHD-400 spectrometer at 300 K. pH values ​​were measured with a SevenEasy pH-meter (Mettler Toledo, Giessen, Germany).

[0082] Synthesis Protocol 1) Solid-phase peptide synthesis following the Fmoc strategy TCP-Resin Load (GP1) Trityl chloride polystyrene (TCP) resin was loaded with Fmoc-protected amino acids (AA) by stirring a solution of TCP resin (1.95 mmol / g) and Fmoc-AA-OH (1.5 equiv.) in anhydrous DCM with DIPEA (4.5 equiv.) at room temperature for 2 h. The remaining trityl chloride was capped by adding methanol (2 mL / g resin) for 15 min. The resin was subsequently filtered, washed with DCM (2 × 5 mL / g resin), DMF (2 × 5 mL / g resin), and methanol (5 mL / g resin), and dried in vacuo. The final loading of Fmoc-AA-OH was determined by the following equation:

[0083]

number

[0084] After resin amide bond formation (GP2) For conjugation of the building blocks to resin-bound peptides, a mixture of TBTU and HOBT was used as a base in DMF (10 mL / g resin) for 5 min prior to activation with DIPEA or 2,4,6-trimethylpyridine. The exact stoichiometry and reaction time for each conjugation step are given in the synthesis protocol. After the reaction, the resin was washed with DMF (6 × 5 mL / g resin).

[0085] Resin Fmoc-after deprotection (GP3) The resin-bound Fmoc-peptide was treated with 20% piperidine in DMF (v / v, 8 mL / g resin) for 5 min, followed by 15 min. Afterwards, the resin was washed extensively with DMF (8 × 5 mL / g resin).

[0086] After resin Dde-deprotection (GP4) The Dde-protected peptide (1.0 equiv.) was dissolved in 2% hydrazine monohydrate in DMF (v / v, 5 mL / g resin) and shaken for 20 min (GP4a). In the case of the Fmoc group, Dde-deprotection was carried out by adding a solution of imidazole (0.92 g / g resin), hydroxylamine hydrochloride (1.26 g / g resin) in NMP (5.0 mL) and DMF (1.0 mL) for 3 h at room temperature (GP4b). After deprotection, the resin was washed with DMF (8 × 5 mL / g resin).

[0087] Peptide cleavage from resin with simultaneous deprotection of acid-labile protecting groups (GP5) The fully protected resin-bound peptide was dissolved in a mixture of TFA / TIPS / water (v / v / v; 95 / 2.5 / 2.5) and shaken for 30 min. The solution was filtered, and the resin was treated in the same manner for another 30 min. Both filtrates were combined, stirred for another 5 h, and concentrated under a stream of nitrogen. The crude peptide was obtained after dissolving the residue in a mixture of tert-butanol and water, followed by lyophilization.

[0088] natGa-complex formation (GP6) nat For Ga-conjugation, the peptide (1.0 equiv.) was dissolved in a 3:1 (v / v) mixture of tBuOH in HO, and an aqueous solution of Ga(NO) (3.5 equiv.) was added. After heating the resulting mixture at 75 °C for 30 min, the peptide was purified by RP-HPLC.

[0089] 2) Synthesis of PSMA binding motif Glu-Urea-Glu((tBuO)EuE(OtBu)2)

[0090] [ka]

[0091] The tBu-protected Glu-urea-Glu binding motif (EuE) was synthesized according to the previously published procedure for tBu-protected Glu-urea-Lys (EuK) (Scheme 1).

[0092] Di-tert-butyl(1H-imidazole-1-carbonyl)-L-glutamate (i) A solution of 2.0 g (7.71 mmol, 1.0 equiv.) of l-di-tert-butyl-L-glutamate HCl in DCM was cooled on ice for 30 min and subsequently treated with 2.69 mL (19.28 mmol, 2.5 equiv.) of TEA and 3.3 mg (0.3 mmol, 0.04 equiv.) of DMAP. After stirring for an additional 5 min, 1.38 g (8.84 mmol, 1.1 equiv.) of 1,1'-carbonyldiimidazole (CDI) dissolved in DCM was added slowly over 30 min. The reaction mixture was further stirred overnight and allowed to warm to RT. The reaction was quenched with 38 mL of saturated NaHCO3, along with washing steps with water (2x) and brine (2x), and dried over Na2SO4. The remaining solvent was removed in vacuo and the crude product (S)-di-tert-butyl 2-(1H-imidazole-1-carboxamido)pentanedioate (i) was used without further purification.

[0093] 5-Benzyl 1-(tert-butyl)(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (ii) The crude product (S)-di-tert-butyl-2-(1H-imidazole-1-carboxamido)pentanedioate (i), 2.72 g (7.71 mmol, 1.0 equiv.), was dissolved in 1,2-dichloroethane (DCE) and cooled on ice for 30 min. To this solution, 2.15 mL (15.42 mmol, 2.0 equiv.) of TEA and 2.54 g (7.71 mmol, 1.0 equiv.) of HL-Glu(OBzl)-OtBu·HCl were added, and the solution was stirred overnight at 40 °C. The remaining solvent was evaporated, and the crude product was purified using silica gel flash chromatography with an eluent mixture containing ethyl acetate / hexane / TEA (500:500:0.8; v / v / v). After removal of the solvent, 5-benzyl-1-(tert-butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (ii) was obtained as a colorless oil.

[0094] (tBuO)EuE(OtBu)2(iii) To synthesize (tBuO)EuE(OtBu)2, 3.17 g (5.47 mmol, 1.0 equiv.) of 5-benzyl-1-(tert-butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate (ii) was dissolved in 75 mL of EtOH, and 0.34 g (0.57 mmol, 0.1 equiv.) of palladium on activated carbon (10%) was added to the solution. The flask containing the reaction mixture was first purged with H2, and the solution was stirred overnight at room temperature under light H2 pressure (balloon). The crude product was purified on Celite, and the solvent was evaporated in vacuo. Product (iii) was obtained as a hygroscopic solid (84%). HPLC (B 10% to 90% in 15 min): R = 11.3 min. The calculated monoisotopic mass (C 23 H 49 N2O9): 488.3; found: m / z = 489.4 [M+H] +, 516.4[M+Na] + .

[0095] [ka]

[0096] 3) Synthesis of fluorinated silicon acceptors 4-(Di-tert-butylfluorosilyl)benzoic acid (SiFA-BA)

[0097] [ka]

[0098] SiFA-BA was synthesized according to a previously published procedure (Scheme 2). All reactions were carried out in a dry reaction vessel under argon using a vacuum gas manifold. ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane(i) To a stirred solution of 4-bromobenzyl alcohol (4.68 g, 25.0 mmol, 1.0 equiv) in anhydrous DMF (70 mL) were added imidazole (2.04 g, 30.0 mmol, 1.2 equiv) and TBDMSCl (4.52 g, 30.0 mmol, 1.2 equiv), and the resulting mixture was stirred at room temperature for 16 h. The mixture was then poured into ice-cold HO (250 mL) and extracted with EtO (5 × 50 mL). The combined organic fractions were washed with saturated aqueous NaHCO (2 × 100 mL) and brine (100 mL), dried, filtered, and concentrated in vacuo to give the crude product, which was purified by flash column chromatography (silica, 5% EtOAc / petrol) to give i as a colorless oil (7.18 g, 95%). 1 H NMR (400 MHz, CDCl3): δ [ppm] = 0.10 (6H, s, SiMe2t-Bu), 0.95 (9H, s, SiMe2tBu), 4.69 (2H, s, CH2OSi), 7.21 (2H, d), 7.46 (2H, d).HPLC (B50~100% in 15 minutes):t R=15 minutes.

[0099] Di-tert-butyl{4-[(tert-butyldimethylsilyloxy)methyl]phenyl}fluorosilane(ii) A solution of tBuLi in pentane (7.29 mL, 1.7 mol / L, 12.4 mmol, 2.4 equiv.) was added to a solution of ((4-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i) (1.56 g, 5.18 mmol, 1.0 equiv.) in dry THF (15 mL) under magnetic stirring at −78°C. After stirring the reaction mixture at −78°C for 30 minutes, the resulting suspension was added dropwise over 30 minutes to a cooled (−78°C) solution of di-tert-butyldifluorosilane (1.12 g, 6.23 mmol, 1.2 equiv.) in dry THF (10 mL). The reaction mixture was allowed to warm to room temperature over 12 h and then hydrolyzed with saturated aqueous NaCl (100 mL). The organic layer was separated, and the aqueous layer was extracted with diethyl ether (3 × 50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to give ii as a yellowish oil (1.88 g, 95%) This was used for the subsequent reaction without further purification. NMR spectra were obtained according to the literature. [2] HPLC (B 50-100% in 20 min): R =19 minutes.

[0100] 4-(di-tert-butylfluorosilanyl)benzyl alcohol (iii) A catalytic amount of concentrated aqueous HCl (0.5 mL) was added to a suspension of ii (1.88 g, 4.92 mmol, 1.0 equiv) in methanol (50 mL). The reaction mixture was stirred at room temperature for 18 h, then the solvent and volatiles were removed under reduced pressure. The residue was redissolved in diethyl ether (40 mL) and the solution was washed with aqueous NaHCO3. The aqueous layer was extracted with diethyl ether (3 x 50 mL). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to yield iii as a solidified yellowish oil (1.29 g, 98%). The product was used without further purification. NMR spectra were obtained from the literature.[2] The data reported in

[2012] were followed. HPLC (B 50-100% in 15 min): R =8.2 minutes.

[0101] 4-(Di-tert-butylfluorosilyl)benzaldehyde(iv) A solution of alcohol iii (1.37 g, 5.10 mmol, 1.0 equiv.) in dry dichloromethane (20 mL) was added dropwise to a stirred, ice-cooled suspension of pyridinium chlorochromate (2.75 g, 12.8 mmol, 2.5 equiv.) in dry dichloromethane (60 mL). After the reaction mixture was stirred at 0 °C for 30 min and at room temperature for 2.5 h, anhydrous diethyl ether (40 mL) was added and the supernatant solution was decanted from the black, gummy material. The insoluble material was washed thoroughly with diethyl ether, and the combined organic phases were passed through a short pad of silica gel (10 cm per gram of crude product) for filtration. The solvent was removed in vacuo to yield aldehyde iv as a yellowish oil (1.31 g, 96%). NMR spectra were obtained from the literature. [2] The data reported in

[2012] were followed. HPLC (B 50-100% in 15 min): R =10.5 minutes.

[0102] 4-(Di-tert-butylfluorosilyl)benzoic acid (v) At room temperature, 1 M aqueous KMnO4 (30 mL) was added to a mixture of iv (1.31 g, 4.92 mmol, 1.0 equiv.), tert-butanol (30 mL), dichloromethane (3.3 mL), and 1.25 M NaH2PO4·H2O buffer (20 mL) at pH 4.0–4.5. After stirring for 25 min, the mixture was cooled to 5 °C, and excess KMnO4 (0.78 g, 4.92 mmol, 1.0 equiv.) was added. The reaction was then quenched by adding saturated aqueous Na2SO3 (50 mL). After adding 2 M aqueous HCl, all of the MnO2 was dissolved. The resulting solution was extracted with diethyl ether (3 × 100 mL). The combined organic layers were washed with saturated aqueous NaHCO3, dried over MgSO4, filtered, and concentrated under reduced pressure to give a white solid, which was purified by recrystallization from Et2O / n-hexane (1:3, 12 h) to give v (0.84 g, 60%). NMR spectra were obtained from the literature. [2] The data reported in

[2012] were followed. HPLC (B 50-100% in 15 min): R =8.5 minutes.

[0103] [ka]

[0104] 4) Synthesis of rhPSMA-7.1 to 7.4 The first synthetic steps for the preparation of the four different isomers of rhPSMA-7 are identical and performed together. The standard Fmoc-SPPS protocol described above was applied, starting from resin-bound Fmoc-D-Orn(Dde)-OH. After cleavage of the Fmoc group with 20% piperidine in DMF (GP3), (tBuO)EuE(OtBu)2 (2.0 equiv.) was conjugated with HOAt (2.0 equiv.), TBTU (2.0 equiv.), and DIPEA (6.0 equiv.) in DMF for 4.5 h. After cleavage of the Dde-group (GP4a) with a mixture of 2% hydrazine in DMF, a DMF solution of succinic anhydride (7.0 equiv.) and DIPEA (7.0 equiv.) was added and the reaction was allowed to proceed for 2.5 h. Conjugation of Fmoc-D-Lys(OtBu)·HCl (2.0 equiv.) was achieved by adding a mixture of HOAt (2.0 equiv.), TBTU (2.0 equiv.), and DIPEA (6.0 equiv.) in DMF to the resin. After 5 min of preactivation, Fmoc-D-Lys(OtBu)·HCl (2.0 equiv.) dissolved in DMF was added and the reaction was allowed to proceed for 2.5 h (GP2). Subsequent cleavage of the Fmoc group was achieved by adding a mixture of 20% piperidine in DMF (GP3). Finally, the resin was split to synthesize rhPSMA-7.1–7.4 (Scheme 3).

[0105] rhPSMA-7.1(D-Dap-(R)-DOTA-GA):

[0106] [ka]

[0107] Fmoc-D-Dap(Dde)-OH (2.0 equiv.) was preactivated in a mixture of HOAt (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF and added to the resin-bound peptide for 2.5 h. Subsequent orthogonal Dde-deprotection was carried out using imidazole and hydroxylamine hydrochloride dissolved in a mixture of NMP and DMF for 3 h. SiFA-BA (1.5 equiv.) was reacted with the free amine of the side chain with HOAt (1.5 equiv.), TBTU (1.5 equiv.), and DIPEA (4.5 equiv.) as activating agents in DMF for 2 h. After Fmoc-deprotection with piperidine (GP3), (R)-DOTA-GA(tBu)4 (2.0 equiv.) was added to the resin-bound peptide for 2 h in DMF. The peptide was conjugated with HOAT (2.0 equiv.), TBTU (2.0 equiv.) and 2,4,6-trimethylpyridine (6.7 equiv.) for 0.5 h. Cleavage from the resin with simultaneous deprotection of the acid-labile protecting groups was carried out with TFA according to GP5. nat Ga-complexation was performed as described in GP6.

[0108] rhPSMA-7.2(L-Dap-(R)-DOTA-GA):

[0109] [ka]

[0110] Fmoc-L-Dap(Dde)-OH (2.0 equiv.) was preactivated with a mixture of HOAt (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF for 2.5 h. After orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage were performed as described for rhPSMA-7.1. (R)-DOTA-GA(tBu)4 (2.0 equiv.) was conjugated with HOAT (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of the acid-labile protecting group was performed with TFA according to GP5. The peptidenat Ga-complexation was performed as described in GP6.

[0111] rhPSMA-7.3(D-Dap-(S)-DOTA-GA):

[0112] [ka]

[0113] Fmoc-D-Dap(Dde)-OH (2.0 equiv.) was preactivated with a mixture of HOAt (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF for 2.5 h. After orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage were performed as described for rhPSMA-7.1. (S)-DOTA-GA(tBu)4 (2.0 equiv.) was conjugated with HOAT (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of the acid-labile protecting group was performed with TFA according to GP5. The peptide nat Ga-complexation was performed as described in GP6.

[0114] rhPSMA-7.4(L-Dap-(S)-DOTA-GA):

[0115] [ka]

[0116] Fmoc-L-Dap(Dde)-OH (2.0 equiv.) was preactivated with a mixture of HOAt (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF for 2.5 h. After orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage were performed as described for rhPSMA-7.1. (S)-DOTA-GA(tBu)4 (2.0 equiv.) was conjugated with HOAT (2.0 equiv.), TBTU (2.0 equiv.), and 2,4,6-trimethylpyridine (6.7 equiv.) in DMF for 2.5 h. Cleavage from the resin with simultaneous deprotection of the acid-labile protecting group was performed with TFA according to GP5. The peptide nat Ga-complexation was performed as described in GP6.

[0117] rhPSMA-7.1: HPLC (B 10-70% in 15 min): R =10.5 minutes. HPLC (B 25-35% in 40 min): R =31.4 minutes.

[0118] rhPSMA-7.2: HPLC (B 10-70% in 15 min): R =10.4 minutes. HPLC (B 25-35% in 40 min): R =27.9 minutes.

[0119] rhPSMA-7.3: HPLC (B 10-70% in 15 min): R =10.4 minutes. HPLC (B 25-35% in 40 min): R =28.1 minutes.

[0120] rhPSMA-7.4: HPLC (B 10-70% in 15 min): R =10.5 minutes. HPLC (B 25-35% in 40 min): R =29.1 minutes.

[0121] rhPSMA-7.1 to 7.4: Calculated monoisotopic mass (C63H96FGaN12O25Si): 1536.6. Found: m / z = 1539.4 [M+H]+, 770.3 [M+2H]2 + .

[0122] [ka]

[0123] 5) 18 F-labeling 18 For F-labeling, a previously published procedure was applied, which was slightly modified. 18 F - was passed through a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light), which had been preconditioned with 10 mL of water. After drying with 10 mL of air, the water was removed by rinsing the cartridge with 10 mL of anhydrous acetonitrile, followed by 20 mL of air. 18 F was dissolved in 500 μL of anhydrous acetonitrile [K + ⊂2.2.2]OH - The tracer was eluted at 100 μmol. Prior to labeling, 30 μmol of oxalic acid in anhydrous acetonitrile (1 M, 30 μL) was added. This mixture was used either as a whole or as an aliquot for the fluorination of 10–25 nmol of PSMA-SiFA (1 mM in anhydrous DMSO). The resulting reaction mixture was incubated at room temperature for 5 min. For tracer purification, the tracer was transferred to a Sep-Pak C18 light cartridge preconditioned with 10 mL of EtOH followed by 10 mL of HO. The labeling mixture was diluted with 9 mL of PBS (pH 3), passed through the cartridge, and then diluted with 10 mL of HO. The peptide was eluted with 500 μL of a 4:1 mixture of EtOH in water (v / v). The radiochemical purity of the labeled compound was confirmed by radio-RP-HPLC and radio-TLC (silica gel 60 RP-18 F 254s, determined with mobile phase: a 3:2 mixture (v / v) of MeCN in H2O supplemented with 10% 2 M aqueous NaOAc and 1% TFA).

[0124] 6) 125 I-labeling In vitro test ([ 125 The reference ligand for [I]I-BA)KuE was prepared according to a previously published procedure. Briefly, 20.1 mg of the stannylated precursor (SnBu3-BA)(OtBu)KuE(OtBu) was dissolved in 20 μL of peracetic acid and [ 125 5.0 μL (21 MBq) of [I]NaI (74 TBq / mmol, 3.1 GBq / mL, 40 mM NaOH, Hartmann Analytic, Braunschweig, Germany) was dissolved in a solution containing 20 μL of MeCN and 10 μL of acetic acid. The reaction solution was incubated at RT for 10 min, loaded onto a cartridge, and rinsed with 10 mL of water (C18 Sep Pak Plus cartridge preconditioned with 10 mL of MeOH and 10 mL of water). After elution with 2.0 mL of a 1:1 mixture (v / v) of EtOH / MeCN, the radioactive solution was evaporated to dryness under a gentle stream of nitrogen and treated with 200 μL of TFA for 30 min, followed by subsequent evaporation of the TFA. ([ 125 The crude product of [I]I-BA)KuE was purified by RP-HPLC (B 20%-40% in 20 min): R =13.0 minutes.

[0125] In vitro experiments 1) IC 50 Decision PSMA-positive LNCaP cells were grown in Dulbecco's modified Eagle's medium / Nutrition Mixture F-12 (1:1) containing Glutamax-I (Invitrigon) supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified 5% CO atmosphere. 50 For determination of ), cells were harvested 24 ± 2 hours before the experiment and plated in 24-well plates (1.5 × 10 in 1 mL / well). 5After removing the medium, the cells were treated once with 500 μL of HBSS (Hank's Balanced Salt Solution with 1% bovine serum albumin (BSA), Biochrom, Berlin, Germany) and left on ice for 15 min to equilibrate with 200 μL of HBSS (1% BSA). -10 ~10 -4 Addition of 25 μL per well of a solution containing HBSS (1% BSA, control) or the respective ligand in increasing M was followed by addition of 25 μL of ([ 125 25 μL of [I]I-BA)KuE (2.0 nM) was added. All experiments were performed at least three times for each concentration. After 60 min of incubation on ice, the experiment was terminated by removing the medium and rinsing successively with 200 μL of HBSS. The medium from both steps was combined in one fraction, representing the amount of free radioligand. Afterwards, the cells were lysed with 250 μL of 1 M NaOH and combined with 200 μL of HBSS in the next washing step. Quantification of bound and free radioligand was achieved with a γ-counter.

[0126] 2) Internalization For internalization studies, LNCaP cells were harvested 24±2 hours before the experiment and plated in 24-well plates (1.25×10 in 1 mL / well). 5 After removing the medium, the cells were washed once with 500 μL of DMEM-F12 (5% BSA) and equilibrated in 200 μL of DMEM-F12 (5% BSA) at 37°C for at least 15 minutes. Each well was treated with 25 μL of either DMEM-F12 (5% BSA) or 100 μM PMPA solution for blocking. Next, 68 Ga / 18 25 μL of F-labeled PSMA inhibitor (5.0 nM) was added, and the cells were incubated at 37°C for 60 minutes. The experiment was terminated by placing the 24-well plate on ice for 3 minutes, and the medium was subsequently removed. Each well was then washed with HBSS2 The cells were rinsed with 50 μL of ice-cold PMPA (10 μM in PBS) and the fractions obtained from these first two steps were combined and represented the amount of free radioligand. Removal of surface-bound activity was achieved by incubating the cells for 5 minutes with 250 μL of ice-cold PMPA (10 μM in PBS) solution, and again rinsed with 250 μL of ice-cold PBS. Internalized activity was determined by incubating the cells with 250 μL of 1 M NaOH and in combination with fractions from a subsequent washing step with 250 μL of 1.0 M NaOH. Each experiment (control and blocked) was performed in triplicate. Free, surface-bound, and internalized activity were quantified with a γ-counter. All internalization studies were performed using a ([ 125 This was achieved by reference experiments using [I]I-BA)KuE (c = 0.2 nM) and these were performed similarly. Data were corrected for nonspecific internalization and normalized to the specific internalization observed for the radioiodinated reference compound.

[0127] 3) Octanol-water partition coefficient Approximately 1 MBq of labeled tracer was dissolved in 1 mL of a 1:1 (by volume) mixture of phosphate-buffered saline (PBS, pH 7.4) and n-octanol in an Eppendorf tube. After vigorously mixing the suspension for 3 min at room temperature, the vial was centrifuged at 15,000 g for 3 min (Biofuge 15, Heraus Sepatech, Osterode, Germany), and 100 μL aliquots of the two layers were measured in a gamma counter. The experiment was repeated at least six times.

[0128] 4) HSA binding To determine HSA binding, a Chiralpak HSA column (50 × 3 mm, 5 μm, H13H-2433) was used at a constant flow rate of 0.5 mL / min. The mobile phase (A: 50 mM NHOAc in water, pH 7 and B: isopropanol) was freshly prepared for each experiment and used for only one day. The column was maintained at room temperature, and each run was stopped after signal detection to reduce acquisition time. All substances were dissolved at a concentration of 0.5 mg / mL in 50% 2-propanol and 50% 50 mM ammonium acetate buffer (pH 6.9). Because a wide range of albumin binding was assumed for the peptides, the selected reference substances exhibited a range of HSA binding from 13% to 99%. All nine reference substances were injected sequentially to establish nonlinear regressions in OriginPro 2016G.

[0129] [Table 1]

[0130] In vivo experiments All animal experiments must comply with the German General Animal Welfare Regulations. Animals were cultured in accordance with the animal welfare regulations and the institutional guidelines for the care and use of animals. To establish tumor xenografts, LNCaP cells (10 7 Cells (200 μL) were suspended in a 1:1 mixture (v / v) of Dulbecco's modified Eagle's medium / Glutamax-I (1:1) and Nutrition Mixture F-12 containing Matrigel (BD Biosciences, Germany) and inoculated subcutaneously into the right shoulder of 6- to 8-week-old CB17-SCID mice (Charles River, Sulzfeld, Germany). Mice were used when tumors grew to a diameter of 5-8 mm (3-4 weeks after inoculation).

[0131] 1) Distribution in the body 18 Approximately 1-2 MBq (<0.2 nmol) of F-labeled PSMA inhibitor was injected into the tail vein of male LNCaP tumor-bearing CB-17 SCID mice, which were sacrificed 1 h post-injection (n = 4-5). Selected organs were removed, weighed, and measured in a γ-counter.

[0132] 2) Metabolic testing a) Analytical equipment Analytical reversed-phase high-pressure chromatography (RP-HPLC) was performed using a Shimadzu gradient system (Shimadzu Deutschland GmbH, Neufahrn, Germany) equipped with an SPD-20A UV / Vis detector (220 nm, 254 nm). A Multospher 100 RP18 (125 × 4.6 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany) was used for analytical measurements at a flow rate of 1 mL / min. The eluents for all HPLC runs were water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. Radioactivity was measured using a HERM LB 500 detector (Berthold Technology). Detection was by connection of the UV-photometer outlet to a UV-photometer (Is GmbH, Bad Wildbad, Germany). The gradient for all HPLC runs was as follows: isocratic 5% B 0-3 min, 25-35% B 3-43 min, 95-95% B 43-48 min.

[0133] For radio-thin layer chromatography, silica gel 60 RP-18 F 254 A thin aluminum plate coated with HCl was used with a mobile phase consisting of a 3:2 mixture (v / v) of MeCN in HO supplemented with 10% 2 M aqueous NaOAc and 1% TFA. Analysis was performed using a Scan-RAM radio-TLC detector (LabLogic Systems Ltd., Sheffield, United Kingdom) or a CR 35 This was performed using a BIO phosphorimager (Duerr Medical GmbH, Bietigheim-Bissingen, Germany).

[0134] b) Determination of metabolic stability of rhPSMA-7.1 to 7.4 For in vivo μ metabolism testing, 18 8-12 MBq (<0.6 nmol) of F-labeled ligand (rhPSMA-7.1-7.4) was injected into the tail vein of healthy female CB17-SCID mice (n=4). Mice were anesthetized for 30 minutes, and urine was collected using a bladder catheter. Urine samples were pooled and centrifuged at 9000 rpm for 5 minutes to remove suspended solids. The supernatant was used directly for radio-HPLC analysis using the conditions described above. 19 F peptide bond 18 To demonstrate that isotope exchange with F occurs in urine, each compound was incubated with urine samples from healthy female CB-17-SCID mice at several time intervals, which were then analyzed by radio-HPLC and / or radio-TLC. 19 Add F (0.5 μmol) 18 F-labeled rhPSMA-7.3 was incubated for 2 h.

[0135] c) Determination of the in vivo distribution of rhPSMA-7.1 to 7.4 To quantify the relative uptake of each isomer (rhPSMA-7.1–7.4), male tumor-bearing CB-17-SCID mice were injected with a racemic mixture of rhPSMA-7 (180–280 MBq, S AThe animals were injected with 1000-247-349 GBq / μmol (produced at the Klinikum rechts der Isar (Technical University Hospital Munich) using a fully automated procedure). The animals were kept under anesthesia for 30 minutes and then sacrificed. Urine, blood, liver, kidney, and tumor samples were collected and processed as described below. Urine samples were centrifuged at 9000 rpm for 5 minutes to produce a clear solution, which was then directly subjected to radio-HPLC analysis. Blood was diluted to 1 mL with HO and centrifuged twice at 13000 g for 5 minutes. The supernatant was collected and loaded onto a Strata X cartridge (500 mg of 33 μm polymer reversed-phase cartridge, preconditioned with 5 mL of MeOH, followed by 5 mL of HO). After washing with 5 mL of HO, the cartridge was eluted with a 6:4 mixture (v / v) of MeCN in HO supplemented with 1% TFA. The eluate was diluted with water and analyzed by radio-HPLC. Tumor, kidney, and liver were homogenized using a Potter-Elvehjem tissue grinder (Kontes Glass Co, Vineland, USA) or a MM-400 ball mill (Retsch GmbH, Haan, Germany).

[0136] I) Potter Elvehjem Tissue Grinder Tumors and kidneys were homogenized separately in a tissue homogenizer with 1 mL of extraction buffer (850 μL of 1 M HEPES (pH 7.4), 100 μL of 20 mM PMPA, and 100 μL of 1 M NaCl) for 30 min. The resulting homogenates were collected and centrifuged at 13,000 g for 5 min. Subsequently, the supernatant was collected, centrifuged again (13,000 g, 5 min), and loaded onto a Strata X cartridge (500 mg of 33 μm polymer reversed-phase, preconditioned with 5 mL of MeOH, followed by 5 mL of HO). After washing with 5 mL of HO, the cartridge was washed with a 6:4 mixture (v / v) of MeCN in HO supplemented with 1% TFA. The eluate from each organ was diluted with water and analyzed by radio-HPLC.

[0137] II) MM-400 Ball Mill Organs (tumor, kidney, and liver) were homogenized separately in 2-mL tubes with three grinding balls (3 mm diameter) and 1 mL of extraction buffer (850 μL of 1 M HEPES (pH 7.4), 100 μL of 20 mM PMPA, and 100 μL of 1 M NaCl) at 30 Hz for 10 min. The homogenates were centrifuged at 13,000 g for 5 min, and the supernatants were collected. Subsequently, the pellets were suspended in 1 mL of extraction buffer and homogenized again in a ball mill at 30 Hz for 10 min. After centrifugation (13,000 g, 5 min), both supernatants were combined and loaded onto a Strata X cartridge (500 mg of 33 μm polymer reversed-phase gel, preconditioned with 5 mL of MeOH and then 5 mL of HO). After washing with 5 mL of HO, the cartridge was eluted with a 6:4 mixture (v / v) of MeCN in HO supplemented with 1% TFA. The eluate from each organ was diluted with water and analyzed by radio-HPLC. To demonstrate that breakthrough during cartridge loading was not the result of unbound F-18, the supernatant was also examined by radio-TLC after centrifugation.

[0138] Finally, the ratios of the individual isomers were determined from the HPLC profiles of the extracted samples and compared with the isomer ratios obtained from quality control of the racemic mixture of rhPSMA-7. Decay-corrected extraction and cartridge loading efficiencies, as well as the total extraction activity of the tested samples, are shown in Table 2. Cartridge elution efficiencies were >99% for all experiments. [Example]

[0139] Example 2: Results Chromatographic Peak Assignment Chromatographic peak assignments are a) rhPSMA7-rac mixture b) By comparing the UV profiles of each enantiopure rhPSMA7 compound with a co-injected rhPSMA7-rac mixture.

[0140] The following names are used to denote different isomers: rhPSMA-rac:[19 F][ nat Ga]D / L-Dap-R / S-DOTAGA-rhPSMA7 rhPSMA-7-1:[ 19 F][ nat Ga]D-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-2:[ 19 F][ nat Ga]L-Dap-R-DOTAGA-rhPSMA7 rhPSMA-7-3:[ 19 F][ nat Ga]D-Dap-S-DOTAGA-rhPSMA7 rhPSMA-7-4:[ 19 F][ nat Ga]L-Dap-S-DOTAGA-rhPSMA7.

[0141] [Table 2]

[0142] binding affinity The first set of values ​​(rhPSMA-7.1 and rhPSMA-7.2; Figure 6a) was calculated using the respective ligands nat In the second data set (Fig. 6b), the complexed ligand was compared with the uncomplexed ligand by using it for a dilution series of the solution obtained directly after Ga-complexation. nat To separate the Ga-salt, purification was performed by RP-HPLC. As no significant differences were observed, both series were combined and used to calculate the mean values ​​(±SD).

[0143] [Table 3]

[0144] [Table 4]

[0145] Internal Migration Test

[0146] [Table 5]

[0147] [Table 6]

[0148] Lipophilicity (octanol-water partition coefficient) The logP value was determined using phosphate-buffered saline (PBS, pH 7.4) and n-octanol (=logP oct / PBS ) was conducted.

[0149] [Table 7]

[0150] [Table 8]

[0151] Binding of PSMA inhibitors to human plasma proteins

[0152] [Table 9]

[0153] At 1h pi [ 18 F][ nat Biodistribution of Ga]rhPSMA7.1-7.4

[0154] [Table 10]

[0155] At 1 h pi due to competition [ 18 F][ nat Biodistribution of Ga]rhPSMA7.1-7.4

[0156] [Table 11]

[0157] [ 18 Quantification of the relative changes in the amount of each rhPSMA7.x isomer in blood, kidney, liver, urine, and tumor after application of [F]rhPSMA7-rac. [ 18 To quantify the relative changes in each rhPSMA7 isomer in blood, liver, kidney, urine, and tumor 30 minutes after injection of [F]rhPSMA7-rac into LNCaP tumor-bearing mice, two different homogenization methods (Potter and ball mill) were used to extract the tracer from kidney, liver, and tumor tissues (see Materials and Methods).

[0158] Table 12 summarizes the observed efficiency of both homogenization methods and the effectiveness of the subsequent solid phase extraction procedure (to separate the tracer from the protein fraction).

[0159] [Table 12]

[0160] Extraction of activity from samples using the potter was quite efficient, whereas the use of a ball mill was disappointing. Nevertheless, ball milling achieved extraction efficiencies of >60%.

[0161] Considering the possible species that can be formed by metabolic cleavage of the amide bond of rhPSMA7, only species a) that significantly increase the lipophilicity of F18-Fluoride b) seem plausible. Therefore, in principle, it seems possible that the "iL" species shown in Figure 12 would not be extracted from tissue samples (water extraction) and therefore would not appear in the final analysis. However, it should be noted that such species should appear in vivo in the liver and intestine (hepatobiliary excretion of lipophilic compounds) or be bound to plasma proteins (resulting in high activity levels in the blood, while demonstrating excellent extraction efficiency).

[0162] For quantification of each isomer in a racemic mixture, and particularly for the poorly resolved first and second peaks (rhPSMA 7.2 and rhPSMA 7.3), a graphical approximation was first used. This approach is based on the assumptions that a) each isomer elutes from the HPLC column with the same peak shape, and b) the different peak heights can be used as a first approximation to calculate the linearity factor for the small, resolved peaks (i.e., rhPSMA 7.2 and rhPSMA 7.3).

[0163] Based on these assumptions, the first analysis is 18 F][ nat This was performed using one LNCaP tumor-bearing mouse co-injected with [Ga]rhPSMA7-rac. The Systat software package "PeakFit" was used in three experiments to validate these experiments and to improve the graphical analysis with a more valid procedure. PeakFit allows for the automated nonlinear separation, analysis, and quantification of HPLC elution profiles by a deconvolution procedure using a Gaussian response function with a Fourier deconvolution / filtering algorithm (https: / / systatsoftware.com / products / PeakFit / ).

[0164] Comparing the graphical analysis of the first experiment revealed that the graphical analysis overestimated the second peak (rhPSMA7.3) and underestimated the first peak, therefore all data sets were reanalyzed and quantified by PeakFit.

[0165] HPLC-analysis of four independent experiments 30 min p.i. in tumor-bearing mice 1. Evaluation of Peaks 3 and 4 (rhPSMA7.4 and rhPSMA7.1) by Radio-HPLC We first examined whether deconvolution techniques would reveal similar data for the final two peaks (rhPSMA7.4 and 7.1), with good separation (albeit without baseline separation).

[0166] 2. Evaluation of all peaks (rhPSMA7.1, 7.2, 7.3, and 7.4) by radio-HPLC Figures 14a and 14b show the injection solution ([ 18 F][ nat The percentage change of each rhPSAM7.n isomer in a given sample relative to its percentage in [rhPSMA7.n]rhPSMA7-rac) is summarized; results for individual experiments are shown in Figure 14. The proportion of each isomer was quantified by analysis of the HPLC elution profile by Systat "PeakFit." The percentage change of each isomer in a given sample relative to its percentage in the injection solution was then calculated.

[0167] 3. HPLC Data Analysis Radio-HPLC analysis of radioactivity extracted from homogenized (kidney, liver, tumor) or diluted (blood) tissues and subsequently immobilized on and eluted from solid-phase extraction cartridges showed no signs of metabolic instability. Thus, no lipophilic metabolic fragments were observed. It should be noted that F-18-fluoride cannot be accurately detected by HPLC under the conditions used for sample preparation (see TLC analysis).

[0168] Although there is a clear trend with the D-Dap-derivatives rhPSMA7.1 and 7.3, the total changes are low (maximum 15%). It is also important to emphasize in this context that Figures 15 and 16 show "relative changes" without taking into account absolute uptake values.

[0169] Although rhPSMA7.1 has the weakest affinity and internalization of all rhPSMA7 compounds, it shows the greatest percentage positive changes in blood, liver, kidney, and tumor. The reason for this result is unclear, but it can be speculated that homogenization of tissue samples using a ball mill did not result in quantitative cell disruption. Therefore, the rhPSMA7 tracer, which has the highest internalization (rhPSMA7.2: 191.83% ± 15.54%, rhPSMA7.4: 207.33 ± 4.06%, and rhPSMA7.3: 161.41% ± 8.88%), may have been extracted inefficiently, while rhPSMA7.1, which has a lower internalization rate of only 69.55% ± 5.29%, was extracted efficiently and therefore overestimated in the HPLC analysis.

[0170] Furthermore, rhPSMA compounds 7.2 and 7.4 appear to be excreted somewhat more rapidly (see urine values). Compared to rhPSMA7.1, both compounds exhibit higher affinity and internalization rates, but these compounds generally exhibit negative changes in solid tissues and blood. Because no metabolites, i.e., lyophilic metabolites, were detected, it is unclear whether this may be caused by metabolic degradation of 7.2 and 7.4 (both L-Dap derivatives). However, due to their high logP values ​​(see Figure 11), such metabolites may not be extractable in aqueous buffers. In this case, they should appear in the liver (see biodistribution) and possibly in blood samples (likely due to high serum protein binding). Because no increase in activity accumulation was observed in liver tissue during biodistribution studies and activity extraction from blood was very efficient (see Table 3), we hypothesize that significant degradation of rhPSMA7.2 and 7.4 did not occur. This assumption supports the prediction of the efficacy of rhPSMA7.2 and 7.4 in humans. 18 This is supported by unquestionable SUV-values ​​for liver tissue (gallbladder, intestine) in the context of clinical use of [F]rhpsma-rac.

[0171] TLC analysis of tumor-bearing mice 30 min p.i. Radio-TLC analysis was performed a) on urine samples by applying a small amount directly to a TLC strip, b) by analyzing a small amount of non-immobilized activity during the SPE process (the "breakthrough fraction"), and c) by analyzing a small amount of cartridge eluate.

[0172] [Table 13]

[0173] Consideration of TLC data By RP-18 chromatography (due to the free Si-OH groups of the matrix interacting with nca fluoride), 18Since it is very difficult to detect F-fluoride, thin layer chromatography was performed to investigate the quantification of F-18-fluoride in the extracted solution.

[0174] Because none of the reagents and salts typically used for protein precipitation were tested for cold fluoride and to avoid possible release of F-18-fluoride from the tracer by isotope exchange—although such protein loading often limits peak separation, peak tailing, and activity stuck at the starting line—protein precipitation was not performed in the sample preparation process. The resulting solution was used directly for TLC analysis.

[0175] Although the analytically available activity was fairly low in all samples, TLC results indicated that the total F-18-fluoride content was - A urine sample obtained on July 30, 2018 (17.49% free fluoride), - With the exception of a liver sample obtained on August 2, 2018 (25.85% free fluoride), the tissues examined contained approximately 6% or less free fluoride.

[0176] While the analysis of urine by TLC is considered to be reasonable (see profile in Figure 20), the results obtained with liver samples are caused by extensive tailing of the peak representing the unchanged tracer (see Figure 18). Furthermore, due to the peak tailing obtained during QK and the release of [F-18]rhPSMA7-rac in PBS for clinical application (Figure 18), which shows tailing of the product peak, it can be concluded that the aforementioned maximum 6% free fluoride represents an overestimation. As demonstrated by phosphorimaging, this tailing is observed in almost all TLC analyses and contributes to the integrated area of ​​F-18-fluoride.

[0177] It is important to note that neither the human biodistribution study nor clinical PET scans (status as of July 2018: approximately 1400 scans with [F-18]rhPSMA7-rac) resulted in any suspected or identifiable F-18 accumulation in bone. To further investigate the release of F-18 fluoride from [F-18]rhPSMA7-rac (as observed in one urine sample), we investigated the occurrence of F-18 fluoride in additional urine samples (normal mice) by RP-18 HPLC (column with a new RP-18 endcap) and TLC analysis.

[0178] Radio-TLC-analysis of F-18-fluoride formation in normal mice at 30 min pi For this purpose, normal mice were used. Urine samples were collected via catheter over a 30-minute period. The urine was centrifuged and directly subjected to HPLC and TLC.

[0179] As shown in Figure 21, left column, free F-18-fluoride was found in urine samples of all isomers and was also formed when fresh urine was incubated with [F-18]rhPSMA7.4 (right column). Identification of F-18-fluoride was performed by demonstrating that a) this species was retained on the QMA cartridge (data not shown), b) eluted in the dead volume of the RP-18 column, and c) could not be retained or mobilized on the RP-18 column or RP-18 TLC plate, respectively, regardless of the mobile phase used.

[0180] The fact that such high doses of F-18 fluoride were not detected in HPLC analysis of blood or organs, such as kidney, tumor, liver, etc., that no increase in active uptake in bone was observed in biodistribution studies in mice, and that [F-18]rhPSMA7-rac is scheduled for clinical scanning termination in 2017 at TUM (status termination July 2018: approximately 1400 PET scans in patients with prostate cancer), and that no increase in active uptake in bone was observed during clinical PET scans with [F-18]rhPSMA7-rac compound, led the inventors to conclude that [F-18]fluoride may be formed downstream from glomerular filtration of the tracer, resulting in the formation and subsequent excretion of [F-18]fluoride without detectable uptake of F-18-fluoride in blood, organs, or bone.

[0181] This assumption is supported by a number of studies on fluoride toxicology that describe the relevant amounts of fluoride in the kidneys and urine. This finding is supported by the literature. A normal urinary fluoride level of 0.3 ppm was observed in mice (Bouaziz H et al., Fluoride 2005; 38(1): 23-31). Another publication determined that the average fluoride concentration in the urine of normal mice was 0.13-0.14 μg / mL (Poesina ND et al., Rom J Morphol Embryol 2014; 55(2): 343-349), and Inkielewicz I et al. found that the fluoride content in rat serum was approximately 5% of the concentration in the kidney (serum: 0.051 μg / mL, kidney: 0.942 μg / mL) (Fluoride; 36(4): 263-266). Considering that the majority of the tracer is absorbed, particularly by the kidney, and is also physiologically eliminated by the kidney, the elevated fluoride levels in the kidney, combined with a body temperature of 36.6°C, may result in the continuous excretion of F-18-fluoride from the rhPSMA-compound in the kidney.

[0182] Therefore, fresh and non-radioactive urine samples collected from normal mice were incubated with [F-18]rhPSMA7.x for various periods of time (see legend to Figure 21). Figure 22, right column, clearly demonstrates that ex vivo incubation of urine with [F-18]rhPSMA7.x results in the formation of free [F-18]fluoride to various extents, which is promoted by different concentrations of cold F-19-fluoride in the urine samples and increases over time.

[0183] To further support our hypothesis, we added 500 nmol of cold F-19-fluoride to fresh, non-radioactive mouse urine, followed by [F-18]rhPSMA7.3 and incubation for 2 h. According to our hypothesis, high concentrations of [F-19]fluoride should result in the formation of significant amounts of [F-18]fluoride. Figure 23 shows that under these conditions, 98.5% of the radioactivity was exchanged to form [F-18]fluoride within 2 h (Figure 23).

[0184] Since the isotope exchange rate depends on the concentrations of the four related species at equilibrium ([F-18]fluoride, [F-19]fluoride, [F-18]rhPSMA7.3 and [F-19]rhPSMA7.3), it was also investigated whether the addition of [F-18]fluoride to fresh and radioactive urine (20.6% [F-18]fluoride, 79.4% [F-18]rhPSMA7.3) followed by the addition of cold [F-19]rhPSMA7.3 tracer would also label the radiopharmaceutical [F-18]rhPSMA7.3. Unexpectedly, even the small amount of [F-19]rhPSMA7-3 (5 nmol) added to the urine increased [F-18]rhPSMA7-3 by 79.4%–85.8% at room temperature (F-18]fluoride decreased by 20.6%–14.2%).

[0185] The results obtained by isotope exchange in urine are considered representative of all tracers conjugated with the 4-(di-tert-butyl[(18)F]fluorosilyl)-benzyl)oxy moiety and therefore representative of all rhPSAM7 isomers.

[0186] Preclinical dosimetry, human biodistribution and tumor lesion uptake The next 18F-rhPSMA-7 is nat Ga- 18 F-rhPSMA7-rac and 18F-rhPSMA-7.3 nat Ga- 18 Please note that this refers to F-rhPSMA7.3.

[0187] A) Preclinical dosimetry of 18F-rhPSMA-7 and 18F-rhPSMA-7.3 in mice The objective was to investigate the efficacy and safety of steroids at different time points up to 300 minutes after a single intravenous administration in mice. 18 F-rhPSMA-7 and 18 The objectives of this study were to assess the distribution and excretion of F-rhPSMA-7.3 and to perform calculations for internal dosimetry.

[0188] method Three to five mice were injected per time point, each receiving an average of 25.6 ± 3.6 MBq of 18F-rhPSMA-7 and 28.5 ± 4.8 MBq of 18F-rhPSMA-7.3. Severe combined immunodeficiency (SCID) mice were used for the experiments. All animal experiments were performed in accordance with the general animal welfare regulations and the institutional guidelines for the care and use of animals in Germany.

[0189] Mice were sacrificed at the following time points: 18 F-rhPSMA-7: 10, 20, 40, 60, 120 and 180 minutes after administration. 18 F-rhPSMA-7.3: 10, 60, 120, 180 and 300 minutes after administration. Based on initial experiments, 18 Note that the prolonged renal kidney uptake for the late time point (300 min) of F-rhPSMA-7.3a is used for the final experiment.

[0190] The following tissues / fluids were collected: Urine, blood, heart, lung, spleen, pancreas, liver, stomach (fasting), small intestine (fasting), large intestine (fasting), kidney, bladder, testis, fat, muscle (partial, femur), femur, tail, and brain. Urine was collected with a pipette in a CO2 gas chamber. If urination escaped in the chamber, the bladder was aspirated with an insulin syringe. Blood was removed from the heart with an insulin syringe immediately after sacrifice. All other tissues and organs were dissected and transferred directly to plastic containers.

[0191] The weight of the sample in the plastic container was measured using an electronic balance. The weight of the empty and pre-labeled plastic container for the dedicated sample was measured in advance. The tare weight of the plastic container was subtracted from the weight of the measurement sample with the plastic container. The weight calculated above was represented as the weight of the measurement sample.

[0192] The plastic containers containing the samples were placed in specific test tube racks in an automatic gamma counter (PerkinElmer-Wallac, Waltham, USA) to measure the count rate (counts per minute = cpm) for 60 seconds. In addition, 1% (v / v) standards (n = 5) with known radioactivity were measured alongside the samples to convert the count rates of the organ samples to activity.

[0193] Data analysis The count rates of the measurement samples were automatically corrected for decay. The radioactivity distribution ratio (unit: %ID) as a percentage of the injected dose in the measurement sample was determined using the following equation: The sum of the count rates from all measurement samples obtained from one mouse was expressed as the count rate for the administered radioactivity.

[0194]

number

[0195] The radioactivity distribution ratio per unit weight of the measurement sample (unit: %ID / g), excluding urine and fecal samples, was determined using the following equation: The weight of the measurement sample was determined by subtracting the empty measurement container from the container containing the sample.

[0196]

number

[0197] Dosimetry Analysis For consistency of statistical calculations for each radiotracer, 18 F-rhPSMA-7 and 18 The same number of time points for F-rhPSMA-7.3 were used. 18 For F-rhPSMA-7, the 10 and 20 minute time points were combined to create a 15 minute endpoint.

[0198] Time integrals of activity for accumulation in significant source organs (AUCs) were generated by both numerical integration and physical collapse according to J Juan et al., Journal of Pharmaceutical Sciences, 1993, 82:762-763.

[0199] Kirshner et al. established a method using linear scaling of percent injected volume in animals by the ratio of organ weight to total body weight in the phantom in both species. - Kirschner AS, Ice RD, Beierwaltes WH.Radiation Dosimetry of 131I-19-Iodocholesterol. J Nucl Med.1973 Sep 1;14(9):713-7. - Kirschner A, Ice R, Beierwaltes W. Letters to the editor. J Nucl Med. (1975):248-9.

[0200] Briefly, to calculate human dosimetry from biodistribution in mice, extrapolation was necessary to account for differences between animals and humans. Normal organ radiation doses were estimated for a 70-kg standard adult anatomical model using time-dependent organ activity concentrations (in percent of injected dose per gram, %ID / g) and systemic activity measured in biodistribution studies in mice.

[0201] Tissue activity concentrations in mice were converted to tissue fractional activity in a 70-kg standard adult using the relative fractional organ masses in a standard adult and a "standard" 25-gram mouse. The time-dependent systemic activity was fit to an exponential function, and because activity concentrations in the liver and GI tracer were low at all time points tested, the difference between injected activity and systemic activity was assumed to be excreted in the urine.

[0202] The organ residence time was calculated by the trapezoidal method and numerical integration, and compared with other parts of the body. 18 The F retention time was calculated as the difference between the total body retention time and the sum of the organ and urine retention times. The bladder content retention time was estimated using a dynamic voiding model in the OLINDA / EXM1.0 dosimetry software. Finally, the standard adult average organ dose equivalent (mSv / MBq) and effective dose (also mSv / MBq) were then calculated using OLINDA / EXM1.0.

[0203] Final calculation of radiation absorbed dose and dosimetry from biodistribution in mice: The tissues or organs where significant accumulation of radioactivity occurred (i.e., source organs) were kidney, spleen, lung, liver, and heart. Regarding activity accumulation and clearance, rapid clearance from blood and clearance into urine was found, but relatively slow accumulation in the kidney. result

[0204] [Table 14]

[0205] [Table 15]

[0206] [Table 16]

[0207] [Table 17]

[0208] conclusion The radioactivity distribution ratios were calculated for all examined time points in mice. 18 F-rhPSMA-7 and 18 After administration of F-rhPSMA-7.3, radioactivity distribution ratios were highest in the kidney. Furthermore, radioactivity distribution ratios were higher in the spleen and bladder for both radiotracers compared with all other evaluated tissues, with activity ratios lower than 8% ID / g.

[0209] 18 F-rhPSMA-7 / 18 The main elimination route is defined by the kidney and urinary system, as the majority of F-rhPSMA-7.3 activity is enriched in the kidney and excretion by the bladder reveals high activity.

[0210] Using bladder emptying intervals of 3.5 h and 1.0 h, the extrapolated total effective doses were: 18 For F-rhPSMA-7, it was 2.66E-02 and 1.22E-02 mSv / MBq; 18 For F-rhPSMA-7.3, it was 2.17E-02 and 1.28E-02 mSv / MBq. Injection of up to 370 MBq (10 mCi) for clinical scans should result in a favorable radiation exposure of less than 5 mSv for both agents assuming a 1 h voiding interval.

[0211] The notable differences between both radiotracers are: 18Renal uptake is only evident because F-rhPSMA-7.3 tends to accumulate more gradually with longer retention. Furthermore, radiation exposure is comparable between both agents.

[0212] B) Human biodistribution and tumor lesion uptake of 18F-rhPSMA-7 and 18F-rhPSMA-7.3 The following section describes the biodistribution of 18F-rhPSMA-7 and 18F-rhPSMA-7.3. A proof-of-concept evaluation was performed under compassionate use. The drugs were applied in accordance with the German Medicinal Products Act, AMG §13 2b, and in accordance with the responsible regulatory authority (Government of Oberbayern).

[0213] All subjects were examined with a Biograph mCT scanner (Siemens Medical Solutions, Erlangen, Germany). All PET scans were acquired in 3D mode with an acquisition time of 2–4 min per bed position. Emission data were corrected for randomness, dead time, scatter, and attenuation and iteratively reconstructed with a regular subset expectation-maximization algorithm (4 iterations, 8 subsets), followed by a postreconstruction smoothing Gaussian filter (5-mm full-width at half maximum).

[0214] method Human biodistribution was evaluated in 47 and 32 patients, respectively, according to clinical 18 F-rhPSMA-7- and 18 This was estimated by analyzing F-rhPSMA-7.3-PET / CT scans. 18 F-rhPSMA-7 vs. 18For F-rhPSMA-7.3, the mean injected activity was 324 (range 236-424) MBq versus 345 (range 235-420) MBq, and the uptake time was 84 (range 42-166) minutes versus 76 (range 59-122) minutes, respectively.

[0215] The mean and maximum standardized uptake values ​​(SUVmean / SUVmax) were determined for background (gluteal muscle), normal organs (salivary gland, blood pool, lung, liver, spleen, pancreas, duodenum, kidney, bladder, bone) and three representative tumor lesions. Tumor uptake was calculated as: 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3 was analyzed in 89 lesions (26 primary tumors / local recurrences, 23 bone, 38 lymph nodes, and 2 visceral metastases) and 63 lesions (14 primary tumors / local recurrences, 30 bone, 18 lymph nodes, and 1 visceral metastasis), respectively.

[0216] For SUV calculation, the circular region of interest is defined as the region with a focal increase in uptake in the transaxial slice. A 50% contour was drawn around the area of ​​interest and automatically adapted to the 3D volume of interest (VOI). Organ-to-background and tumor-to-background ratios were calculated.

[0217] result 18 F-rhPSMA-7 and 18 The human biodistribution of F-rhPSMA-7.3 showed the typical pattern known from other PSMA-ligands. 18 F-rhPSMA-7 and 18 The uptake parameters for F-rhPSMA-7.3 were: 18 The lower retention of activity in the bladder and higher uptake in tumor lesions was very similar to that of F-rhPSMA-7.3, i.e. 18 F-rhPSMA-7 vs. 18 The SUV average for F-rhPSMA-7.3 was 18 F-rhPSMA-7 vs. 18For F-rhPSMA-7.3, the ratios were 16.9 vs. 16.0 (parotid gland), 19.6 vs. 19.6 (submandibular gland), 2.0 vs. 1.9 (blood pool), 0.7 vs. 0.7 (lung), 7.0 vs. 7.3 (liver), 9.1 vs. 8.5 (spleen), 32.4 vs. 35.5 (kidney), 2.5 vs. 2.8 (pancreas), 10.9 vs. 11.0 (duodenum), 1.1 vs. 1.3 (non-diseased bone), and 10.2 vs. 2.0 (bladder), respectively. 18 F-rhPSMA-7.3 vs. 18 F-rhPSMA-7 uptake values ​​were significantly lower in bladder retention (2.0±0.8 vs. 6.3±21.2, p<0.05) and significantly higher in tumor lesions (32.5±42.7 vs. 20.0±20.2, p<0.05).

[0218] [Table 18]

[0219] [Table 19]

[0220] [Table 20]

[0221] [Table 21]

[0222] Conclusion: The distribution in humans is as follows for most normal organs: 18 F-rhPSMA-7 and 18 F-rhPSMA-7.3. However, 18 For F-rhPSMA-7.3, tracer retention in the bladder was significantly lower and tumor lesion uptake was significantly higher, providing clear advantages for clinical imaging. 18An imaging example of F-rhPSMA-7.3 with favorable human biodistribution and high uptake in tumor lesions is shown in FIG.

Claims

1. The following formula: 【Chemistry 1】 {wherein F is １８ F or １９ F and Ga 3+ is nat Ga 3+ , or a pharmaceutically acceptable salt thereof.

2. F, １８ The compound of claim 1 , wherein:

3. F, １９ The compound of claim 1 , wherein:

4. 10. A method of synthesizing a compound of claim 1, comprising the steps of: 【Chemistry 2】 In the formula, a, b, c, d, e, f １ , g, h, i ２ , j and k are as follows: a) 20% Piperidine, (DMF); bi(tBuO)EuEttu) ２ ,HOAt,BT!,,,6666!!!;; c) 2% hydrazine (DMF); d) Succinic anhydride, DIPEA, (DMF); e) Fmoc-D-Lys(OtBu)・HCl, HOAt, TBTU, DIPEA, (DMF); f １ ) Fmoc-D-Dap(Dde)-OH, HOAt, TBTU, 2,4,6-collidine, (DMF); g) Imidazole, Hydroxylamine Hydrochloride, (NMP, DMF); h) SiFA-BA, HOAt, TBTU, DIPEA (DMF); i ２ ) (S)-DOTA-GA(tBu)4, HOAt, TBTU, 2,4,6-collidine (DMF); j) Cleavage and deprotection: TFA, TIPS, H ２ O; k) Ga(NO ３ ) ３ ,(tBuOH,H ２ O)。

5. continuation １８ 5. The method of claim 4, further comprising the step of F-labeling.

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