RU2787105C2 - New psa-binding agents and their use - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a compound of the general formula (1a), or its pharmaceutically acceptable salts, or complexes marked with a radioactive mark, which have a capability of selective binding to membrane of a prostate-specific antigen (hereinafter – PSA), and which can be used as radio-pharmaceutical agents, imaging agents, as well as for the treatment of cancer. In the formula (1a), D is a chelating residue selected from a group including 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (hereinafter – DOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (hereinafter – NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (hereinafter – DOTAGA), or their derivatives, X, each independently, is selected from O, R¹ and R², each independently, is H, F, Cl, Br, I, or C₁-C₁₂ alkyl, Y is selected from a group including a direct bond or C₁-C₁₂ alkyl, R³, R⁴, and R⁵, each independently, is selected from -CO₂H, a spacer has the formula (3a), (3b), or (3c), where m is an integer 1 or 2, n is an integer selected from 1, 2, 3, 4, or 5, o is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, a linker has the structural formula (6), where X, each independently, is O, Q is selected from C₅-C₁₄ cycloalkyl, W is selected from -(CH₂)_c-aryl, where c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and a, b, each independently, is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The invention also relates to the use of the specified compounds for the production of complexes marked with a radioactive mark, to complexes marked with a radioactive mark, pharmaceutical compositions, and kits containing these compounds, options of the use of the specified compounds, complexes, compositions, and kits, including for diagnostics, treatment and/or prevention of prostatic cancer, pancreatic cancer, kidney cancer, or bladder cancer, a method for in vitro determination of the presence of cells and/or tissues expressing PSA.

EFFECT: obtainment of PSA-binding compounds for the treatment of cancer.

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36 cl, 27 dwg, 15 tbl, 6 ex

Description

The present invention relates to novel compounds and radiolabeled complexes comprising a chelating agent, a PSA-binding molecule (PSA - Prostate Specific Membrane Agent - PSA Prostate Specific Membrane Antigen) and an albumin-binding molecule, interconnected by suitable linkers and spacers, and intended for use as diagnostic and/or therapeutic radiopharmaceuticals. In particular, the compounds and complexes of the present invention can be used as (theragnostic) tracers, imaging agents, and therapeutic agents for the detection of PSA-expressing target cells and tissues, as well as the treatment and diagnosis of cancer.

Prostate cancer (PC - Prostate cancer - PR - Prostate Cancer) occupies a leading position among all cancers in the US and Europe. At least 1-2 million men in the eastern hemisphere suffer from prostate cancer, with the disease estimated to affect one in six men between the ages of 55 and 85. According to the American Cancer Society, approximately 161,000 new cases of prostate cancer are diagnosed each year in the United States. The five-year survival rate for patients with stage IV metastatic prostate cancer is only 29%.

As metastatic prostate cancer becomes hormone resistant, few therapeutic options remain, often with very low success rates. Current medical guidelines generally recommend antimitotic chemotherapy with docetaxel. However, treatment is almost always associated with serious adverse reactions and only modestly improves survival rates. Thus, early diagnosis and accurate monitoring of potential recurrences is an important task. Diagnosis of prostate cancer is based on the examination of histopathological or cytological samples of the prostate. Existing imaging modalities for treatment management and monitoring of advanced or recurrent prostate cancer include computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound, but are often insufficient to effectively control and treat the disease. Accordingly, there is a great clinical need to develop more effective tools for both early diagnosis and treatment of PD.

It is well known that tumor cells may express unique proteins having a modified structure due to a mutation, or may overexpress normal (ie, non-mutated) proteins that are normally produced at extremely low levels by non-tumor cells. Tumor-specific antigens (TSA), which are present only on the surface of tumor cells and absent on healthy cells, and tumor-associated antigens (TAA) Antigens) that are present on some tumor cells as well as healthy cells. Tumor-specific antigens result from mutations in proto-oncogenes and tumor suppressors, resulting in abnormal protein production, while expression of tumor-associated agents is generally due to mutations in other genes unrelated to tumor formation.

Expression of such proteins on the surface of tumor cells makes it possible to diagnose and type the disease by detecting these tumor markers. Protein-binding agents or small drug molecules that carry imaging labels and are capable of recognizing such tumor markers are commonly used for non-invasive diagnosis and imaging of cancers.

A promising new series of small molecular weight imaging agents bind to prostate-specific membrane antigen (PSA). PSA, also known as folate hydrolase I (FTG1 - Folate hydrolase I - FTG1), is a type II transmembrane glycoprotein consisting of 750 amino acids. The PSA gene is located on the short arm of chromosome 11 and functions as both a folate hydrolase and a neuropeptidase. It has neuropeptidase properties equivalent to those of glutamate carboxypeptidase II (GCPII), which is referred to as brain PSA, and can modulate glutamatergic transport by cleavage of N -acetyl-aspartyl-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (Nan, F .; et al J Med Chem 2000, 43, 772-774). D.F.

PSA is (i) mainly restricted to the prostate (although found in small amounts in the neovascular network of some other solid tumors, including the bladder, pancreas, lungs, and kidneys, but never in normal vessels), (ii) is abundantly expressed as a protein on various stages of prostate cancer (in amounts up to 10 ⁶ PSA molecules per cancer cell), (iii) is present on the cell surface but does not circulate, and (iv) is associated with enzymatic and signaling activity. In addition, PSA expression is also over-regulated in poorly differentiated androgen-insensitive or metastatic cancer cells, with expression generally correlated with disease progression.

The unique expression of PSA makes it an important marker for prostate cancer (and some other cancers). In addition, PSA is a good extracellular target for imaging agents. PSA is taken up by the cell after binding to a ligand and thus is not only an excellent target for targeted radioisotope therapy (using particle-emitting radioisotopes), but can also be used for other treatment strategies, including cell-specific delivery of immunotoxins to a tumor, reorientation of immune cells, prodrug activation, PSA vaccines, and DNA immunization with plasmids and adenoviruses. Since it is poorly expressed in healthy tissues, PSA is also a potential target for high-dose therapy with minimal side effects.

et al (JNM 2015, 56: 914-920 и EP 2862857 A1), который распространяется по организму и быстро выводится из кровотока (J Nucl Med. 2015; 56(11): 1697-705). Однако, несмотря на то, что быстрый и системный доступ значительно усиливает связывание агента с опухолевыми клетками и проникновение в них, у доступных в настоящее время ПСА-связывающих агентов имеется риск того, что они будут опосредовать неспецифические ненаправленные взаимодействия в нормальных тканях, экспрессирующих мишень, а также риск накопления радиофармсредств в экскреторных органах (таких, как почки). Таким образом, неопухолевые ткани могут подвергаться воздействию радиоактивных доз, которые рано или поздно приведут к необратимым повреждениям тканей. Было показано, что различные меченые радиоактивными изотопами небольшие ПСА-связывающие агенты (включая PSMA-617) накапливаются в слезных и слюнных железах пациентов и могут приводить к повреждению железистой ткани, в особенности при использовании альфа-излучающих радионуклидов (Zechmann et al. Eur J Nucl Med Mol Imaging, 2014; 41(7):1280-92 и Kratochwil et al. J Nucl Med. 2017 Apr 13. pii: jnumed.117.191395, doi: 10.2967/jnumed.117.191395 [Epub]). Одним из возможных решений является использование ПСА-связывающих агентов с высокой аффинностью к ПСА (Kratochwil et al. J Nucl Med. 2015; 293-298 и Chatalic et al. Theragnostics. 2016; 6: 849-861).Several anti-PSA antigens have already been developed in the past, carrying therapeutic or diagnostic molecules. The US Food and Drug Administration (FDA) has approved the anti-PSA monoclonal body radioimmunoconjugate (mAb) 7E11, also known as PROSTASCINT®, which is used to diagnose metastasis and recurrence of prostate cancer. The successful use of this radiopharmaceutical agent is limited due to the fact that this antibody binds to the intracellular domain of PSA, therefore, can only attack dead cells. In addition, the use of monoclonal antibodies and antibody fragments as imaging fragments is often limited due to their slow renal clearance, heterogeneous distribution, poor penetration into tumor tissues, and immunogenic potential. To overcome these difficulties, small molecules of PSA-binding agents have been developed that can bind to the extracellular domain of PSA for PET/CT and SPECT/CT imaging, including radiolabeled N-[N-[(S)-1,3-dicarboxypropyl]- carbamoyl]-S-[11C]methyl-l-cysteine (DCFBC) and several urea-based peptimimetic PSA inhibitors (see Bouchelouche et al. Discov Med. 2010 Jan; 9(44): 55-61), including PSA- MIP-1095 ligand (Hillier et al. Cancer Res. 2009 Sep 1; 69(17): 6932-40), currently in clinical trials, and a DOTA-conjugated PSMA-617 inhibitor developed by

et al (JNM 2015, 56: 914-920 and EP 2862857 A1), which is distributed throughout the body and rapidly cleared from the circulation (J Nucl Med. 2015; 56(11): 1697-705). However, while rapid and systemic access greatly enhances agent binding to and penetration into tumor cells, currently available PSA binding agents have the risk of mediating non-specific non-targeted interactions in normal tissues expressing the target. and the risk of accumulation of radiopharmaceuticals in excretory organs (such as the kidneys). Thus, non-tumor tissues can be exposed to radioactive doses that will sooner or later lead to irreversible tissue damage. Various radiolabelled small PSA-binding agents (including PSMA-617) have been shown to accumulate in the lacrimal and salivary glands of patients and can cause damage to glandular tissue, especially when alpha-emitting radionuclides are used (Zechmann et al. Eur J Nucl Med Mol Imaging, 2014;41(7):1280-92 and Kratochwil et al J Nucl Med 2017 Apr 13 pii: jnumed.117.191395, doi: 10.2967/jnumed.117.191395 [Epub]). One possible solution is to use PSA binding agents with high PSA affinity (Kratochwil et al. J Nucl Med. 2015; 293-298 and Chatalic et al. Theragnostics. 2016; 6: 849-861).

Recently, Kelly et al (J Nucl Med. 2017 pii: jnumed.116.188722. doi: 10.2967/jnumed.116.188722. [Epub ahead of print]) studied agents with affinity for both PSA and human serum albumin - (HSA - Human Serum Albumine - HSA). The ligands developed by Kelly et al include a para-(iodophenyl)butyric acid molecule for binding to HSA, and a urea-based PSA-binding molecule. In the compounds developed by Kelly et al, the radiotherapeutic iodine ( ¹³¹ I) molecule is covalently linked to the HSA binding molecule, which in turn is directly linked to the PSA binding molecule via a hydrocarbon chain. However, these compounds have limitations associated with the ability to use only one radionuclide - iodine. In addition, no significant uptake/internalization of the agent by target cells was demonstrated.

Another approach has been proposed by Choy et al, Theranostics 2017; 7(7): 1928-1939 who evaluated a ¹⁷⁷ Lu-labeled phosphoramidate-based PSA inhibitor with an albumin-binding molecule in the formulation. The DOTA chelating agent combined with the ¹⁷⁷ Lu radionuclide has also been linked to the irreversible PSA inhibitor CTT1298 (EP 2970345 A1). The PSA-binding molecule based on phosphoramidate alone exhibits extremely poor stability, especially at elevated temperatures (elevated temperatures under conditions of prolonged acidity lead to hydrolysis of the phosphoramidate PN bond), which are required for the coordinative radiolabeling reaction via chelating agents such as DOTA. Therefore, a direct radiolabeling reaction cannot be carried out and a multi-step prelabeling approach must be used. Thus, ¹⁷⁷ Lu-DOTA-azide must first be obtained as a precursor, then the precursor must be linked to the benzocyclooctyne-modified PSA molecule. Finally, a thorough HPLC purification of the combined compound must be carried out; processing HPLC-eluent (in an atmosphere of N ₂ ) and dissolution in a physiological environment. This procedure is unlikely to be carried out in clinical practice, since it requires work with highly radioactive compounds. Preclinical biodistribution data demonstrate the unsatisfactory effect of the radiolabeled agent, in particular with regard to the ratio of tumor cells/kidneys, which did not exceed 1.

Despite significant progress in recent years, the diagnosis and treatment of prostate cancer still remains a challenge. For early diagnosis and treatment of prostate cancer, new diagnostic or imaging agents are needed that can highly selectively bind to prostate tumor cells and demonstrate good pharmacokinetic properties that allow rapid and non-invasive imaging of the tumor and provide a therapeutic effect.

Thus, the object of the present invention is to overcome the shortcomings of the means known from the field of technology to meet the needs of medicine.

This problem is solved with the help of the present invention, described below, and presented in more detail in the claims.

General remarks

While the present invention is detailed below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein as they may vary. It should also be clear that the terminology used in the description in no way limits the essence of the present invention, which is within the scope of the claims. Unless otherwise specified, all technical and scientific terms used in the description have the generally accepted meanings known to specialists in these fields.

The following discloses aspects of the present invention. These elements are listed within the various embodiments of the present invention, however, it should be clear that they can be combined in various ways and in any number and form new embodiments of the invention. The variously described examples and preferred embodiments of the present invention should not be construed as limiting the invention to specific embodiments.

The present description is to be understood as supporting and covering embodiments of the invention that are clearly disclosed aspects in combination with any number of other disclosed and/or preferred elements. Moreover, any combinations and permutations of all the above elements should be considered disclosed in the description of the present application, unless its context indicates otherwise.

Throughout the description and claims, unless otherwise indicated in the description, the term "comprise", as well as its derivatives "comprises" and "comprising" should be understood as referring to the inclusion of any component, constituent or step, but not to the exclusion of any other component, constituent or step not specified. The term "consists of" refers to a particular embodiment of the term "comprises" and any other components, constituents or steps not specified are excluded. In the context of the present invention, the term "comprises" encompasses the concept of "consists of". The term "comprising" thus encompasses the concept of "comprising" in the same way as "consisting", for example, a composition containing X may consist solely of X, and may include any other additional ingredients, say X+Y.

The articles "a" and "an" and "the" and similar indicating speech units used in the context of the present invention to describe it (especially in the claims section) refer to both the singular and the plural, unless otherwise specified or otherwise does not follow directly from the context ( note: articles are not used in Russian ). The description of a range of values is here intended to act as a simplified method for referring to each individual value within a given range. Except as otherwise indicated in the present description, each individual value is included in the description as if it were indicated in the present description individually. Nothing in this specification should be construed as indicating any undeclared element essential to the practice of the present invention.

The phrase “practically” does not exclude the term “completely” i.e. a composition that is "substantially" free of compound Y may be completely free of compound Y. Where necessary, the phrase "substantially" may be omitted from the specification.

The term "about" used in relation to the numerical value x means x±10%.

According to the present invention, unless otherwise specified, various alternative embodiments of the invention can be combined with each other.

For greater clarity and readability, definitions are provided throughout the description. Any technical features mentioned in these definitions can be considered in the context of almost every embodiment of the invention. Additional definitions and explanations may be provided specifically in the context of these embodiments of the invention.

DEFINITIONS

The term "hydrocarbon radical" refers to hydrocarbon groups, ie. to hydrocarbon chains preferably independently selected from the group of alkyl, alkenyl, alkynyl, aryl or aralkyl.

The term "alkyl" includes linear compounds ("straight chain"), branched chain compounds, as well as cyclic radicals containing from 1 to 13 carbohydrate atoms, preferably 1-20, 1-15, 1-10, 1-8 , 1-6, 1-4, 1-3 or 1-2 carbon atoms. In particular, the term "C _1-12 alkyl" refers to hydrocarbon radicals whose hydrocarbon chain is straight or branched or cyclic and contains from 1 to 12 carbon atoms. Specific examples of alkyl residues are methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, octyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, genicosyl, docosyl, tricosyl, tetracosyl , pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl or triacosyl, including their various branched and/or cyclic isomers, for example t -butyl or isopeptyl, etc. Cyclic isomers may also be referred to in the description by the term "cycloalkyl", which is used in relation to saturated alicyclic hydrocarbon residues containing 3 ring carbon atoms. "Saturated" linear, branched and cyclic alkyl groups are also united by the specified term. The term also encompasses “heteroalkyls,” which include alkyl groups in which one or more carbon atoms in the carbon chain have been replaced by a heteroatom such as, but not limited to, N, O, and S. Heterocyclic groups include unsaturated, partially saturated, and saturated cyclic systems such as, for example, imidazolinyl and imidazolidyl groups. Heterocyclic groups include, without limitation, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, isoxazolyl, oxazolyl thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyronyl, oxatian, dioxyl, diathianyl, pyranyl, pyridyl, pyrimidinyl, pyradazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydropyridylylone, diquinuthynuclyinyl, indolyl, indolinyl, isoindolyl, azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotriazolyl, benzaimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiazolyl, benzothiazolyl , imidazopyridium л (азабензимидазолил), триазолопиридил, изоксазолопиридил, пуринил, ксантинил, аденинил, гуанинил, хинолинил, изохинолинил, хинолизинил, хиноксалинил, хиназолинил, циннолинил, фталазинил, нафтиридинил, птеридинил, тианафталенил, дигидробензотиазинил, дигидробензофуранил, дигидроиндолил, дигидробензодиоксинил, тетрагидроиндолил, тетрагидроинлазолил, тетрагидробензимидазолил , tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Heterocyclic groups may be substituted or unsubstituted. Typical substituted heterocyclic groups may be monosubstituted or substituted by more than one atom, examples include, but are not limited to, groups such as pyridyl or morpholinyl which are 2-, 3-, 4-, 5- or 6-substituted, or disubstituted various substituents such as those listed above.

The term "cyclic" combines the term "polycyclic", which describes structures with one or more rings in the composition. In particular, the term "cyclic" also refers to spirocyclic structures in which two or more rings share one atom, as well as to 5 fused polycyclic compounds in which two or more rings share two atoms.

The term "alkenyl" as used in the present invention includes linear, branched and cyclic radicals containing from 2 to 30 carbon atoms, preferably 2-20, 2-15, 2-10, 2-8, 2-6, 2-4 or 2-3 carbon atoms including at least one double bond between carbon atoms. Specific examples of "alkenyl" groups are the various unsaturated equivalents of alkenes, such as those described in relation to alkyl groups, named according to conventional conventions known to those skilled in the art, depending on the number and arrangement of double bonds between carbon atoms, for example, butanediylidene ( butanediylidene), 1-propanyl-3-ylidene. "Alkenyl" groups preferably have at least 1, more preferably 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 double bonds, with the double bond preferably located in the following positions of the hydrocarbon chain: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, or 29. Alkenyl groups may be either substituted or unsubstituted.

The term “alkynyl” as used herein includes linear, branched and cyclic radicals containing from 2 to 30 carbon atoms, preferably 2-20, 2-15, 2-10, 2-8, 2-6, 2-4 or 2-3 carbon atoms, including at least one triple bond between carbon atoms. Specific examples of "alkynyl" groups are a variety of unsaturated alkyl equivalents, such as those described with respect to alkyl groups, named according to conventional conventions known to those skilled in the art, depending on the number and arrangement of triple bonds between carbon atoms. "Alkynyl" groups preferably have at least 1, more preferably 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 triple bonds, with the triple bond preferably located in the following positions of the hydrocarbon chain: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, or 29. Alkynyl groups may be either substituted or unsubstituted.

The term "aryl" refers to monocyclic or polycyclic or fused polycyclic aromatic ring systems. The term includes monocyclic or polycyclic or fused polycyclic aromatic "heteroaryl" ring systems in which at least one carbon atom in the ring is replaced by a heteroatom. In most cases, the terms "aryl" and "heteroaryl" refer to groups containing 3-30 carbon atoms, for example 3-10 atoms, more often 2-6 carbon atoms.

The terms "arylalkyl" or "aralkyl" are used interchangeably and refer to groups containing at least one alkyl group and at least one aryl group as defined above. In an aralkyl group, as the definition implies, the aralkyl group is connected to another molecule of the substance or conjugate according to the invention via an alkyl group, for example a benzyl group.

The term “halogen” or “halo” as used herein includes fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

The term "heteroatom" includes N, O, S and P, preferably N and O.

The term "substituted" refers to a hydrocarbon group as defined herein (eg, an alkyl or alkenyl group) in which one or more bonds to a carbon atom present in the group are replaced by a bond to a non-carbon, non-hydrogen atom. Substituted groups also include groups in which one or more bonds to a carbon or hydrogen atom are replaced by one or more bonds, including a double or triple bond to a heteroatom. Thus, a “substituted” group will be substituted with one or more substituents, unless otherwise specified. Thus, a "substituted" group will be substituted with one or more substituents, unless the description further states otherwise. According to some embodiments of the invention, the substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups are halogens (ie F, Cl, Br and I); hydroxyl, alkoxy, alkoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; urea compounds; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e. CN), haloalkyl; aminoalkyl; hydroxyalkyl; cycloalkyl and the like.

Conjugates

The present invention relates to novel plasma protein-binding PSA ligands with improved targeted antitumor properties and a favorable pharmacokinetic profile. As used herein, the term “pharmacokinetics” preferably includes stability, biodistribution, half-life and/or clearance of a drug or diagnostic agent in a subject. The present invention relates to novel conjugates formed by covalently linking urea-based PSA peptidomimetic binding molecules by means of suitable spacers and linkers with a chelating agent capable, on the one hand, of forming complexes with drug/diagnostic molecules of radioactive substances, and on the other hand with molecules binding human serum albumin (HSA). The spacer and linker groups connecting binding molecules and chelating agents have been shown to be key determinants of the targeting and pharmacokinetic properties of the final conjugates. The new conjugates preferably have improved and specific properties, namely better uptake and internalization by cells. The inventors demonstrated that PSA-binding molecules (1) better compartmentalized the conjugates in the blood (with limited effects on healthy non-target tissues without compromising access to the tumor vasculature), (2) had longer plasma clearance, and (3) were better absorbed by tumor cells and retained in them (due to an increase in the number of passages through the tumor bed). Thus, the introduction of a PSA-binding molecule significantly improves the biodistribution and, ultimately, the therapeutic efficacy of the compounds of the present invention.

In particular, it has been demonstrated that the claimed conjugates are more intensively captured by tumors compared to other PSA ligands known from the technical field. Such improved characteristics of the conjugates associated with their increased uptake by tumor cells, in particular, allow to reduce the administered activity, but at the same time maintain the desired dosage necessary to achieve a therapeutic effect or sufficient uptake by the cells if they are used for imaging (diagnosis). In this regard, the conjugates are presented in the form of radioactive labeled complexes with a chelating agent that combines a drug and/or diagnostic radioactive isotope (usually a metal isotope). Reducing the required dose of new conjugates (and in particular their radiolabeled (metal) labeled complexes) has, among others, the following advantages: (1) less radioactive material is required (radioactivity is reduced), which ultimately leads to lower production costs and greater availability - both are significant for alpha-active substances such as ²²⁵ Ac, for example, which are difficult and expensive to produce and are advantageous in extending the shelf life of substances due to less self-irradiation, which usually leads to the decay of radioactive complexes (i.e., radiolysis); (2) the patient receives a lower total absorbed dose of radiation (which preferentially allows for outpatient treatment and also has a less harmful environmental impact).

Thus, the conjugates claimed in accordance with the present invention are promising drugs with optimal characteristics for both radionuclide imaging and endoradiotherapy.

In general, the new PSA ligands of the present invention (also referred to herein as “conjugates” or “compounds”) thus comprise a first end group (chelating agent), a second end group (albumin-binding molecule), and a third end group. a group (PSA-binding molecule) that are covalently linked or interconnected by suitable linkers or spacers.

According to the first aspect, the present invention relates to a compound of general formula (1):

where

D is a chelating agent, preferably as disclosed in the description,

Abm is an albumin binding molecule, preferably as disclosed in the description,

Pbm is a PSA-binding molecule, preferably as disclosed in the description,

the spacer includes at least one C-N bond,

the linker has the general formula (6), as disclosed in the description,

a is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and

-CH- group in the general formula (1) is a "branch point" connecting the PSA-binding molecule (Pbm) and the albumin-binding molecule (Abm);

or to its pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

Preferred values for D, Abm, Pbm, linker and spacer are disclosed in the present description.

In particular, the present invention relates to compounds of general formula (1)(i) or (1)(ii):

where

Abm is an albumin binding molecule, preferably as disclosed in the description,

Pbm is a PSA-binding molecule, preferably as disclosed in the description,

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA), N,N" -bis [2-hydroxy-5-( carboxyethyl)benzyl]ethylenediamine-N,N"-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7- bis (carboxymethyl) -1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10- tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid ( DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7- bis [methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N'- {5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO) and diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,

X is each independently selected from O, N, S, or P,

R¹ and R² each independently is H, F, Cl, Br, I, branched, straight or cyclic C₁-C₁₂ hydrocarbon, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, OR⁶,OCOR⁶, CHO, COR⁶, CH₂OR⁶ _, NR⁶R⁷, CONR⁶R⁷, COOR⁶, CH₂NR⁶R⁷, SR⁶, =O, =S or =NH, or R¹ and R² interconnected to form a cyclic structure, including branched, unbranched or cyclic C₁-C₁₀ a hydrocarbon group, wherein said hydrocarbon group is optionally interrupted by at most 2 heteroatoms and optionally substituted by at most 3 groups independently selected from F, Cl, Br, I, OR⁶,OCOR⁶, COOR⁶, CHO, COR⁶, CH₂OR⁶, NR⁶R⁷, CH₂NR⁶R⁷, and SR⁷, =O, =S and =NH,

Y is selected from the group consisting of a direct link or a linear, branched or cyclic optionally substituted C₁-C₁₂ alkyl optionally interrupted by no more than two heteroatoms, OR⁶,OCOR⁶, CHO, COR⁶, CH₂OR⁶ _, NR⁶R⁷, COOR⁶, CH₂NR⁶R⁷, SR⁶, =O, =S or =NH, with one or more non-adjacent CH₂-group can be independently substituted with -O-, -CO-, -CO-O-, -O-CO-, -NR⁶-, -NR⁶-CO-, -CO-NR⁶-, -NR⁶-COO-, -O-CO-NR⁶-, -NR⁶-CO-NR⁶-, -CH=CH-, -C≡C-, -O-CO-O-, SR⁶-, SO₃R⁶-,

R ⁶ and R ⁷ are each independently selected from H or a branched, straight or cyclic C _1-12 hydrocarbon,

R ³ , R ⁴ and R ⁵ are each independently selected from the group consisting of -COH, -CO ₂ H, -SO ₂ H, -SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H, -PO ₄ H ₂ , C(O)-(C ₁ -C ₁₀ )alkyl, -C(O)-O(C ₁ -C ₁₀ )alkyl, -C(O)-NHR ⁸ or -C(O)-NR ⁸ R ^9, wherein R ⁸ and R ⁹ are each independently selected from the group consisting of H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, Br, I, C(O), C(S), - C(S)-NH-benzyl-, -C(O)-NH-benzyl, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH, -(CH ₂ ) _p -(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH-C(O)-(CH ₂ ) _q , -(CH _r CH ₂ ) _t -NH-C(O)-(CH ₂ ) _p , -(CH ₂ ) _p -CO-COH, -(CH ₂ ) _p -CO-CO ₂ H, -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -COH] ₃ , -C[(CH ₂ ) _p -COH] ₃ , -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -CO ₂ H] ₃ , -C[(CH ₂ ) _p -CO ₂ H] ₃ or -(CH ₂ ) _p -(C ₅ -C ₁₄ )heteroaryl,

the spacer includes at least one C-N bond,

the linker has the general formula (6), as disclosed in the description,

a, b, p, q, r, t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

or to their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

Separately, it is emphasized that the structure disclosed in formula (1) below includes at least one peptide bond:

The conjugates claimed in the present invention are ligands with affinity for PSA and serum albumin. The term “ligand” as used herein refers to a compound capable of interacting (attaching, binding) with a target (here PSA or HSA). The conjugates of the present invention may be functionally defined as "PSA targeting agents". Preferably, the "ligands" are capable of selectively binding to their target. The term "selective binding" means that a compound binds with greater affinity to its intended target than to non-target molecules.

“Binding affinity” is the strength of the interaction between a ligand (ie a small organic molecule, protein or nucleic acid) and its target/binding partner. Typically, binding affinity is measured and labeled using the equilibrium binding constant (K _D ), which is the ratio between the rate of dissociation (k _off ) and the rate of association (k _on ), which are used to evaluate and rank the strengths of biomolecular interactions. The association rate (K _on ) indicates how quickly the ligand binds to the target, and the dissociation rate (K _off ) indicates how quickly the ligand detaches from its target. K _D (K _off /K _on ) and binding affinity are inversely related. Thus, the term "selective binding" preferably means that the ligand binds to its intended target with a K _D that is less than the K _D of that ligand binding to another molecule that is not its target. There are many methods for measuring binding affinity and dissociation constant, such as ELISA, gel electrophoretic shift delay assay, affinity absorption assay, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance, and spectroscopic analysis.

In the context of the present invention, the K _D value of a PSA binding molecule (HSA binding molecule) with a non-target molecule is at least 1.5 times, preferably at least 2-, 3-, 5-, 10-, 15 -, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 100- 200-, 300-, 400-, 500-, 750 -, or 1000 times the K _D value for binding the claimed conjugate or molecule to human PSA (HSA).

In the context of the present invention, it is further preferred that conjugates bind to PSA with high binding affinity and nanomolar K _D value (nM), and moderate affinity and micromolar K _D value (μM (micromolar)) bind to HSA.

In particular, it is preferable to balance the binding affinities for PSA and HSA in such a way as to enhance uptake and retention of the substance by tumor cells, as well as increase plasma clearance, while simultaneously reducing the potential deleterious effect on non-target cells. In particular, the conjugates of the present invention may exhibit a higher binding affinity for PSA than for HSA.

In particular, the present invention relates to compounds of general formula (1)(i):

where

Abm is an albumin-binding molecule,

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA), N,N''- bis [2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7- bis ( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10- tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7- bis [methyl(2-hydroxymethyl )phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N '-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO ) and diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,

X is each independently selected from O, N, S, or P,

R ³ , R ⁴ and R ⁵ are each independently -COH, -CO ₂ H, -SO ₂ H, -SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H, -PO ₄ H ₂ , -C(O)-(C ₁ -C ₁₀ )alkyl, C(O)-O(C ₁ -C ₁₀ )alkyl, -C(O)-NHR ⁸ or -C(O)-NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are each independently H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, Br, I, C(O), C(S), -C(S)-NH -benzyl-, -C(O)-NH-benzyl, C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH, -(CH ₂ ) _p -(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH-C(O)-(CH ₂ ) _q , -(CH _r CH ₂ ) _t -NH-C(O)-(CH ₂ ) _p , -(CH ₂ ) _p -CO-COH, -(CH ₂ ) _p -CO-CO ₂ H, -(CH2) _p -C (O)NH-C [(CH ₂ ) _q -COH] ₃ , -C [(CH ₂ ) _p -COH] ₃ , -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -CO ₂ H] ₃ , -C[(CH ₂ ) _p -CO ₂ H] ₃ or -(CH ₂ ) _p -(C ₅ -C ₁₄ )heteroaryl,

the spacer includes at least one C-N bond,

the linker has the general formula (6) as disclosed here,

a, b, p, q, r, t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

or to their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

In particular, the present invention relates to the most preferred conjugates of general formula (11):

where

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA), N,N''- bis [2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7- bis ( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10- tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7- bis [methyl(2-hydroxymethyl )phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N '-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO ) and diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,

X is each independently selected from O, N, S, or P,

R ¹ and R ² are each independently H, F, Cl, Br, I, branched, straight or cyclic optionally substituted C ₁ -C ₁₂ hydrocarbon, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, OR ⁶ , OCOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ _, NR ⁶ R ⁷ , CONR ⁶ R ⁷ , COOR ⁶ , CH ₂ NR ⁶ R ⁷ , SR ⁶ , =O, =S or =NH, or R ¹ and R ² are interconnected to form a cyclic structure comprising a branched, straight or cyclic C ₁ -C ₁₀ hydrocarbon group, wherein said hydrocarbon group is optionally interrupted by at most 2 heteroatoms and optionally substituted by at most 3 groups independently selected from F, Cl, Br , I, OR ⁶ , OCOR ⁶ , COOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , CH ₂ NR ⁶ R ⁷ , and SR ⁷ , =O, =S and =NH,

Y is selected from the group consisting of a direct link or a linear, branched or cyclic optionally substituted C₁-C₁₂ alkyl optionally interrupted by no more than two heteroatoms, OR⁶,OCOR⁶, CHO, COR⁶, CH₂OR⁶ _, NR⁶R⁷, COOR⁶, CH₂NR⁶R⁷, SR⁶, =O, =S or =NH, with one or more non-adjacent CH₂-group can be independently substituted with -O-, -CO-, -CO-O-, -O-CO-, -NR⁶-, -NR⁶-CO-, -CO-NR⁶-, -NR⁶-COO-, -O-CO-NR⁶-, -NR⁶-CO-NR⁶-, -CH=CH-, C≡C-, -O-CO-O-, SR⁶-, SO₃R⁶-,

R ⁶ and R ⁷ are each independently selected from H or a branched, straight or cyclic C ₁₁₂ hydrocarbon,

R ³ , R ⁴ and R ⁵ are each independently selected from the group consisting of -COH, -CO ₂ H, -SO ₂ H, -SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H, -PO ₄ H ₂ , C(O)-(C ₁ -C ₁₀ )alkyl, -C(O)-O(C ₁ -C ₁₀ )alkyl, -C(O)-NHR ⁸ or -C(O)-NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are each independently selected from the group consisting of H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, Br, I, C(O), C(S), - C(S)-NH-benzyl-, -C(O)-NH-benzyl, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH, -(CH ₂ ) _p -(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH-C(O)-(CH ₂ ) _q , -(CH _r CH ₂ ) _t -NH-C(O)-(CH ₂ ) _p , -(CH ₂ ) _p -CO-COH, -(CH ₂ ) _p -CO-CO ₂ H, -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -COH] ₃ , -C[(CH ₂ ) _p -COH] ₃ , -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -CO ₂ H] ₃ , -C[(CH ₂ ) _p -CO ₂ H] ₃ or -(CH ₂ ) _p -(C ₅ -C ₁₄ )heteroaryl,

the spacer includes at least one C-N bond,

the linker has the structural formula (6):

where X is each independently O, N, S, or P,

Q is selected from substituted or unsubstituted alkyl, alkylaryl and cycloalkyl, preferably from substituted or unsubstituted C ₅ -C ₁₄ aryl, C ₅ -C ₁₄ alkylaryl or C ₅ -C ₁₄ cycloalkyl,

W is selected from -(CH ₂ ) _c -aryl or -(CH ₂ ) _c -heteroaryl, wherein c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and

a, b, p, q, r, t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10,

or to their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

Albumin binding molecule

The conjugates claimed in the present invention include (when compared with already known PSA ligands) an albumin-binding element (also referred to as an "albumin-binding molecule"), as disclosed in the description, which preferably has the ability to selectively bind to human serum albumin ( HSA). The term "selective binding" is disclosed above.

The albumin binding element (Abm) may be any element capable of binding to albumin.

The most preferred albumin binding elements are those described below. The albumin-binding element may preferably bind non-covalently to serum albumin, preferably human serum albumin, typically with a binding affinity of less than about 100 μM (micromoles), such as from about 3 μM (micromoles) to 50 μM (micromoles).

Human Serum Albumin (HSA) is the highest content in blood plasma and makes up approximately half of the volume of all serum proteins. The term “Human Serum Albumin” or “HSA” as used herein preferably refers to human serum albumin encoded by the ALB gene. More preferably, the term refers to a protein listed in the UniProt database as P02768 (accession 240, last modified May 10, 2017), as well as its functional variants, isoforms, fragments, or (post-translational or otherwise modified) derivatives. .

Without wishing to be bound by any particular theory, the authors hypothesized that the albumin binding element (Abm) in the claimed conjugates preferentially increases their plasma half-life and influences blood compartmentalization of the claimed conjugates, improving their access to PSA-expressing ( tumor) cells or target tissues, resulting in an increase in the ratio of tumor targets/non-tumor targets for PSA-expressing normal (non-tumor) organs (such as kidneys, lacrimal and salivary glands).

Thus, the albumin binding element is expected to provide an improved pharmacokinetic profile for the subject conjugates, preferably without affecting (reducing or destroying) the desired function of the chelating agent and the PSA binding element.

From a structural point of view, exemplary albumin binding elements according to the present invention may preferably contain linear and branched groups containing 1-40 carbon atoms and a distal acid group. Suitable albumin binding elements, among others, are described in US 2010/172844 A1, WO 2013/024035 A1 and WO 2008/053360 A2, which are incorporated herein by reference in their entirety.

As stated above, in the conjugates of the present invention, the albumin-binding element has the general formula (2):

where

R¹ and R² each independently selected from the group consisting of H, F, Cl, Br, I, branched, straight or cyclic C₁-C₁₂ hydrocarbon, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, OR⁶,OCOR⁶, CHO, COR⁶, CH₂OR⁶ _, NR⁶R⁷, CONR⁶R⁷, COOR⁶, CH₂NR⁶R⁷, SR⁶, =O, =S or =NH, or R¹ and R² interconnected to form a cyclic structure, including branched, unbranched or cyclic C₁-C₁₀ a hydrocarbon group, wherein said hydrocarbon group is optionally interrupted by at most 2 heteroatoms and optionally substituted by at most 3 groups independently selected from F, Cl, Br, I, OR⁶,OCOR⁶, COOR⁶, CHO, COR⁶, CH₂OR⁶, NR⁶R⁷, CH₂NR⁶R⁷, and SR⁷, =O, =S and =NH,

Y is selected from the group consisting of a direct link or a linear, branched or cyclic optionally substituted C₁-C₁₂ alkyl optionally interrupted by no more than two heteroatoms, OR⁶,OCOR⁶, CHO, COR⁶, CH₂OR⁶ _, NR⁶R⁷, COOR⁶, CH₂NR⁶R⁷, SR⁶, =O, =S or =NH, with one or more non-adjacent CH₂-group can be independently substituted with -O-, -CO-, -CO-O-, -O-CO-, -NR⁶-, -NR⁶-CO-, -CO-NR⁶-, -NR⁶-COO-, -O-CO-NR⁶-, -NR⁶-CO-NR⁶-, -CH=CH-, -C≡C-, -O-CO-O-, SR⁶-, SO₃R⁶-,

R ⁶ and R ⁷ are each independently selected from H or a branched or straight or cyclic C _1-12 hydrocarbon, and

X is O, N, P and S.

The radicals R ¹ and R ² may be in the ortho, meta or para position.

If R ¹ and R ² are combined together in such a way that they form a cyclic structure, then said cyclic structure is preferably a linear or branched hydrocarbon chain of 3-12, preferably 3-10, even more preferably 3-9, 3-8, 3-7, 3-6, 3-5, 3-4 or 4 carbon atoms, which are attached to the phenyl ring in two positions, i.e. form two bonds to said phenyl ring, thus forming a ring structure fused to said phenyl ring. In particular, said ring structure may be selected from (substituted or unsubstituted) adamantyl compounds. Preferably, said two bonds are located at the meta (3-) and para (4-) positions, at the ortho-(2-) and meta positions, or at the ortho and para positions of said phenyl ring. Said ring structure is optionally interrupted by no more than 2, preferably 1 or no heteroatoms. Preferably, said ring structure may be a C ₄ (1,4-diradical) chain fragment attached at its 1 and 4 atoms to said phenyl ring to form a six-membered ring connected to said phenyl ring, preferably at the meta and para positions of said phenyl ring, i.e. preferably to form a meta- and para-fused six-membered ring.

Preferably, R ¹ and R ² are each independently selected from H, halogen, preferably iodine or bromine, and C _1-6 alkyl, preferably C _1-3 alkyl, even more preferably methyl. More preferably, R ¹ is H and R ² is selected from halogen, preferably iodine or bromine, and C _1-6 alkyl, preferably C _1-3 alkyl, even more preferably methyl. Even more preferably, R ¹ is H and R ² is H or para and is selected from iodine, bromine and methyl.

Preferably, Y may be a branched optionally substituted C ₁ -C ₁₂ hydrocarbon, more preferably a linear or branched optionally substituted C ₁ -C ₁₀ hydrocarbon, even more preferably a linear or branched optionally substituted C ₁ -C ₆ hydrocarbon, most preferably a linear or branched optionally substituted C ₁ -C ₃ hydrocarbon.

More preferably, Y may be -(CH ₂ ) ₃ -.

Preferably, X may be O.

Thus, the albumin binding molecule of formula (2) may preferably include or consist of one of the formulas (2a)-(2c):

Other possible, potentially less preferred, albumin-binding molecules are disclosed in US 2010/0172844 A1, among others.

According to preferred embodiments of the present invention, the compounds have any of the general formulas (11.1)-(11.3):

where D, linker, X, R ₁ -R ₅ , a and b have the meanings as defined for the General formula (11).

spacer

In the claimed conjugates, the albumin-binding element is conjugated (ie, covalently linked or connected) to the -CH- “branch point” via a “spacer”. The term "spacer" as used herein is used to specifically designate a group that connects and fills the space between the albumin-binding element and -CH- "branch point" and/or "separation" of these groups from other groups/elements of the conjugate.

Preferably, the spacer avoids spatial interference between the albumin-binding element and the remaining groups or elements of the inventive conjugate and provides sufficient mobility and plasticity. Furthermore, preferably, the spacer can be designed to provide, maintain and/or permit sufficient HSA binding, high affinity PSA binding, and rapid and desirable selective entry of the PSA-conjugated complex into PSA-positive target cells.

The authors of the present invention have determined that preferably the spacer should contain at least one CN bond. Suitable spacers should preferably be stable in vivo . The design of the spacer is most often dependent on the structure of the entire conjugate and is preferably chosen to provide the functionality of the rest of the conjugate (eg, PSA binding, HSA binding, internalization, etc.). Accordingly, spacers can be, in particular, rigid or plastic, affect the lipophilicity or hydrophilicity of the whole conjugate, and so on.

Preferably, the spacer may include a linear or branched optionally substituted C ₁ -C ₂₀ hydrocarbon containing no more than 5 heteroatoms, more preferably a C ₁ -C ₁₂ hydrocarbon, even more preferably a C ₂ -C ₆ hydrocarbon, most preferably a C ₂ -C ₄ hydrocarbon . Preferably, the hydrocarbon comprises at least 4 heteroatoms, preferably N.

Preferably, the spacer is a -[CHR ¹⁰ ] _u -NR ¹¹ - group, where R ¹⁰ and R ¹¹ are each independently selected from H and a branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon, where u is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. More preferably, R ¹⁰ and R ¹¹ may be H and u is an integer selected from 2, 3, or 4. More preferably , R ¹⁰ and R ¹¹ are H and u is 4.

Thus, preferably the claimed conjugates may include a spacer of formula (3a)

Accordingly, the preferred conjugates of the present invention (for example, PSA-ALB-03 and PSA-ALB-06 disclosed in the examples) contain an albumin-binding element of formula (2a)-(2b) attached to the "branch point" with using a spacer of formula (3a).

Alternatively or additionally, the spacer may contain at least one amino acid residue. As used herein, the term “amino acid residue” refers to a specific amino acid monomer as part of a molecule within a spacer.

An "amino acid" is any organic molecule that contains both an acidic (usually carboxyl (-COOH)) and an amino (-NH ₂ ) functionality. One or both of these groups may optionally be modified. The amino group and the acid group may be in any position relative to each other, however, amino acids are usually 2-aminocarboxylic acids, 3-aminocarboxylic acids, 4-aminocarboxylic acids, and so on. The amino group can be attached to ^1mu , ^2mu , ^3mu , ^4mu , ^5mu , ^6mu , ^7mu , ^8mu , ^9mu , ^10mu (etc.) but not more than to the ^20th hydrocarbon atom amino acid(s). In other words, the amino acid(s) may be alpha, beta, gamma, delta, epsilon (etc.), but no more than omega amino acid(s). Preferably, the acid group is a carboxy (-COOH) group. However, other acid groups such as -OPO ₃ H, -PO ₃ H, -OSO ₃ H or -SO ₃ H are also acceptable.

Preferably, the amino acid residue is derived from naturally occurring amino acids or derivatives thereof. In addition, it is preferred that the amino acid residue be derived from alpha(α-)amino acids, wherein said acid may be a D- or L-amino acid.

More preferably, said amino acids are the D or L enantiomers of amino acids selected from the group consisting of arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan. , tyrosine and/or valine.

More preferably, said amino acids are (D-/L-) aspartic acid, glutamic acid or lysine. The spacer may include 1, 2, 3, 4 or 5 amino acid residues, in particular D-aspartate, D-glutamate or L-lysine. Claimed conjugates containing the D-enantiomer may have the additional beneficial effect of reducing the rate of metabolism and thus clearance from blood plasma. Preferably, the spacer may contain 2-3 of the indicated amino acid residues, in particular D-aspartate or D-glutamate residues. In other words, the spacer may include a peptide that is preferably 2-5 amino acids, more preferably 2-3 amino acids. Alternatively, the spacer may contain 1-2 amino acid residues, preferably L-lysine.

Thus, claimed conjugates may include a spacer of formula (3b)

where

m is an integer 1 or 2,

n is an integer selected from 1, 2, 3, 4 or 5, preferably 2 or 3.

Alternatively, the spacer may include an amino acid residue connected to the "branch point" with a linear or branched optionally substituted C ₁ -C ₂₀ hydrocarbon group containing at least one N heteroatom.

Thus, claimed conjugates may include a spacer of formula (3c):

where o is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, o is 5.

According to preferred embodiments of the present invention, the claimed conjugates may have one of the following general formulas (12.1)-(12.4) or (13.1)-(13.4):

where the values of D, spacer, linker, X, R ¹ -R ⁵ , a, b, m, n in the general formulas (12.1)-(12.4) and (13.1)-(13.4) are the same as defined for the general formulas (1) and (11), and d is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 1, 2, 3, 4, 5, or 6 .

Chelating agent

The conjugates of the present invention also contain a chelating agent.

The terms “chelating agent” or “chelating residue” are used interchangeably herein to refer to polydentate (multiple bonds) ligands capable of forming two or more separate coordination bonds to a central (“coordination”) (metal) ion. In particular, such molecules, or molecules capable of donating one pair of electrons, may also be referred to as “Lewes bases”. The central (metal) ion is usually coordinated by two or more electron pairs of the chelating agent. The terms "bidentate chelating agent", "tridentate chelating agent" and "tetradentate chelating agent" are well known in the art and refer to chelating agents having two, three or four pairs of electrons, respectively, which the agent can easily and simultaneously donate to a metal ion, which is coordinated by a chelating agent. Usually, the electron pairs of the chelating agent form coordination bonds with one central metal ion; however, in specific examples, the chelating agent may form coordination bonds with one or more metal ions, and various bonding options are possible.

The terms “coordinating” or “coordinating” refer to interactions in which one donor of multiple electron pairs coordinates (or “coordinates”) with a central metal ion, i.e. donates two or more pairs of electrons.

Preferably, the chelating agent is selected based on the ability to coordinate with the desired central metal ion, usually radioactive, as detailed in the specification.

Thus, the chelating agent D may have one of the following formulas (4a)-(4jj):

Preferably, the chelating agent may be DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid which has the formula (4a)), NODAGA (2-(4,7- bis (carboxymethyl )-1,4,7-triazonan-1-yl)-pentanedioic acid which has the formula (4c)), or derivatives thereof.

According to some preferred embodiments of the present invention, the chelating agent may be DOTA. According to other preferred embodiments of the present invention, the chelating agent is NODAGA.

Preferably, DOTA effectively complexes diagnostic (eg ⁶⁸ Ga) and therapeutic (eg ⁹⁰ Y or ¹⁷⁷ Lu) radioactive substances and thus allows the use of the same conjugate for both imaging and treatment purposes, those. as a therapeutic agent. DOTA derivatives capable of complexing with radioactive isotopes of scandium ( ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc), including DO3AP (which has the formula (4hh)), DO3AP ^PrA (which has the formula (4ii)) or DO3AP ^ABn (which has the formula ( 4jj)) are also preferred and are described in Kerdjoudj et al, Dalton Trans., 2016, 45, 1398-1409.

Other preferred chelating agents in the context of the present invention are N,N''- bis [2-hydroxy-5-(carboxyethyl)-benzyl]ethylenediamine-N,N''-diacetic acid ( HBED-CC ), ), 1,4 ,7-triazacyclononane-1,4,7-triacetic acid ( NOTA ), 2-(4,7,10- tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA) , 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7- bis [methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N'-{5 -[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO) and diethylenetriaminepentaacetic acid (DTPA), or their derivatives.

The chelating group, for example DOTA, can complex with the central metal ion, in particular with a radioactive substance, as defined in the description. Alternatively, the chelating group, for example DOTA, may not complex with the central metal ion, in particular with the radioactive substance, as defined in the description, and thus be in free form. Where the chelating agent (DOTA) is not complexed with said metal ion, the carboxylic acid group of the chelating agent may be in either the free acid form or the salt form.

PSA binding element

The conjugates of the present invention contain a PSA-binding element (also referred to in the description as "PSA-binding molecule"), which is preferably capable of selectively binding to human PSA. The term "selective binding" is disclosed above.

In particular, the present invention relates to compounds of general formula (1)(ii):

where

Pbm is a PSA-binding molecule,

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA), N,N''- bis [2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7- bis ( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10- tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA), 1,4,7-triazacyclononanephosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7- bis [methyl(2-hydroxymethyl) phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N' -{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO) and diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,

X is O, N, S or P,

R ¹ and R ² are each independently H, F, Cl, Br, I, branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, OR ⁶ , OCOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ _, NR ⁶ R ⁷ , CONR ⁶ R ⁷ , COOR ⁶ , CH ₂ NR ⁶ R ⁷ , SR ⁶ , =O, =S or =NH, or R ¹ and R ² are connected with each other to form a cyclic structure comprising a branched, straight or cyclic C ₁ -C ₁₀ hydrocarbon group, wherein said hydrocarbon group is optionally interrupted by at most 2 heteroatoms and optionally substituted by at most 3 groups independently selected from F, Cl, Br, I, OR ⁶ , OCOR ⁶ , COOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , CH ₂ NR ⁶ R ⁷ , and SR ⁷ , =O, =S and =NH,

Y is selected from the group consisting of a direct link or a linear, branched or cyclic optionally substituted C₁-C₁₂ alkyl optionally interrupted by no more than two heteroatoms, OR⁶,OCOR⁶, CHO, COR⁶, CH₂OR⁶ _, NR⁶R⁷, COOR⁶, CH₂NR⁶R⁷, SR⁶, =O, =S or =NH, with one or more non-adjacent CH₂-group can be independently substituted with -O-, -CO-, -CO-O-, -O-CO-, -NR⁶-, -NR⁶-CO-, -CO-NR⁶-, -NR⁶-COO-, -O-CO-NR⁶-, -NR⁶-CO-NR⁶-, -CH=CH-, C≡C-, -O-CO-O-, SR⁶-, SO₃R⁶-,

R ⁶ and R ⁷ are each independently selected from H or a branched, straight or cyclic C _1-12 hydrocarbon,

the spacer includes at least one C-N bond,

the linker has the general formula (6) as defined in the description, and

a, b, p, q, r, t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or to their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

The PSA binding element binds reversibly or irreversibly to PSA, typically with a binding affinity of less than about 100 μM (micromoles).

Human prostate-specific membrane antigen (PSA) (also known as glutamate carboxypeptidase II (GCPII), folate hydrolase I, folipoly-gamma-glutamate carboxypeptidase (FGCP), and N-acetylated-alpha-linked acid dipeptidase I (NAALADase I)) is a transmembrane zinc type II metallopeptidase, which is highly expressed in the nervous system, prostate, kidneys and small intestine. It is considered a tumor marker in prostate cancer. The term “human prostate-specific membrane antigen” or “PSA” as used herein preferably refers to the protein encoded by the human FOLH1 gene. More preferably, the term refers to a protein that is listed in the UniProt database as number. Q04609 (accession number 186, last modified May 10, 2017), as well as its functional variants, isoforms, fragments or (post-translational or otherwise modified) derivatives.

A PSA binding element may, in general, be a binding element capable of selectively (if necessary, irreversibly) binding to a Prostate-Specific Membrane Antigen (Human) (see Chang Rev Urol. 2004; 6(Suppl 10): S13-S18).

The PSA binding element is preferably selected based on the ability to exhibit selective affinity for PSA. Preferred PSA binding elements are described in WO2013/022797A1, WO2015/055318A1 and EP 2862857 A1, which are incorporated herein by reference in their entirety.

Thus, in the conjugates of the present invention, the PSA-binding element preferably has the general formula (5):

where

X is O, N, S or P,

R³, R^four and R^five each independently selected from the group consisting of -COH, -CO₂H, -SO₂H, -SO₃H, -SO_fourH, -PO₂H, -PO₃H, -PO_fourH₂, C(O)-(C₁-C₁₀)alkyl, -C(O)-O(C₁-C₁₀)alkyl, -C(O)-NHR⁸ or -C(O)-NR⁸R^nine, while R⁸ and R^nine each independently selected from the group consisting of H, bond, (C₁-C₁₀)alkylene, F, Cl, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl, -C(O)-(C₁-C₁₀)alkylene, -(CH₂)_p-NH, -(CH₂)_p-(C₁-C₁₀)alkylene, -(CH₂)_p-NH-C(O)-(CH₂)_q, -(CH_rCH₂)_t-NH-C(O)-(CH₂)_p, -(CH₂)_p-CO-COH, -(CH₂)_p-CO-CO₂H, -(CH2)_p-C(O)NH-C[(CH₂)_q-COH]₃, -C[(CH₂)_p-COH]₃, -(CH2)_p-C(O)NH-C[(CH₂)_q-CO₂H]₃, -C[(CH₂)_p-CO₂H]₃or -(CH₂)_p-(C_five-C_fourteen)heteroaryl, and

b, p, q, r, t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In preferred PSA binding elements, b is an integer selected from 1, 2, 3, 4 or 5, R ³ , R ⁴ and R ⁵ are each CO ₂ H and X is O.

Linker

In the claimed conjugates, the PSA-binding element is attached/attached to the -CH- “branch point” via a suitable linker. The term “linker” is used herein to specifically designate a group that connects or links and thus increases the distance between the PSA binding element and the -CH- “branch point”, and/or “separates” the PSA binding element from the remaining conjugate.

Preferably, the linker avoids spatial interference between the PSA-binding element and the remaining groups or elements of the inventive conjugate, and provides sufficient mobility and plasticity. Furthermore, preferably, the linker can be designed to provide, maintain and/or permit sufficient PSA binding, high affinity PSA binding, and rapid and desirable selective entry of the PSA-conjugated complex into PSA-positive target cells.

PSA-binding elements, in particular, the preferred PSA-binding elements of General formula (5), can be attached to the claimed conjugate through a suitable linker, as described, for example, in the application EP2862857A1. The specified linker can give the claimed conjugate optimal lipophilic properties in order to increase PSA binding and cellular uptake with subsequent internalization. The linker may preferably contain at least one cyclic group and at least one aromatic group (in particular in group Q and W).

Thus, in the claimed conjugates, the preferred linkers may have the general formula (6)

where

X is each independently selected from O, N, S, or P,

Q is selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl, preferably from substituted or unsubstituted C ₅ -C ₁₄ aryl, C ₅ -C ₁₄ alkylaryl or C ₅ -C ₁₄ cycloalkyl,

W is selected from -(CH ₂ ) _c -aryl or -(CH ₂ ) _c -heteroaryl, where c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Without wishing to be bound by any particular theory, the inventors believe that hydrophilic or polar functional groups in the linker or as side chains (in particular, Q, W) can advantageously increase the PSA-binding properties of the claimed conjugate.

Wherein the Q group is a substituted aryl, alkylaryl, or cycloalkyl, where possible substituents are listed above in the "Definitions" section and include, without limitation, halogens (ie, F, Cl, Br, and I); hydroxyls; alkoxy, alkeneoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy and heterocyclylalkoxy groups, carbonyls (oxo); carboxyls; esters; urea compounds; oximes, hydroxylamines; alkoxyamines; aralkoxyamines, thiols, sulfides, sulfoxides, sulfones, sulfonyls, amines, N-oxides, hydrazines, hydrazides, hydrazones, azides, amides, urea compounds, amidines, guanidines, enamines, imides, isocyanates, isothiocyanates, cyanates, thiocyanates, imines, nitro groups, nitriles (ie CN), haloalkyls, aminoalkyls, hydroxyalkyls, cycloalkyls.

Preferably, Q is selected from substituted or unsubstituted C ₅ -C ₇ cycloalkyl.

Preferably, W is selected from -(CH ₂ ) _c -naphthyl, (CH ₂ ) _c -phenyl, -(CH ₂ ) _c -biphenyl, -(CH ₂ ) _c -indolyl, -(CH ₂ ) _c -benzothiazolyl, where c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. More preferably, W is selected from -(CH ₂ )-naphthyl, -(CH ₂ )- phenyl, -(CH ₂ )-biphenyl, -(CH ₂ )-indolyl or -(CH ₂ )-benothiazolyl.

Preferably, each X is O.

Thus, the most preferred linker connecting the PSA-binding element with the claimed conjugate may have the following structural formula (6a)

In the conjugates claimed in the present invention and described in any of the structural formulas presented here, substituents or groups identified as provisionally substituting may be (if applicable) defined as follows.

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N''-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl )phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N '-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO ) and diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof. More preferably, D may be selected from DO TA, NODAGA or their derivatives.

X is each independently selected from O, N, S, or P. More preferably, each X is O.

R ¹ and R ² are each independently H, F, Cl, Br, I, branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, OR ⁶ , OCOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , CONR ⁶ R ⁷ , COOR ⁶ , CH ₂ NR ⁶ R ⁷ , SR ⁶ , =O, =S or =NH, or R ¹ and R ² are connected with each other to form a cyclic structure comprising a branched, straight or cyclic C ₁ -C ₁₀ hydrocarbon group, wherein said hydrocarbon group is optionally interrupted by at most 2 heteroatoms and optionally substituted by at most 3 groups independently selected from F, Cl, Br, I , OR ⁶ , OCOR ⁶ , COOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , CH ₂ NR ⁶ R ⁷ and SR ⁷ , =O, =S and =NH, while R ⁶ and R ⁷ are each independently selected from H or a branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon. More preferably, R ¹ may be H and R ² is selected from halogen, preferably iodine or bromine, and C ₁ -C ₆ alkyl, preferably C ₁ -C ₃ alkyl, even more preferably methyl. Even more preferably, R ¹ is H and R ² is H, or may be in the para position and represent iodine, bromine or methyl.

Y is preferably selected from the group consisting of a direct bond and a linear, branched or cyclic, C ₁ -C ₁₂ alkyl optionally interrupted by no more than two heteroatoms, optionally substituted with at least one halogen, branched or straight or cyclic C ₁ -C ₁₀ hydrocarbon , OR ⁶ , OCOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , COOR ⁶ , CH ₂ NR ⁶ R ⁷ , SR ⁶ , =O, =S or =NH, wherein one or more is not the adjacent CH ₂ group can be independently substituted with -O-, -CO-, -CO-O-, -O-CO-, -NR ⁶ -, -NR ⁶ -CO-, -CO-NR ⁶ -, - NR ⁶ -COO-, -O-CO-NR ⁶ -, -NR ⁶ -CO-NR ⁶ -, -CH=CH- , -C≡C-, -O-CO-O-, SR ⁶ -, SO ₃ R ⁶ -, while R ⁶ and R ⁷ each independently selected from H or a branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon. More preferably, Y may be a linear or branched optionally substituted C ₁ -C ₁₂ hydrocarbon, more preferably a linear or branched optionally substituted C ₁ -C ₁₀ hydrocarbon, even more preferably a linear or branched optionally substituted C ₁ -C ₆ hydrocarbon, most preferably a linear or a branched optionally substituted C ₁ -C ₃ hydrocarbon. More preferably Y is -(CH ₂ ) ₃ -.

R ³ , R ⁴ and R ⁵ are each independently selected from the group consisting of -COH, -CO ₂ H, -SO ₂ H, -SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H, -PO ₄ H ₂ , -C(O)-(C ₁ -C ₁₀ )alkyl, -C(O)-O(C ₁ -C ₁₀ )alkyl, -C(O)-NHR ⁸ or -C(O)- NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are each independently selected from the group consisting of H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH, -(CH ₂ ) _p -(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH-C(O)-(CH ₂ ) _q , (CH _r CH ₂ ) _t -NH-C(O)-(CH ₂ ) _p , -(CH ₂ ) _p -CO-COH, -(CH ₂ ) _p -CO-CO ₂ H, -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -COH] ₃ , -C[(CH ₂ ) _p -COH] ₃ , -(CH2) _p -C(O)NH-C[(CH ₂ ) _q -CO ₂ H] ₃ , C[(CH ₂ ) _p -CO ₂ H ] ₃ or -(CH ₂ ) _p -(C ₅ -C ₁₄ )heteroaryl, more preferably R ³ , R ⁴ and R ⁵ is -CO ₂ H.

The spacer may preferably contain one C-N bond. More preferably, the spacer may have formulas (3a), (3b) or 3(c) as disclosed here.

The linker preferably has the general formula (6) as disclosed here. More preferably, the linker may have formula (6a) as disclosed here.

Q is preferably selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl, preferably substituted or unsubstituted C ₅ -C ₁₄ aryl, C ₅ -C ₁₄ alkylaryl or C ₅ -C ₁₄ cycloalkyl.

W is preferably selected from -(CH ₂ ) _c -aryl or -(CH ₂ ) _c -heteroaryl, where c is preferably an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

A is preferably an amino acid residue. More preferably, A is selected from (D-) Aspartate, (D-) Glutamate or (L-Lysine).

V is preferably selected from a direct bond, N, or an optionally substituted C ₁ -C ₁₂ hydrocarbon containing no more than three heteroatoms, where said heteroatom is preferably N.

n is preferably an integer selected from 1, 2, 3, 4 or 5, preferably 1, 2 or 3.

m is preferably 0 or 1.

a, b, p, q, r, t are preferably each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In accordance with the above, the preferred conjugates according to the present invention have the general formula (1a):

where

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N''-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl )phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N '-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO ) and diethylenetriaminepentaacetic acid (DTPA), or derivatives thereof,

X is each independently selected from O, N, S, or P,

R ¹ and R ² are each independently H, F, Cl, Br, I, branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, OR ⁶ , OCOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , CONR ⁶ R ⁷ , COOR ⁶ , CH ₂ NR ⁶ R ⁷ , SR ⁶ , =O, =S or =NH, or R ¹ and R ² are connected with each other to form a cyclic structure comprising a branched, straight or cyclic C ₁ -C ₁₀ hydrocarbon group, wherein said hydrocarbon group is optionally interrupted by at most 2 heteroatoms and optionally substituted by at most 3 groups independently selected from F, Cl, Br, I , OR ⁶ , OCOR ⁶ , COOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , CH ₂ NR ⁶ R ⁷ and SR ⁷ , =O, =S and =NH,

Y is preferably selected from the group consisting of a direct bond or linear, branched or cyclic, C ₁ -C ₁₂ alkyl, optionally interrupted by no more than two heteroatoms, OR ⁶ , OCOR ⁶ , CHO, COR ⁶ , CH ₂ OR ⁶ , NR ⁶ R ⁷ , COOR ⁶ , CH ₂ NR ⁶ R ⁷ , SR ⁶ , ═O, ═S, or ═NH, wherein one or more non-adjacent CH ₂ groups may be independently substituted with -O-, -CO-, -CO -O-, -O-CO-, -NR ⁶ -, -NR ⁶ -CO-, -CO-NR ⁶ -, -NR ⁶ -COO-, -O-CO-NR ⁶ -, -NR ⁶ -CO -NR ⁶ -, -CH=CH-, -C≡C-, -O-CO-O-, SR ⁶ -, SO ₃ R ⁶ ,

R ⁶ and R ⁷ are each independently selected from H or a branched or straight or cyclic C ₁ -C ₁₂ hydrocarbon,

R ³ , R ⁴ and R ⁵ are each independently selected from the group consisting of -COH, -CO ₂ H, -SO ₂ H, -SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H, -PO ₄ H ₂ , -C(O)-(C ₁ -C ₁₀ )alkyl, -C(O)-O(C ₁ -C ₁₀ )alkyl, -C(O)-NHR ⁸ or -C(O)- NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are each independently selected from the group consisting of H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, Br, I, C(O), C(S), -C(S)-NH-benzyl-, -C(O)-NH-benzyl, C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH, -(CH ₂ )p -(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH-C(O)-(CH ₂ ) _q , -(CH _r CH ₂ ) _t -NH-C(O)-(CH ₂ ) _p , -(CH ₂ ) _p -CO-COH, -(CH ₂ ) _p -CO-CO ₂ H, -(CH ₂ ) _p -C(O)NH-C[(CH ₂ ) _q -COH] ₃ , -C[(CH ₂ ) _p -COH] ₃ , -(CH ₂ ) _p -C(O)NH-C[(CH ₂ ) _q -CO ₂ H] ₃ , -C[(CH ₂ ) _p - CO ₂ H] ₃ or -(CH ₂ ) _p -(C ₅ -C ₁₄ )heteroaryl,

the spacer contains at least one C-N bond,

the linker has the general formula (6) as defined above, and

a, b, p, q, r, t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

More preferably, the conjugate of the present invention may have the general formula (12.4) or (13.4):

where

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N''-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl )phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N '-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)-(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide ( DFO) and diethylenetriaminepentaacetic acid (DTPA), or their derivatives,

R ¹ and R ² are each preferably independently selected from H, halogen, preferably iodine or bromine, or C ₁ -C ₆ alkyl, preferably C ₁ -C ₃ alkyl, even more preferably methyl;

the linker has the general formula (6) as defined above, more preferably the linker has the general formula (6a) as defined above,

a, b, d, m, n are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably a and b are each independently an integer a number selected from 0, 1, 2, 3, 4, 5, or 6;

b, d and m are each independently an integer selected from 1, 2, 3, 4, 5, or 6.

More preferably, the claimed conjugates have the general formula (1b)

where

D is a chelating agent, preferably selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N''-bis[2-hydroxy-5- (carboxyethyl)benzyl]ethylenediamine-N,N''-diacetic acid (HBED-CC), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7-bis( carboxymethyl)-1,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid acid (DOTAGA), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacidononan-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl )phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9,3,1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N '-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)-(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide ( DFO) and diethylenetriaminepentaacetic acid (DTPA), or their derivatives,

Q is selected from substituted or unsubstituted aryl, alkylaryl or cycloalkyl,

W is -(CH ₂ ) _d -aryl or -(CH ₂ ) _d - heteroaryl,

R ¹ and R ² are each independently selected from the group consisting of H, F, Cl, Br, I, a C ₁ -C ₁₂ branched, linear or cyclic hydrocarbon optionally containing 2 heteroatoms and optionally substituted with no more than 3 groups independently selected from F, Cl, Br, I, branched, straight or cyclic C ₁ -C ₁₂ hydrocarbons, OR ⁷ , OCOR ⁷ , COOR ⁷ , CHO, COR ⁷ , CH ₂ OR ⁷ , NR ⁷ R ⁸ , CH ₂ NR ⁷ R ⁸ and SR ⁸ ═O, ═S and ═NH, where R ⁷ and R ⁸ are each independently H or a branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon; preferably R ¹ and R ² each independently represents H, Br, I and linear C ₁ -C ₁₂ alkyl;

R ³ , R ⁴ and R ⁵ are each independently -COH, CO ₂ H, -SO ₂ H, -SO ₃ H, -SO ₄ H, -PO ₂ H, -PO ₃ H, -PO ₄ H ₂ . -C(O)-(C ₁ -C ₁₀ )alkyl, C(O)-O(C ₁ -C ₁₀ )alkyl, -C(O)-NHR ⁸ or -C(O)-NR ⁸ R ⁹ , where R ⁸ and R ⁹ are each independently selected from the group consisting of H, bond, (C ₁ -C ₁₀ )alkylene, F, Cl, Br, I, C(O), C(S), -C(S)- NH-benzyl-, -C(O)-NH-benzyl, -C(O)-(C ₁ -C ₁₀ )alkylene, -(CH ₂ ) _p -NH, -(CH ₂ ) _p -(C ₁ - C ₁₀ )alkylene, -(CH ₂ ) _p -NH-C(O)-(CH ₂ ) _q , -(CH _r CH ₂ ) _t -NH-C(O)-(CH ₂ ) _p , -(CH ₂ ) _p -CO-COH, -(CH ₂ ) _p -CO-CO ₂ H, (CH ₂ ) _p -C (O)NH-C [(CH ₂ ) _q -COH] ₃ , -C [(CH ₂ ) _p -COH] ₃ , -(CH ₂ ) _p -C(O)NH-C[(CH ₂ ) _q -CO ₂ H] ₃ , -C[(CH ₂ ) _p -CO ₂ H] ₃ or -(CH ₂ ) _p -(C ₅ -C ₁₄ )heteroaryl,

a, b, d, p, q, r, s, and t are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and

the spacer contains at least one C-N bond,

or their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

In preferred conjugates of general formula (1b) to the values “D”, “Q”, “W”, “a”, “b”, “R ¹ ”, “R ² ”, R ³ ”, “R ⁴ ” and/ or “R ⁵ ” any of the definitions below apply, preferably at least two, more preferably at least three, even more preferably at least four, or most preferably all of the definitions below.

D may be any suitable chelating agent (eg as disclosed herein), more preferably D is DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA, or derivatives thereof. More preferably D is DOTA, NODAGA, DO3AP, DO3APPrA or DO3APABn.

Q may be selected from substituted or unsubstituted C ₅ -C ₇ cycloalkyl. W may be selected from -(CH ₂ )-naphthyl, -(CH ₂ )-phenyl, -(CH _{2 )-biphenyl, -(CH 2 )} -indolyl or -(CH ₂ )-benzothiazolyl, more preferably W is -(CH ₂ )-naphthyl.

a, b are each independently an integer selected from 0, 1, 2, 3, 4, 5, or 6.

R ¹ and R ² are each independently selected from H, iodine and C ₁ -C ₃ alkyl, and R ³ , R ⁴ and R ⁵ are each CO ₂ H.

Such preferred conjugates may have the general formula (1c):

[ ] _a

where

any one, at least two, more preferably at least three, most preferably all of the definitions below may be applicable to “D”, “a”, “R ¹ ” and/or “R ² ”:

D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof, more preferably D may be selected from DOTA, NODAGA, DO3AP, DO3APPrA or DO3APABn,

a may be an integer selected from 0, 1, 2, 3, 4, 5, or 6,

R ¹ and R ² are each independently H, iodine, or C ₁ -C ₃ alkyl, and

the spacer contains at least one C-N bond,

or their pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes.

In preferred conjugates of general formula (1c),

a can be 0, and

the spacer can be -[CHR ¹⁰ ]u-NR ¹¹ - where R ¹⁰ and R ¹¹ can each be independently selected from H and a branched, straight or cyclic C ₁ -C ₁₂ hydrocarbon, where u can be an integer selected of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In preferred conjugates of general formula (1a), the spacer has formula (3a). Accordingly, said preferred conjugates have the general formula (7a):

where

D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof, more preferably D may be selected from DOTA, NODAGA, DO3AP, DO3APPrA or DO3APABn,

R ¹ and R ² each independently be selected from H, iodine or C ₁ -C ₃ alkyl,

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

In particular, preferred conjugates according to the present invention may have the formula (7a)(i), (7a)(ii) or (7a)(iii):

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

The conjugates of formula (7a)(i) are also referred to here as "PSA-06" or "PSA-ALB-06".

The conjugates of formula (7a)(ii) are also referred to here as "PSA-03" or "PSA-ALB-03". The conjugates of formula (7a)(iii) are also referred to here as "PSA-89" or "PSA-ALB-89".

Alternatively, in preferred conjugates of general formula (1c), the spacer contains at least one amino acid residue selected from (D-/L-) aspartate, glutamate or lysine. Preferably, the spacer may contain at least 1, 2, 3, 4 or no more than 5 amino acid residues, preferably independently selected from (D-/L-) aspartate, glutamate or lysine amino acid residues.

Such conjugates may preferably contain a spacer of general formula (3b) or (3c). Thus, said preferred conjugates may have the general formula (7b):

where

D may be selected from DOTA, DOTA, HBED-CC, NOTA, NODAGA, DOTAGA, TRAP, NOPO, PCTA, DFO, DTPA or derivatives thereof, more preferably D may be selected from DOTA, NODAGA, DO3AP, DO3APPrA or DO3APABn,

R ¹ and R ² are each independently selected from H, iodine, or C ₁ -C ₃ alkyl,

A is an amino acid residue, preferably selected from (D-)Aspartate, (D)-Glutamate or (L-Lysine),

V is selected from a direct bond, N or an optionally substituted C ₁ -C ₁₂ hydrocarbon containing not more than 3 heteroatoms, where said heteroatom is preferably N,

n is an integer selected from 1, 2, 3, 4 or 5, preferably 1, 2 or 3,

and a is an integer selected from 1, 2, 3, 4, 5, or 6,

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

In particular, these conjugates may have the formula (7b)(i), (7b)(ii) or (7b)(iii):

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

The conjugates of formula (7b)(ii) are also referred to here as "PSA-05" or "PSA-ALB-05".

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

The conjugates of formula (7b)(ii) are also referred to in the description as "PSA-07" or "PSA-ALB-07".

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

The conjugates of formula (7b)(iii) are also referred to here as "PSA-08" or "PSA-ALB-08".

or their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes.

The conjugates of formula (7b)(iv) are also referred to here as "PSA-04" or "PSA-ALB-04".

The present invention also relates to conjugates of structural formula (14), (15) and (16):

Pharmaceutically acceptable salts

The present invention also relates to pharmaceutically acceptable salts of the claimed conjugates.

The process for preparing a pharmaceutical composition is well known to those skilled in the art. Pharmaceutically acceptable salts of the claimed conjugates can be prepared using standard techniques, for example, by reacting any free base and/or acid of the claimed conjugate and a stoichiometric amount of the desired salt-forming substance, acid or base, respectively.

Pharmaceutically available salts according to the present invention include salts with inorganic cations such as sodium, potassium, calcium, magnesium, zinc and ammonium, as well as salts with organic bases. Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine, diolamine, olamine, procam and tromethamine. Pharmaceutically acceptable salts according to the present invention also include salts derived from organic or inorganic acids. Suitable anions include acetate, adipate, besylate, bromide, camsylate, chloride, citrate, edisylate, estolate, fumarate, gluceptate, gluconate, glucoronate, hippurate, hyclate, hydrobromide, hydrochloride, iodine, isethionate, lactate, lactobionate, maleate, mesylate, methyl bromide, methyl sulfate, napsilate, nitrate, oleate, pamoate, phosphate, polygalacturonate, stearate, succinate, sulfate, sulfosalicylate, tannate, tartrate, terephthalate, tosylate, and triethiodide.

Complex / Non-complex forms

The present invention also relates to the conjugates described here, where the chelating agent D may be complexed with a metal ion (eg, radioactive metal), and may remain free.

The term “radionuclide” (or “radioisotope”) refers to isotopes of natural or artificial elements with an unstable neutron-proton ratio, so the isotope decays to emit particulate particles (i.e. protons (alpha radiation) or electrons (beta radiation) or electromagnetic field (gamma radiation).In other words, radionuclides undergo radioactive decay.Chelating agent D may complex with any known radionuclide.Such a radioisotope may preferably be useful for imaging or treating cancer.Such radionuclides include, but are not limited to, ⁹⁴ Tc , ⁹⁹ mTc, ⁹⁰ In, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵¹ Tb, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁶⁴ Cu, ⁶⁷ Cu, ⁵⁵ Co, ⁵⁷ Co, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ²²⁵ Ac, ²¹³ Bi, ²¹² Bi, ²¹² Pb, ²²⁷ Th, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁵² Gd, ¹⁵³ Gd, ¹⁵⁷ Gd or ¹⁶⁶ Dy. factors such as the chemical structure and chelating ability of the chelate agent D, as well as what field of application is planned for the final (complex) conjugate (i.e. diagnostic or therapeutic). In particular, beta emitters such as ⁹⁰ Y, ¹³¹ I, ¹⁶¹ Tb and ¹⁷⁷ Lu can be used for concomitant systemic radiotherapy. The use of DOTA as a chelating agent makes it possible to effectively use such radioisotopes as ⁶⁸ Ga, ^43,44,47 Sc, ¹⁷⁷ Lu, ¹⁶¹ Tb, ²²⁵ Ac, ²¹³ Bi, ²¹² Bi, ²¹² Pb.

According to some preferred embodiments of the present invention, the radionuclide is ¹⁷⁷ Lu. According to some preferred embodiments of the present invention, the radionuclide is ⁴⁴ Sc. According to some embodiments of the present invention, the radionuclide is ⁶⁴ Cu. According to some preferred embodiments of the present invention, the radionuclide is ⁶⁸ Ga.

The choice of an appropriate combination of conjugates and radionuclides is within the knowledge and competence of a person skilled in the art. In particular, according to some preferred embodiments of the present invention, the chelating agent may be DOTA and the radionuclide ¹⁷⁷ Lu. According to other preferred embodiments of the present invention, the chelating agent may be DOTA and the radionuclide ⁶⁸ Ga. According to other preferred embodiments of the present invention, the chelating agent may be DOTA and the radionuclide ⁴⁴ Sc. According to another preferred embodiment of the present invention, the chelating agent may be DOTA and the radionuclide ⁶⁴ Cu. According to another preferred embodiment of the present invention, the chelating agent may be NODAGA and the radionuclide ⁶⁴ Cu.

Esters and Prodrugs

The present invention also includes the claimed conjugates in their esterified form, in particular when the free carboxyl groups are esterified. Such esterified compounds may be prodrugs of the claimed conjugates. Suitable prodrugs in the form of esters are various alkyl esters, including saturated and unsaturated C ₈ -C ₁₈ fatty acids.

Enantiomers

The claimed conjugates may exist in certain geometric or stereoisometric forms. In addition, the compounds may also be optically active. The claimed conjugates may also include cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (L) isomers, racemic mixtures of such compounds, and other mixtures thereof. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. If, for example, a particular enantiomer of a group or conjugate is desired, it can be obtained by asymmetric synthesis or by reaction with a chiral auxiliary element, whereby the resulting diastereomeric mixture is separated and the auxiliary group is removed to obtain the pure desired enantiomer. Alternatively, if the group or conjugate contains a basic functional group, such as an amino group, or an acidic functional group, such as carboxyl, then diastereomeric salts are prepared with an appropriate optically active base or acid, followed by separation of the diastereomers by fractional crystallization or chromatography and reduction of the pure enantiomers which is well known to those skilled in the art.

A “stereoisomer” is one stereoisomer of a compound substantially free of the other stereoisomers of that compound. Therefore, a stereoisometrically pure compound having one chiral center will be completely free of the opposite enantiomer of that compound. A stereoisometrically pure compound with two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisometrically pure compound contains more than about 80% by weight of one stereoisomer and less than about 20% by weight of other stereoisomers, for example, more than 90% by weight of one stereoisomer and less than about 10% by weight of other stereoisomers, or more than the weight of one stereoisomer and less than about 5% by weight of other stereoisomers, or more than 97% by weight of one stereoisomer and less than about 3% by weight of other stereoisomers of the compound.

Thus, all formulas presented here include their enantiomers and/or stereoisomers.

Radiolabeled complexes

According to an additional aspect, the present invention also relates to the use of the claimed conjugates to obtain radiolabeled complexes. Such complexes preferably include a conjugate according to the present invention and a radionuclide. Chelating agent D preferably coordinates the radionuclide to form a radioactive complex. Suitable radionuclides may be selected from the teragnostic metal isotopes and include, but are not limited to, ⁹⁴ Tc, ^99m Tc, ⁹⁰ In, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵¹ Tb, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁶⁴ Cu, ⁶⁷ Cu, ⁵⁵ Co, ⁵⁷ Co, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ²²⁵ Ac, ²¹³ Bi, ²¹² Bi, ²¹² Pb, ²²⁷ Th, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁵² Gd, ¹⁵³ Gd, ¹⁵⁷ Gd or ¹⁶⁶ Dy.

According to an additional aspect, the present invention also relates to complexes comprising a radionuclide (preferably selected from the group presented above) and a conjugate according to the present invention.

pharmaceutical composition

According to an additional aspect, the present invention also relates to a pharmaceutical composition comprising the claimed conjugate (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes, as described here), as well as a pharmaceutically acceptable carrier and/or excipient.

The term "pharmaceutically acceptable" refers to a substance or agent that is compatible with the claimed conjugate and does not interfere with or inhibit its diagnostic and therapeutic activity. Pharmaceutically acceptable carriers preferably have a sufficiently high degree of purity and sufficiently low toxicity, which makes them suitable for administration to the patient.

Dosage forms, carriers and excipients

Pharmaceutically acceptable excipients may serve a variety of functions and include, but are not limited to, solvents, fillers, bulking agents, carriers, disintegrants, binding agents, lubricants, glidants, coating agents, solvents and co-solvents, buffering agents, preservatives, adjuvants, antioxidants. , wetting agents, defoamers, thickeners, sweeteners, flavoring agents and wetting agents.

Suitable pharmaceutically acceptable excipients are usually selected based on the dosage form (pharmaceutical) composition.

For (pharmaceutical) compositions in liquid form, suitable pharmaceutically available excipients are solvents, diluents and carriers such as (non-pyrogenic) water (isotonic) saline solutions, e.g. phosphate or citrate buffer solution, fatty oils, vegetable oils such as eg peanut oil, cottonseed oil, sesame oil, olive oil, corn oil; ethanol, polyols (eg glycerol, propylene glycol, polyethylene glycol and the like); lecithin; surfactants; preservatives such as benzyl alcohol, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like; isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride; aluminum monostearate or gelatin; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and tonicity adjusting agents such as sodium chloride or dextrose. The pH value can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Buffers can be hypertonic, isotonic, or hypotonic depending on the specific reference medium, i.e. the buffer contains a greater, equal or lesser amount of salt relative to the main medium, while preferably such concentrations of the salts mentioned above are used that do not lead to damage to the cells as a result of osmosis or other concentration effects. The reference medium is a fluid that exists in vivo , such as blood, lymph, cytosolic fluids, or other body fluids, or, for example, fluids that can be used as an in vitro reference medium, such as known buffers or fluids. Such conventional buffers or liquids are well known to those skilled in the art.

Liquid (pharmaceutical) compositions that are administered by injection, and in particular by intravenous injection, should preferably be sterile and stable during manufacture and storage. Such compositions are typically formulated as parenteral solutions which are pyrogenic free, have a suitable pH, are isotonic and maintain stability of the main ingredient(s).

For liquid pharmaceutical compositions, suitable pharmaceutically available excipients and carriers are water, usually pyrogen-free water; isotonic saline or buffered (aqueous) solutions, such as phosphate, citrate and other buffer solutions. In particular, for injectable dosage forms of the claimed (pharmaceutical) compositions, water or preferably a buffer can be used, and even more preferably an aqueous buffer, which may contain a sodium salt, for example, at least 50 mm sodium salt; a calcium salt, for example at least 0.01 mM calcium salt; and optionally a potassium salt, eg at least 3 mM potassium salt.

Salts of sodium, calcium and optionally potassium may be in the form of halides, such as chlorides, iodides or bromides; in the form of hydroxides, carbonates, hydrocarbonates or sulfates, etc. Without limitation, examples of sodium salts are NaCl, NaI, NaBr, Na ₂ CO ₃ , NaHCO ₃ , Na ₂ SO ₄ , examples of possible potassium salts are, for example, KCl, KI, KBr, K ₂ CO ₃ , KHCO ₃ , K ₂ SO ₄ , and examples of calcium salts are, for example, CaCl ₂ , CaI ₂ , CaBr ₂ , CaCO ₃ , CaSO ₄ , Ca(OH) ₂ . In addition, the organic anions of the cations mentioned above may be buffered.

Buffers suitable for the injectable dosage forms listed above may contain salts such as sodium chloride (NaCl), calcium chloride (CaCl ₂ ) and optionally potassium chloride (KCl), and additional anions may be present in addition to the chlorides. CaCl ₂ may also be substituted with another salt such as KCl. Typically, the salts in the injection buffer are present at a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl), and at least 0.01 mM calcium chloride (CaCl ₂ ).

The injection buffer may be hypertonic, isotonic or hypotonic, taking into account the specific reference environment, i.e. the buffer contains a greater, equal or lesser amount of salt relative to the main medium, while, preferably, such concentrations of the salts mentioned above are used that do not lead to damage to the cells as a result of osmosis or other concentration effects.

For (pharmaceutical) compositions in (semi-)solid dosage forms, suitable pharmaceutically available excipients and carriers are binding agents such as microcrystalline cellulose, gum tragacanth or gelatin; starch or lactose; sugars such as, for example, lactose, glucose and sucrose; starches such as, for example, corn starch or potato starch; cellulose and its derivatives; such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; disintegrants such as alginic acid; lubricants such as magnesium stearate; glidants such as steric acid, magnesium stearate; calcium sulfate, colloidal silicon dioxide and the like; sweeteners such as sucrose or saccharin; and/or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

In general, (pharmaceutical) compositions for topical administration may be in dosage forms such as creams, ointments, gels, pastes or powders. (Pharmaceutical) compositions for oral administration may be presented in dosage forms such as tablets, capsules, liquids, powders or sustained release formulations. However, in accordance with the preferred embodiments of the present invention, the claimed (pharmaceutical) compositions are administered parenterally, in particular by intravenous or intratumoral injection, therefore they are made in a liquid or lyophilized dosage form intended for parenteral administration, as described above. Dosage forms for parenteral administration are usually stored in vials, IV bags, ampoules, cartridges or pre-filled syringes and can be administered by injection, inhalation or aerosol, with the injection route being preferred.

The (pharmaceutical) composition may be presented in lyophilized form. Lyophilized (pharmaceutical) compositions are preferably reconstituted in a suitable buffer on an effective aqueous vehicle prior to administration.

The (pharmaceutical) compositions preferably contain a safe and effective amount of the claimed conjugate(s) or radiolabeled complexes(a).

As used herein, the term "safe and effective amount" refers to that amount of the agent(s) that is sufficient to establish a diagnosis and/or produce significant beneficial changes in the course of the disease for which the agent is being administered. At the same time, however, the “safe and effective amount” is small enough to avoid the occurrence of serious adverse reactions, i.e. in fact, allows you to achieve a reasonable balance between benefits and risks. In addition, a "safe and effective amount" will differ depending on the specific condition being diagnosed or treated, as well as the age and physical condition of the patient, the severity of the disease, the duration of treatment, the patient's concomitant therapy, and the nature of the particular used in the composition. a pharmaceutically acceptable excipient or carrier, and the like.

The claimed conjugates are also contemplated for use in the preparation of a medicament, preferably for the treatment of cancer, in particular for the treatment and/or prevention of prostate cancer, pancreatic cancer, kidney cancer or bladder cancer.

Kit

According to a further aspect, the present invention also relates to a kit comprising the claimed conjugate(s) (including pharmaceutically acceptable salts, esters, solvates or radiolabeled complexes) and/or pharmaceutical composition(s) according to the present invention.

If necessary, the kit may contain at least one additional agent from the list given for the pharmaceutical composition, including a radionuclide, an antimicrobial agent, a stabilizing agent, and similar compounds.

The kit may consist of two or more parts, each of which contains any of the components described for example above, in suitable containers. For example, each container may be in the form of vials, bottles, soft bottles, jars, sealed bags, envelopes or pouches, tubes and blister packs, or any other suitable form, such containers preventing premature mixing of the components. Each of the various components may be provided separately, or some of the various components may be provided together (ie, in the same container).

The container may also be a compartment or chamber within the package itself, such as a vial, tube, jar or envelope, or sachet, or blister pack or bottle, such that the contents of one compartment are unable to physically come into contact with the contents of another compartment before they are deliberately mixed by a pharmacist or the attending physician.

The kit or parts of the kit may also contain technical instructions, including information about the introduction and dosage of any of the components.

Therapeutic and diagnostic methods and their uses

According to an additional aspect, the present invention relates to the claimed conjugates (including their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes), pharmaceutical compositions or kits intended for use in medicine and/or diagnostics. Preferably, such claimed conjugates, pharmaceutical compositions or kits are used for human medical purposes. Thus, the present invention also relates to said claimed conjugates, pharmaceutical composition or kit for use as a medicinal product.

The claimed conjugates preferably have the ability to selectively bind to the membrane of the prostate-specific antigen (PSA). According to one embodiment, the present invention also relates to claimed conjugates, pharmaceutical compositions or kits for use in a method for detecting the presence of cells and/or tissues expressing a prostate-specific membrane antigen (PSA).

PSA is expressed mainly by malignant cancer cells. As used herein, the term "cancer" refers to neoplasms characterized by uncontrolled and usually rapid proliferation of cells that tend to invade surrounding tissues and also have the ability to metastasize to distant regions of the human body. The term combines both benign and malignant neoplasms. Malignancy in cancer is usually characterized by features such as anaplasia, invasiveness, and metastasis, while benign tumors usually do not have these properties. The term also refers to neoplasms characterized by tumor growth, as well as cancers of the circulatory and lymphatic systems.

In particular, PSA may be overexpressed in prostate, pancreatic, kidney, or bladder cancer cells.

In accordance with another embodiment, the present invention relates to the claimed conjugates (including their pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes), a pharmaceutical composition or a kit intended for use in a method for the diagnosis, treatment and/or prevention prostate cancer, pancreatic cancer, kidney cancer, or bladder cancer.

The term "diagnosis" or "diagnosis" refers to the process of identifying a disease by its characteristic symptoms and manifestations and/or, as in this case, to the analysis of biological markers (such as proteins or genes) associated with the disease.

The term “treatment” of a disease includes the processes of preventing or preventing the disease (which essentially means preventing the development of clinical symptoms); inhibition of the disease (ie cessation or suppression of the development of clinical symptoms); and/or alleviating the course of the disease (ie, initiating regression of existing symptoms). It should be kept in mind that it is not always possible to distinguish between the processes of "prevention" and "suppression" of a disease or pathological condition until the final indicator signs or events are unknown or hidden. Accordingly, the term "prevention" obviously refers to a type of "treatment" that combines "prevention" and "suppression". Thus, the term "treatment" also includes the term "prophylaxis".

The terms "subject", "patient", or "individual" as used herein generally refer to humans and non-human animals, preferably mammals (e.g., non-human primates, including marmosets, tamarins, spider monkeys, owl-faced monkeys, green monkeys, squirrel monkeys, and baboons). , macaques, chimpanzees, orangutans, gorillas, cows, horses, sheep, pigs, chickens, cats, dogs, mice, rats, rabbits, guinea pigs, etc.), including chimeric and transgenic animals and disease models. In the context of the present invention, the term “subject” preferably refers to non-human primates or humans, more preferably humans.

The methods and methods described herein and related to the diagnosis, treatment or prevention of cancer, in particular prostate cancer, pancreatic cancer, kidney cancer or bladder cancer, preferably may include the steps of (a) administering the claimed conjugate (including its pharmaceutically acceptable salts, esters, solvates, and radiolabeled complexes), pharmaceutical composition or kit to a patient, and (b) obtaining an x-ray image of said patient's body.

The claimed conjugates, pharmaceutical compositions or kits are usually administered parenterally. The introduction can be carried out systemically, in particular by intravenous (in/in), subcutaneous, intramuscular or intradermal injection. Alternatively, the administration may be carried out locally, in particular by intratumoral injection.

The claimed conjugates, pharmaceutical compositions or kits can be administered to a subject in need thereof several times a day, daily, every other day, weekly or monthly. Preferably, the treatment, diagnosis, or prophylaxis is effective at effective doses of the claimed conjugates, pharmaceutical compositions, or kits.

Effective doses of the claimed conjugates can be determined using routine techniques, for example, using animal models. Such animals include, but are not limited to, rabbits, sheep, mice, rats, dogs, and non-human primates. Therapeutic efficacy and toxicity of the claimed conjugates or radiolabelled complexes can be determined using standard pharmaceutical procedures adopted, for example, to determine the LD50 (dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective for 50% of the population) and carried out in cell culture or experimental animals. The ratio of toxic and therapeutic effects can be expressed as a therapeutic index and presented as a ratio of LD50/ED50. Data obtained from cell culture studies or animal studies can be used to determine the range of doses for humans. Doses of the claimed conjugates are preferably in the range of circulating concentrations that include the ED50 with little or no toxicity.

For example, therapeutically or diagnostically effective doses of the claimed conjugates may be in the range of about 0.001 mg to 10 mg, preferably in the range of about 0.01 mg to 5 mg, more preferably in the range of about 0.1 mg to 2 mg per dose unit. or about 0.01 nmol to 1 mmol per dose unit, in particular 1 nmol to 1 mmol per dose unit, preferably 1 micromol to 1 mmol per dose unit. It is also contemplated that therapeutically or diagnostically effective doses of the claimed conjugates may be in the range (per kg of body weight) from about 0.01 mg/kg to 10 g/kg, preferably from about 0.05 mg/kg to 5 g/kg, more preferably from about 0.1 mg/kg to 2.5 g/kg. Due to their favorable pharmacokinetic properties, the claimed conjugates can preferably be administered at lower doses than other PSA ligands, which is their advantage.

As stated above, the present invention allows the claimed conjugates to be used for therapeutic purposes, including targeting PSA-expressing cells. As used herein, the term “theragnostic” refers to “only diagnostic”, “only therapeutic”, as well as “treatment-diagnostic” methods. According to a further aspect, the present invention relates to an in vitro method for detecting the presence of cells and/or tissues expressing a prostate-specific membrane antigen (PSA), comprising (a) contacting said cells and/or tissues expressing a prostate-specific membrane antigen (PSA) with the claimed conjugates (including their pharmaceutically acceptable salts, esters, solvates, and radiolabeled complexes), pharmaceutical compositions or kits, and (b) using imaging techniques to detect said cells and/or tissues, optionally radiological imaging.

In the in vivo and in vitro methods and methods claimed in accordance with the present invention, radiological imaging can be performed using any means and methods known to specialists in this field. Preferably, radiological imaging means positron emission tomography (PET-CT) or single photon emission computed tomography (SPECT = SPECT). Target cells or tissues examined by X-ray imaging using the subject conjugates preferably include (possibly tumor) prostate cells or tissues, spleen cells or tissues (also possibly tumor) or kidney tissues and cells (also possibly tumor).

According to the in vivo and in vitro methods and methods claimed in accordance with the present invention, the detection of PSA-expressing cells or tissues may indicate the presence of prostate tumor(s), metastatic prostate tumor(s), kidney tumor(s), tumor (cells) of the bladder, as well as the presence of a combination of these tumors. Therefore, the claimed conjugates (including pharmaceutically acceptable salts, esters, solvates and radiolabeled complexes), pharmaceutical compositions and kits can be used specifically for the diagnosis (and if necessary treatment) of prostate cancer, kidney cancer, pancreatic cancer and bladder cancer. .

DESCRIPTION OF FIGURES

FIGURE 1:Chromatograms for (A)¹⁷⁷Lu-PSA-ALB-01, (B) ¹⁷⁷Lu-PSA-ALB-03, (B)¹⁷⁷Lu-PSA-ALB-04, (G)¹⁷⁷Lu-PSA-ALB-05, (D)¹⁷⁷Lu-PSA-ALB-06, (E)¹⁷⁷Lu-PSA-ALB-07, i (F)¹⁷⁷Lu-PSA-ALB-08 labeled with activity of 50 MBq/nmol obtained by quality control of compounds using HPLC.

FIGURE 2 : Partition coefficient of n-octanol/PBS for ¹⁷⁷ Lu-PSA-ALB-01 (n=3), ¹⁷⁷ Lu-PSA-ALB-03 (n=3), ¹⁷⁷ Lu-PSA-ALB-04 (n= 1), ¹⁷⁷ Lu-PSA-ALB-05 (n=1), ¹⁷⁷ Lu-PSA-ALB-06 (n=1), ¹⁷⁷ Lu-PSA-ALB-07 (n=1), ¹⁷⁷ Lu-PSA- ALB-08 (n=1) compared to control ¹⁷⁷ Lu-PSA-617 (n=3).

FIGURE 3: UF analysis data for¹⁷⁷Lu-PSA-ALB-01 (n=2), ¹⁷⁷Lu-PSA-ALB-03 (n=2),¹⁷⁷Lu-PSA-ALB-04 (n=1),¹⁷⁷Lu-PSA-ALB-05 (n=1),¹⁷⁷Lu-PSA-ALB-06 (n=2),¹⁷⁷Lu-PSA-ALB-07 (n=2),¹⁷⁷Lu-PSA-ALB-08 (n=2) vs. control¹⁷⁷Lu-PSA-617 (n=2).

FIGURE 4: Capture and internalization¹⁷⁷Lu-PSA-ALB-01 (n=2), ¹⁷⁷Lu-PSA-ALB-03 (n=2),¹⁷⁷Lu-PSA-ALB-04 (n=1),¹⁷⁷Lu-PSA-ALB-05 (n=1),¹⁷⁷Lu-PSA-ALB-06 (n=2),¹⁷⁷Lu-PSA-ALB-07 (n=2),¹⁷⁷Lu-PSA-ALB-08 (n=2) vs. control¹⁷⁷Lu-PSA-617 (n=3). (A&B) Data obtained from PSA-positive PC-3 PIP cells. (B&D) and for PSA-negative PC-3 flu cells.

FIGURE 5: Biodistribution data for compounds ¹⁷⁷ Lu-PSA-ALB-01 and ¹⁷⁷ Lu-PSA-ALB-03 (A), ¹⁷⁷ Lu-PSA-ALB-04 and ¹⁷⁷ Lu-PSA-ALB-05 (B) and ¹⁷⁷ Lu-PSA-ALB-06, ¹⁷⁷ Lu-PSA-ALB-07 and ¹⁷⁷ Lu-PSA-ALB-08 (C) in tissues of PC-3 PIP/flu tumor-bearing mice.

FIGURE 6 : Final data on (A) tumor capture (B), tumor/flow ratio (C), tumor/kidney ratio (D) and tumor/liver for ¹⁷⁷ Lu-PSA-ALB-01-08.

FIGURE 7 : Scintigraphic images at different time points after administration.

FIGURE 8 : SPECT-CT images of various sites after drug administration.

FIGURE 9 : PET-CT images obtained 1 and 3 hours after administration of the compound PSA-ALB-06 labeled with radioactive Gallium-68.

FIGURE 10 : (A) Data on the biodistribution of compound ⁴⁴ Sc-PSA-ALB-06 in PC-3 PIP/flu tumor-bearing mice at 1, 4 and 6 hours after administration. (B) Data on the biodistribution of compound ¹⁷⁷ Lu-PSA-ALB-06 in PC-3 PIP/flu tumor-bearing mice at 1, 4 and 24 hours after administration.

FIGURE 11 : PET/CT images of PC-3 PIP/flu tumor-bearing mice rendered using the maximum intensity projection (MIPs) algorithm with one scale for all time intervals. (A) PET/CT scan taken 1 hour after injection of ⁴⁴ Sc-PSA-ALB-06. (B) PET/CT scan obtained 4 hours after injection of ⁴⁴ Sc-PSA-ALB-06, (C) PET/CT scan obtained 20 hours after injection of ⁴⁴ Sc-PSA-ALB-06.

FIGURE 12 : PET/CT images of PC-3 PIP/flu tumor-bearing mice presented using the maximum intensity projection (MIPs) algorithm with one scale for all time intervals. (A/B) PET/CT scan taken one hour after injection of ⁴⁴ Sc-PSA-ALB-06.

FIGURE 13 : PET/CT images of PC-3 PIP/flu tumor-bearing mice presented using the maximum intensity projection (MIPs) algorithm with one scale for all time intervals. (A/B) PET/CT scan obtained 20 hours after injection of ⁴⁴ Sc-PSA-ALB-06.

FIGURE 14 : Semi-log graphs plotted from ultrafiltration data to calculate B ₅₀ values of ⁶⁴ Cu-PSA-ALB-06 (B ₅₀ =770) and ⁶⁴ Cu-PSA-ALB-89 (B ₅₀ =454) after incubation in human plasma with different protein concentrations (mean ± SD, n≥3).

FIGURE 15: Cellular uptake and internalization (mean ± SD, n=3) of ⁶⁴ Cu-PSA-ALB-89 and ⁶⁴ Cu-PSA-ALB-06 (A) PSA-positive PC-3 PIP cells and (B) PSA- negative PC-3 flu cells.

FIGURE 16: Tissue biodistribution profile of compound ⁶⁴ Cu-PSA-ALB-89 administered to Balb/c nude mice inoculated with PC-3 PIP and PC-3 flu tumor xenografts at 1 h, 4 h and 24 h after injection. Values are mean±SD of values obtained for n=3-6 mice.

FIGURE 17: PET/CT images rendered using the maximum intensity projection algorithm. (A-D) PET/CT images of mice at 1 h, 4 h, 16 h and 24 h after ⁶⁴ Cu-PSA-ALB-89 injection. The scale was built by cutting off 2% of the background for better visualization of tumors, kidneys and liver. (PSA+ = PC-3 PIP tumor xenograft; PSA- = PC-3 flu tumor xenograft; Ki = kidney; Li = liver; Bl = bladder).

FIGURE 18 : PSA-binding precursor used for the synthesis of PSA-ALB-02/-05/-07.

FIGURE 19 : Chemical structure of (A) PSA-ALB-02, (B) PSA-ALB-05, and (C) PSA-ALB-07.

FIGURE 20: Graphs showing the stability of 177Lu-PSA-ALB-02, ¹⁷⁷ Lu-PSA-ALB-05, and ¹⁷⁷ Lu-PSA-ALB-07, and 177Lu-PSA-617 over a 24-hour time period in (A) the absence and (B) the presence of ascorbic acid. Values are mean ± SD of three independent measurements.

FIGURE 21: Capture and internalization of ¹⁷⁷ Lu-PSA-ALB-02, ¹⁷⁷ Lu-PSA-ALB-05, and ¹⁷⁷ Lu-PSA-ALB-07 compared to ¹⁷⁷ Lu-PSA-617. (A) Data obtained from PSA-positive PC-3 PIP cells. The bars show the mean ± SD for three independent measurements taken three times. (B) Data obtained from PSA-negative PC-3 PIP cells. Bars show mean ± SD for three independent measurements taken three times.

FIGURE 22 : Biodistribution data (corrected for radioactive decay) up to 192 hours after compound injection obtained for all three ¹⁷⁷ Lu-PSA albumin-binding ligands as well as ¹⁷⁷ Lu-PSA-617. (A) Biodistribution data for ¹⁷⁷ Lu-PSA-ALB-02, (B) ¹⁷⁷ Lu-PSA-ALB-05, (C) ¹⁷⁷ Lu-PSA-ALB-07, and (D) ¹⁷⁷ Lu-PSA-617. Values are mean±SD of values obtained for n=3-6 mice.

FIGURE 23 : Graphs showing biodistribution data (not corrected for radioactive decay) 192 hours after administration of (A) ¹⁷⁷ Lu-PSA-ALB-02, (B) ¹⁷⁷ Lu-PSA-ALB-05, and (C) ¹⁷⁷ Lu -PSA-ALB-07. Each point on the graph represents the mean ± SD of the values obtained for n = 3-6 mice.

FIGURE 24: SPECT/CT images presented using the maximum intensity projection algorithm (MIPs) of PC-3 PIP/flu tumor-bearing mice 24 h after administration of (A) ¹⁷⁷ Lu-PSA-ALB-02, (B) ¹⁷⁷ Lu -PSA-ALB-05, and (C) ¹⁷⁷ Lu-PSA-ALB-07. PSA ⁺ = PSA-positive PC-3 PIP tumor; PSA ⁻ = PSA-negative PC-3 flu tumor; Ki = kidney; Bl = bladder; Li = liver.

FIGURE 25 : (A/B/C) SPECT/CT images rendered by maximum intensity projection (MIPs) of PC-3 PIP/flu tumor bearing mice at 4 h (A), 24 h (B), and 72 h (B) after injection of ¹⁷⁷ Lu-PSA-ALB-02. (D, E, F) SPECT/CT images rendered using the maximum intensity projection (MIPs) algorithm of PC-3 PIP/flu tumor-bearing mice at 4 h (D), 24 h (E), and 72 h (F) after the introduction of ¹⁷⁷ Lu-PSA-617. PSA ⁺ = PSA-positive PC-3 PIP tumor; PSA ⁻ = PSA-negative PC-3 flu tumor; Ki = kidney; Bl = bladder; Li = liver.

FIGURE 26 : SPECT/CT images presented using the maximum intensity projection (MIPs) algorithm of PC-3 PIP/flu tumor-bearing mice at different time points after administration of ¹⁷⁷ Lu-ALB-03 and ¹⁷⁷ Lu-PSA-ALB-06 ( A - B). MIPs for mice at (A) 4 h, (B) 24 h, and (C) 72 h after administration of ¹⁷⁷ Lu-ALB-03 (25 MBq, 1 nmol). (D-F) MIPs for mice at (D) 24 h, (E) 24 h and (F) 72 h after administration of ¹⁷⁷ Lu-PSA-ALB-06 (25 MBq, 1 nmol). PSA ⁺ = PSA-positive PC-3 PIP tumor; PSA ⁻ = PSA-negative PC-3 flu tumor; Ki = kidney; Bl = bladder;

FIGURE 27: Study of therapy of PC-3 PIP tumor bearing mice with ¹⁷⁷ Lu-PSA-ALB-06 and ¹⁷⁷ Lu-PSA-617. (A) Tumor growth curves versus tumor volume on Day 0 (taken as 1) for mice treated with saline (Group A), mice treated with 2 MBq 177Lu-PSA-617 (Group B), 5 MBq ¹⁷⁷ Lu- PSA-617 (Group C), 2 MBq ¹⁷⁷ Lu-PSA-ALB-06 (Group D), and 5 MBq ¹⁷⁷ Lu-PSA-ALB-06 (Group E). The data were collected for the period of time before the death of the first animal from the respective group. (B) Kaplan-Meir plot for Groups A-E. (C) Relative body weight for Groups A-E.

EXAMPLES

Example 1: Getting and in vivo analysis of DOTA-functionalized Albumin-binding PSA ligands

1.1 MATERIALS AND METHODS

1.1.1 NEW PSA LIGANDS (REVIEW)

All seven variants of PSA ligands with a portable albumin-binding molecule were synthesized using solid-phase synthesis, which has been shown to be the most suitable for obtaining the albumin-affinity PSA ligands described above.

Multi-step synthesis (19 steps for PSA-ALB-01, 17 steps for PSA-ALB-03, 20 steps for PSA-ALB-04 and PSA-ALB-05, 17 steps for PSA-ALB-06, 23 steps for PSA- ALB-07 and PSA-ALB-08) made it possible to obtain these compounds in an isolated state with a total product yield of 26-49%. The crude products were purified by semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC - Reverse-Phase High Performance Liquid Chromatography - HPLC with reversed phase), the degree of purification of the final compounds was >98%. The study of the compounds described above was carried out using analytical RP-HPLC and MALDI-MS (Matrix-assisted laser desorption) or ESI-MS (Spray ionization mass spectrometry), respectively. The results of the analysis are presented in Table 1.1.

^a Mass spectrometry of unlabeled ligand, defined as [M+H]⁺;

^b Retention time of unlabeled ligand in analytical RP-HPLC. An analytical column (100 x 4.6 mm) used a Chromolith RP-18e stationary phase with mobile phases consisting of 0.1% TFA in water (A) and ACN (B). For the assay run, a linear gradient of solvent A (90-10% in 10 min) in solvent B was used at a flow rate of 1 ml/min.

et al, JNM 2015, 56: 914-920. Два указанных промежуточных этапа синтеза, позволяющие получить предшественник ПСА-лиганда, применяли аналогично для всех остальных соединений (этап 1-8). Однако, последний связывающий блок линкерного участка ПСА-АЛБ-01 [транс-4-азидоциклогексанкарбоксильная кислота, этап 9-10] был заменен транс-4-(Fmoc-аминометил) циклогексан-карбоксильной кислотой (этап 9-10) во время синтеза ПСА-АЛБ-03/04/05/06/07/08.A peptidomimetic pharmacopore for PSA (L-Glu-NH-CO-NH-L-Lys binding element; step 1-6) was synthesized in analogy to the synthesis described in Eder et al, Bioconjug. Chem. 2012, 23: 688-697. The linker molecule (2-naphthyl-L-Ala-NH-CO-trans-CHX-N3 or 2-naphthyl-L-Ala-NH-CO-trans-CHX-Me-NH2; step 7-10) was synthesized using a standard Fmoc (9-fluoroenylmethyloxycarbonyl) protocol as previously described in

et al, JNM 2015, 56: 914-920. These two intermediate steps in the synthesis, allowing to obtain the precursor of the PSA ligand, were used similarly for all other compounds (step 1-8). However, the last binding block of the linker region of PSA-ALB-01 [trans-4-azidocyclohexanecarboxylic acid, step 9-10] was replaced by trans-4-(Fmoc-aminomethyl) cyclohexane-carboxylic acid (step 9-10) during the synthesis of PSA -ALB-03/04/05/06/07/08.

PSA-ALB-01

For the synthesis of PSA-ALB-01, an economical head-to-tail combination of a purified PSA precursor with free azido groups and a purified albumin-binding molecule [4-(p-iodophenyl)butyric acid-L-Lys] with the compound propargyl-Glu (step 11-17). After effectively fusing the two precursors via the triazole ring (step 18), further purification was performed to remove excess CuSO4⋅5H2O. As a result, PSA-ALB-01 was obtained by conjugation with the chelating agent DOTA in the form of its active ester (DOTA-NHS ester; step 19).

The structural formula of PSA-ALB-01 is shown below:

PSA-ALB-03

To obtain PSA-ALB-03, direct one-sided synthesis on a polymer substrate was used. Next, Fmoc-L-Lys(Alloc)-OH was added to the PSA precursor, Fmoc was deprotected, tris(tBu)-ester was conjugated with DOTA, Alloc was deprotected, and 4-(p-iodophenyl)butyric acid was conjugated (step 11 -sixteen). As a result, PSA-ALB-03 was obtained by mixing and subsequent separation from the polymer substrate using a mixture of TFA:TIPS:H2O (step 17).

The structural formula of PSA-ALB-03 is shown below:

PSA-ALB-04

To prepare PSA-ALB-04, an economical head-to-tail synthesis was used, involving the addition of a resin-coated PSA precursor to DOTA-conjugated L-Lys and a purified albumin-binding element [4-(para-iodophenyl)butyric acid -L-Liz] via direct conjugation of two secondary amines (steps 11-18). After efficiently combining these two precursors using bis(N-hydroxysuccinimide) suberic acid ester (step 19), PSA-ALB-04 was obtained by mixing and then separating from the resin with TFA:TIPS:H2O (step 20).

The structural formula of PSA-ALB-04 is shown below:

PSA-ALB-05

To obtain PSA-ALB-05, direct one-sided synthesis on a polymer substrate was used. After addition of Fmoc-L-Lys(Alloc)-OH to the PSA precursor, deprotection of Fmoc groups, conjugation of Fmoc-D-Asp-OtBu, deprotection of Fmoc groups and conjugation of 4-(p-iodophenyl)butyric acid, deprotection of Alloc groups and DOTA tris(tBu)-ether conjugation (steps 11-19). PSA-ALB-05 by mixing and then separating from the polymeric substrate with a mixture of TFA:TIPS:H2O (step 20).

The structural formula of PSA-ALB-05 is shown below:

PSA-ALB-06

To obtain PSA-ALB-06, direct one-sided synthesis on a polymer substrate was used. After addition of Fmoc-L-Lys(Alloc)-OH to the PSA precursor, Fmoc deprotection, conjugation, tris(tBu)-ester DOTA, Alloc deprotection, and p-(tolyl)butyric acid conjugation were performed (steps 11- sixteen). As a result, PSA-ALB-06 was obtained by mixing and subsequent separation from the polymer substrate using a mixture of TFA:TIPS:H2O (step 17).

The structural formula of PSA-ALB-06 is shown below:

PSA-ALB-07

To obtain PSA-ALB-07, direct one-sided synthesis on a polymer substrate was used. After addition of Fmoc-L-Lys(Alloc)-OH to PSA precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, deprotection Fmoc, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, 4-(p-iodophenyl)butyric acid conjugation, Alloc deprotection, and DOTA tris(tBu)-ester conjugation (step 11-22). As a result, PSA-ALB-07 was obtained by mixing and subsequent separation from the polymer substrate using a mixture of TFA:TIPS:H2O (step 23).

The structural formula of PSA-ALB-07 is shown below:

PSA-ALB-08

To obtain PSA-ALB-08, direct one-sided synthesis on a polymer substrate was used. After addition of Fmoc-L-Lys(Alloc)-OH to PSA precursor, Fmoc deprotection, Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, second Fmoc-D-Asp-OtBu conjugation, deprotection Fmoc, third Fmoc-D-Asp-OtBu conjugation, Fmoc deprotection, (p-tolyl)butyric acid conjugation, Alloc deprotection, and DOTA tris(tBu)-ester conjugation (step 11-22). As a result, PSA-ALB-08 was obtained by mixing and subsequent separation from the polymer substrate using a mixture of TFA:TIPS:H2O (step 23).

The structural formula of PSA-ALB-08 is shown below:

1.1.2 SYNTHESIS OF PSA-ALB-03-08 (Detailed)

a) Synthesis of the binding element Glutamate-urea-Lysine.

Initially 2-Chlorothrityl chloride resin {(2-CT-Resin; Merck; catalog number 8550170005), 0.30 mmol, displacement power 1.63 mmol/g, 100-200 MESH, 1% DVB, total swelling volume in CH2Cl2 >4.2 ml/ g, [184 mg]} in a 5 ml syringe with a filter and a combi-stopper was stirred with anhydrous dichloromethane (DCM) for 45 min.

The 2-CT resin was then washed three times with anhydrous DCM followed by reaction with 1.2 eq. of Alloc (N-allyloxycarbonyl) as well as Fmoc (N-fluoroenylmethoxycarbonyl)-protected L-Lysine {(Fmoc-Lys(Alloc)-OH; Merck ; catalog number 8521240005), 0.36 mmol, 452.50 g/mol, [163 mg], (1)} and 4.8 equiv. N,N-diisopropylethyleneamine {(DIPEA), 1.44 mmol, 129.24 g/mol, 0.742 g/ml, [251 μL]} in 3 ml anhydrous DCM.

The binding of the first protected amino acid on the resin (2) proceeded for a 16 hour course with light agitation. The resin with immobilized L-lysine (2) was washed three times with DSM and three times with DCM2. The unreacted chlorotrityl groups remaining on the resin were washed five times with a 17:2:1 mixture of DCM, methanol (MeOH) and DIPEA (20 ml).

The Alloc and Fmoc-protected L-lysine resin was washed successively three times with DCM1, three times with DCM2 and three times with N,N-dimethylformamide (DMF1), and finally three times with DMF2. Selective removal of Fmoc-protected groups was carried out by washing a mixture of DMF and piperidine in a ratio of 1:1, first once for 2 minutes, then again for 5 minutes to obtain the product (3). The Alloc-protected L-lysine was then washed three times with DMF1, three times with DMF2, three times with DCM1, and finally three times with DCM2.

At the next stage, 10 equivalents of tBu-protected L-glutamate hydrochloride {(H-Glu(OtBu)-OtBu⋅HCl; Merck; catalog number 8540960005), 3.0 mmol, 295.8 g/mol, [ 887 mg], I} An appropriate amount of tBu-protected L-glutamate was dissolved in 150 ml DCM2 followed by the addition of 3 ml DIPEA.

The resulting solution was added dropwise over 4 h to a flask with 1 mmol of ice-cold bis(trichloromethyl)carbonate {(BTC; Sigma; catalog number 15217-10G), 296.75 g/mol, [297 mg], III} in 5 ml of dry DCM.

Then, one portion of the resin with immobilized L-lysine with one free NH2 group (3) was added to the isocyanate solution with a molecule of glutamyl III and intensively stirred for 16 h to obtain the compound bis(tBu)-Glu-urea-Lys(Alloc ) (four).

The obtained product (4) on a resin support was filtered and washed three times with DCM1 and three times with DCM2.

Cleavage of Alloc-protective groups was carried out by interaction with 0.15 eq TPP Pd {[tetrakis(triphenylphosphine)palladium (0); Sigma; Catalog number 216666-1G], 0.045 mmol, 1155.56 g/mol, [105 mg]} in the presence of 15 equiv morpholine {4.5 mmol, 87.12 g/mol, 0.999 g/ml, [392 µl]} in DCM. The total amount of Pd and morpholine was divided into two portions and a successful reaction was carried out by shaking each reagent for 1 hour. The reaction was carried out in the dark using aluminum foil.

The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. To remove residual palladium, the resin was washed an additional ten times with 1% DIPEA in DMF (300 µl DIPEA in 30 ml DMF2) and then another 10 times for 5 minutes with cupral solution {(sodium diethyldithiocarbamate trihydrate; Sigma; catalog number D3506-100G), 225.31 g/mol} in DMF2 at a concentration of 15 mg/ml (450 mg cupral in 30 ml DMF2). Resin-immobilized and bis(tBu)-protected Glu-urea-Lys (5) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and finally three times with diethyl ether (Et2O) and dried in vacuo.

The prostate-specific membrane antigen (PSA) binding element (5) obtained in this way was then used in the next reaction for the synthesis of all seven compounds (PSA-ALB-01/03/04/05/06/07/08).

A brief description of the entire process for the synthesis of bis(tBu)-protected Glu-urea-Lys pharmacopor described above is shown in Scheme 1.1.

Scheme 1.1: Synthesis of Glutamate-Urea-Lysine binding element for PSA-ALB - 01/03/04/05/06/07/08.

The resin-immobilized bis (tBu)-protected coupler (5) was first stirred in anhydrous DCM for 45 minutes. The pre-swollen pharmacophore was washed three times with DCM2, three times with DMF1, and three times with DMF2.

b) Synthesis of the linker site

Relative to resin (0.1 mmol), 4 eq Fmoc-protected 2-naphthyl-L-alanine {(Fmoc-2Nal-OH; Bachem; catalog number B-2100), 0.40 mmol, 437.50 g/mol, [175.0 mg ]}, corresponding to the first structural block of the linker site, activated 3.96 eq. HBTU {(O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; Sigma; catalog number 12804-25G-F), 0.39 mmol, 379.24 g/mol, [147.9 mg ]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [71 μl]} in anhydrous DMF.

Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected pharmacopore (3) immobilized on the resin preliminarily swollen in DMF and stirred for 1 h.

Sequentially, the resin with bis(tBu)-protected Glu-urea-Lysine and Fmoc-protected 2-naphthyl-L-alanine (6) was washed three times with DMF1 and three times with DMF2.

In the next step, 4 equivalents of the second building block, which is azidocyclohexanecarboxylic acid {(N3-1,4-trans-CHC-OH; Iris Biotech; catalog number HAA2235.0001), 0.40 s, 169.18 g/mol, [67.7 mg ]} for PSA-ALB-01 or for Fmoc-protected tranexamic acid {(trans-4-(Fmoc-aminomethyl)cyclohexane-carboxylic acid; Sigma; catalog number 58446-5G-F), 0.40 mmol, 379.45 g/mol , [151.8 mg]} for PSA-ALB-03/04/05/06/07/08 was activated with 3.96 eq HBTU {(Sigma; Catalog number 12804-25G-F), 0.39 mmol, 379.24 g/mol, [147.9 mg ]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [71 μl]} in anhydrous DMF. Two minutes after adding DIPEA, the solution was added to compound (7) pre-swollen in DMF and stirred for 1 hour.

Successively, the resin with bis (tBu)-protected Glu-urea-Lys-2-nafityl-L-alanine and azidocyclohexanecarboxylic acid (8A) was washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and finally three times with Et ₂ O and dried in a vacuum. The final PSA precursor (9A) was obtained by mixing followed by cleavage from the resin for 2 hours with a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TPS) and H ₂ O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in acetonitrile (ACN) and water in a ratio of 1:1 and purified by RP-HPLC.

Additionally, the resin with bis (tBu)-protected Glu-urea-Lys-2-nafityl-L-alanine and Fmoc-protected tranexamic acid (8B) was washed three times with DMF1 and three times with DMF2. Selective cleavage of Fmoc-protected groups from compound (8B) was carried out by washing off a 1:1 mixture of DMF and piperidine once for 2 min and then again for 5 min to obtain products (9B).

The synthesis process described above is briefly depicted in Scheme 1.2.

Scheme 1.2: Synthesis of the Linker site, precursor for PSA-ALB-03/04/05/06/07/08.

c) Synthesis of PSA-ALB-03

Relative to the amount of lysine-coated PSA precursor (9B), 4 equiv of Fmoc- and also Alloc-protected L-lysine ({(Fmoc-Lys(Alloc)-OH; Merck; Cat. No. 8521240005), 0.40 mmol, 452.50 g /mol, [181 mg]} which was activated with 3.96 equiv HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 ]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF Two minutes after the addition of DIPEA, the solution was added to the bis (tBu)-protected PSA precursor (9B) immobilized on the resin pre-swollen in DMF and stirred for 1 h .

Selective cleavage of the Fmoc protecting groups from the final compound (10B) was carried out by washing with a 1:1 mixture of DMF and piperidine once for 2 minutes and then again for 5 minutes to obtain products (11B).

The conjugation of the chelating agent with compound (11B) immobilized on the resin was carried out with 2 equivalents of DOTA- tris (t-Bu)ether {([2-(4,7,10- tris (2-(t-butoxy)-2-oxoethyl) -1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; catalog number 137076-54-1), 0.20 mmol, 572.73 g/mol [115 mg]}. The chelating agent-binding building block was activated with 1.98 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.198 mmol, 379.24 g/mol, [75 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution is added to the resin-immobilized and pre-swollen in DMF compound (11B). The addition of the DOTA chelating agent was continued for 2 hours with gentle agitation. The resulting compound (12B) was then washed three times with DMF1, three times with DMF2, then three times with DCM1, and finally three times with DCM2.

Cleavage of the Alloc-protecting groups from compound (12B) was carried out by interaction with 0.03 eq. morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/ml, [262 μl]} in 3 ml anhydrous DCM The reaction was carried out for 2 hours in the dark using aluminum foil.

The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. To remove residual palladium, the resin was washed an additional ten times with 1% DIPEA in DMF (300 µl DIPEA in 30 ml DMF2) and then another 10 times for 5 minutes with cupral solution {(Sigma; catalog number D3506-100G), 225.31 g/mol} in DMF2 at a concentration of 15 mg/ml (450 mg cupral in 30 ml DMF2). The resulting compound (13B) was then washed three times in DMF1 and three times in DMF2.

Finally, to combine the albumin-binding element, 4 eq iodophenylbutyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} activated 3.96 HBTU {(Sigma; Catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl] } in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the resin-immobilized and pre-swollen in DMF product (13B) and stirred for 1 h.

The resulting compound (14B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and finally three times with Et2O and dried in vacuo.

The final compound PSA-ALB-03 was obtained by mixing and subsequent cleavage from the resin for 2 hours with a mixture that included TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified by RP-HPLC. The synthesis process described above is briefly depicted in Scheme 1.3.

Схема 1.3: Объединение Альбумин-связывающего элемента, хелатирующего агента DOTA и предшественника ПСА для ПСА-АЛБ-03

Figure 1.3: Combining Albumin Binding Element, DOTA Chelating Agent, and PSA Precursor for PSA-ALB-03

d) Synthesis of PSA-ALB-04

Relative to the amount of lysine-coated PSA precursor (9B), 4 equiv Dde, and Fmoc of protected L-lysine {(Dde-Lys(Fmoc)-OH; Merck; catalog number 8540000001), 0.40 mmol, 532.63 g/mol, [213 mg]} was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g /ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the pre-swollen in DMF and immobilized bis(tBu)-protected PSA precursor (9B) and stirred for one hour.

The resulting compound (10B) was then washed three times with DMF1 and three times with DMF2. Selective cleavage of the Fmoc protecting group from the final compound (10B) was carried out by washing with a 1:1 mixture of DMF and piperidine once for 2 minutes and then again for 5 minutes to obtain product 11(B).

The conjugation of the chelating agent and compound (11B) immobilized on the resin was carried out with 3 equivalents of DOTA-tris(t-Bu)ether {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl) - 1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The building block binding the chelating agent was activated with 2.97 eq HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 eq DIPEA {0.40 mmol, 129.24 g /mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the compound (11B) pre-swollen in DMF and immobilized on the resin. The addition of the DOTA chelating agent occurred within 2 hours with gentle agitation.

The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective cleavage of the Dde-protecting group from the final compound (12B) was carried out by washing the mixture with 2% hydrazine in DMF twice for 5 minutes and then again for 10 minutes to obtain the final product (13B).

Relative to the amount of resin-coated product (13B), 2 eq of disuccinimidyl suberate {([bis(N-hydroxysuccinimide ester of suberic acid)]; Sigma; 68528-80-3), 0.20 mmol, 368.34 g/mol, [74 mg]} activated [ 70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the resin-immobilized and pre-swollen in DMF product (13B) and stirred for 1 hour.

The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2.

The synthesis process described above is briefly depicted in Scheme 1.4.

Scheme 1.4: Combination of chelating agent DOTA, PSA precursor and active ester PSA-ALB-04

The synthesis was followed by a parallel preparation of an albumin-binding precursor, starting with 2-chlorotrityl chloride resin {(2-CT-Resin; Merck; catalog number 8550170005), 0.20 mmol, displacement capacity 1.63 mmol/g, 100-200 MESH, 1% DVB , total swelling volume in CH2Cl2 >4.2 ml/g, [123 mg]} in a 5 ml syringe with filter and combi stopper, which was premixed with anhydrous dichloromethane (DCM) for 45 minutes.

The 2-CT resin was then washed three times with anhydrous DCM and then reacted with 1.2 eq of Dde- and also Fmoc-protected L-lysine {(Dde-Lys(Fmoc)-OH; Bachem; catalog number E-3385.0001), 0.24 mmol, 532.64 g/mol, [128 mg] (15B)} and 4.8 equiv DIPEA {0.96 mmol, 129.24 g/mol, 0.742 g/ml, [167 µl]} in 3 ml anhydrous DCM.

Attachment of the first protected amino acid to the resin (16B) proceeded for 16 hours while gently stirring.

Resin-immobilized L-lysine (16B) was washed three times with DCM1 and three times with DCM2. The unreacted chlorotrityl groups remaining on the resin were washed five times with a 17:2:1 mixture of DCM, MeOH, and DIPEA (20 ml).

Sequentially, the resin with Dde and Fmoc-protected L-lysine was washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. Selective cleavage of the Fmoc protecting groups was carried out by washing with a 1:1 mixture of DMF and piperidine once for 2 minutes and then once again for 5 minutes to obtain the product (17B).

The Dde-protected L-lysine was then washed 3x with DMF1 and 3x with DMF2, 3x with DCM1, 3x with DCM2, and finally 3x with Et ₂ O and dried in vacuo.

The resin-coated Dde-protected L-lysine (17B) thus obtained was divided into two portions, and one of them was used in the next reaction. This resin-coated product was stirred with anhydrous DCM for 45 minutes and then washed three times with DMF and three times with DMF2 successively.

Relative to the amount of resin with lysine immobilized on it, 4 eq iodophenylbutyric acid {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]} activated 3.96 eq HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl ]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the resin-immobilized and pre-swollen in DMF product (17B) and stirred for 1 hour.

The resin with immobilized Dde-protected L-lysine and iodophenyl butyric acid (18B) was washed three times with DMF1 and three times with DMF2. Selective cleavage of the Dde-protecting groups from the final compound (18B) was carried out by washing the mixture with 2% hydrazine in DMF twice for 5 minutes and then again for 10 minutes to obtain the product (19B).

Albumin-binding element (20B) was obtained by mixing and subsequent cleavage from the resin for 2 h using a mixture that included 5% TFA in DCM. The solvent mixture was evaporated from the product, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified using RP-HPLC.

The synthesis process described above is briefly depicted in Scheme 1.5.

Scheme 1.5: Association of the albumin-binding element for PSA-ALB-04

Finally, 3 eq of purified albumin-binding element (20B) was conjugated. Product (20B) was dissolved in dry DMF and 100 μl of DIPEA was added. Two minutes after the addition of DIPEA, the solution (20B) was added to the resin-immobilized and pre-swollen in DMF product (14C) and stirred for 1 hour.

The resulting compound (21B) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and finally three times with Et2O and dried in vacuo.

The final compound PSA-ALB-04 was obtained by mixing and subsequent cleavage from the resin for 2 hours using a mixture that included TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified using RP-HPLC.

The synthesis process described above is briefly depicted in Scheme 1.6.

Scheme 1.6: Combination of albumin-binding element and DOTA-conjugated PSA precursor for PSA-ALB-04.

e) Synthesis of PSA-ALB-05

Relative to the amount of lysine-coated PSA precursor (9B), 4 equiv Fmoc, as well as Alloc protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [181 mg]} was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g /ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (9B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

Then the resulting compound (10b) was washed three times with DMF1 and three times with DMF2. Selective cleavage of the Fmoc protecting group from the final compound (10B) was carried out by washing with a 1:1 mixture of DMF and piperidine once for 2 minutes and then again for 5 minutes to obtain the final product (11B).

Relative to the amount of lysine-coated PSA precursor (11B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g /ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (11B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (12B) was then washed three times with DMF1 and three times with DMF2. Selective cleavage of the Fmoc protecting group from the final compound (12B) was carried out by washing with a 1:1 mixture of DMF and piperidine once for 2 minutes and then again for 5 minutes to obtain the final product (13B).

Relative to the amount of lysine- and aspartate-coated PSA precursor (13B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/ mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (11B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2. Selective cleavage of the Fmoc-protecting group from the final compound (14B) was carried out by washing with a mixture of DMF and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain the final product (15B).

Relative to the amount of resin-coated product (15B), 4 eq iodophenylbutyric acid was used {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]}, which was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the resin-immobilized and pre-swollen in DMF product (15B) and stirred for 1 hour.

The resulting compound (16B) was then washed three times with DMF1 and three times with DMF2, three times with DCM1 and finally three times with DCM2.

Cleavage of Alloc-protecting groups from compound (16B) was carried out by interaction with 0.03 eq. morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/ml, [262 μl]} in 3 ml anhydrous DCM The reaction was carried out for 2 hours in the dark using aluminum foil.

The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. To remove residual palladium, the resin was washed an additional ten times with 1% DIPEA in DMF (300 µl DIPEA in 30 ml DMF2) and then another 10 times for 5 minutes with cupral solution {(Sigma; catalog number D3506-100G), 225.31 g/mol} in DMF2 at a concentration of 15 mg/ml (450 mg cupral in 30 ml DMF2). The resulting compound (17B) was then washed three times with DMF1 and three times with DMF2.

The conjugation of the chelating agent with compound (17B) immobilized on the resin was carried out with 3 equivalents of DOTA-tris(t-Bu)ether {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl) -1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; catalog number 137076-54-1), 0.20 mmol, 572.73 g/mol [171 mg]}. The chelating agent-binding building block was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution is added to the resin-immobilized and pre-swollen in DMF compound (17B). The addition of the DOTA chelating agent was continued for 2 hours with gentle agitation. The resulting compound (18B) was then washed three times with DMF1, three times with DMF2, then three times with DCM1, and finally three times with DCM2, and finally three times with Et2O, and dried in vacuo.

The final compound PSA-ALB-05 was obtained by mixing and subsequent cleavage from the resin for 2 hours using a mixture that included TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified using RP-HPLC.

The synthesis process described above is briefly depicted in Scheme 1.7.

Scheme 1.7: Combining the Albumin Binding Element, DOTA Chelating Agent and PSA Precursor for PSA-ALB-05.

Scheme 1.7 (continued): Combining Albumin Binder, chelating agent DOTA and PSA precursor for PSA-ALB-05.

Relative to the amount of lysine-coated PSA precursor (9), 4 equiv of Fmoc was used as well as Alloc of protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [ 181 mg]} which was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (9) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

Selective removal of the Fmoc-protecting group from the compound (10) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (11).

The conjugation of the chelating agent with compound (11) immobilized on the resin was carried out with 2 equivalents of DOTA-tris(t-Bu)ether {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl) -1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; catalog number 137076-54-1), 0.20 mmol, 572.73 g/mol [115 mg]}. The chelating agent-binding building block was activated with 1.98 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [75 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution is added to the resin-immobilized and pre-swollen in DMF compound (11). The addition of the DOTA chelating agent was continued for 2 hours with gentle agitation. The resulting compound (12) was then washed three times with DMF1, three times with DMF2, then three times with DCM1, and finally three times with DCM2.

Cleavage of the Alloc-protecting groups was carried out by reaction with 0.15 eq. 87.12 g/mol, 0.999 g/ml, [262 µl]} in 3 ml anhydrous DCM The reaction was carried out in the dark using aluminum foil.

The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. To remove residual palladium, the resin was washed an additional ten times with 1% DIPEA in DMF (300 μl DIPEA in 30 ml DMF2) and then another 10 times for 5 minutes with cupral solution {(Sigma; catalog number D3506-100G), 225.31 g/mol} in DMF2 at a concentration of 15 mg/ml (450 mg cupral in 30 ml DMF2). The resulting compound (13) was then washed three times with DMF1 and three times with DMF2.

Finally, 4 eq of tolyl-butyric acid {([4-(p-tolyl)butyric acid]; ABCR; AB119212), 0.40 mmol, 178.23 g/mol, [71 mg]}, which activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the resin-immobilized and pre-swollen in DMF product (13) and stirred for 1 hour.

The resulting compound (14) was then washed three times with DMF1, three times with DMF2, three times with DCM1, three times with DCM2, and finally three times with Et2O, and dried in vacuo.

The final compound PSA-ALB-06 was obtained by mixing and subsequent cleavage from the resin for 2 hours using a mixture that included TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified using RP-HPLC

The synthesis process described above is briefly depicted in Scheme 1.8.

Scheme 1.8: Combining the Albumin Binder, DOTA Chelating Agent and PSA Precursor for PSA-ALB-06.

f) Synthesis of PSA-ALB-07

Relative to the amount of lysine-coated PSA precursor (9B), 4 equiv of Fmoc was used, as well as Alloc of protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [ 181 mg]} which was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (9B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (10B) was then washed three times with DMF1, and three times with DMF2.

Selective removal of the Fmoc-protecting group from the compound (10B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (11B).

Relative to the amount of lysine-coated PSA precursor (11B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/mol, [ 123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (11B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (12B) was then washed three times with DMF1, and three times with DMF2.

Selective removal of the Fmoc-protecting group from the obtained compound (12B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (13B).

Relative to the amount of lysine- and aspartate-coated PSA precursor (13B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/ mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (13B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (14B) was then washed three times with DMF1 and three times with DMF2.

Selective removal of the Fmoc-protecting group from the obtained compound (14B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (15B).

Relative to the amount of lysine- and aspartate-coated PSA precursor (15B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/ mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (15B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (16B) was then washed three times with DMF1, and three times with DMF2.

Selective removal of the Fmoc-protecting group from the obtained compound (14B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (17B).

Relative to the amount of resin-coated product (17B), 4 eq iodophenylbutyric acid was used {([4-(p-iodophenyl)butyric acid]; Sigma; I5634-5G), 0.40 mmol, 290.10 g/mol, [116 mg]}, which was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml , [70 µl]}. Two minutes after the addition of DIPEA, the solution was added to the resin-immobilized and pre-swollen in DMF product (17B) and stirred for 1 h.

The resulting compound (18B) was then washed three times with DMF1 and three times with DMF2, three times with DCM1 and finally three times with DCM2.

Cleavage of Alloc-protecting groups from compound (18B) was carried out by reaction with 0.03 eq. morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/ml, [262 μl]} in 3 ml anhydrous DCM The reaction was carried out in the dark for 2 hours using aluminum foil.

The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. To remove residual palladium, the resin was washed an additional ten times with 1% DIPEA in DMF (300 µl DIPEA in 30 ml DMF2) and then another 10 times for 5 minutes with cupral solution {(Sigma; catalog number D3506-100G), 225.31 g/mol} in DMF2 at a concentration of 15 mg/ml (450 mg cupral in 30 ml DMF2). The resulting compound (19B) was then washed three times with DMF1 and three times with DMF2.

The conjugation of the chelating agent with compound (19B) immobilized on the resin was carried out with 3 equivalents of DOTA-tris(t-Bu)ether {([2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl) -1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelating agent-binding building block was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution is added to the resin-immobilized and pre-swollen in DMF compound (17B). The addition of the DOTA chelating agent was continued for 2 hours with gentle agitation.

The resulting compound (20B) was then washed three times with DMF1, three times with DMF2, then three times with DCM1, and finally three times with DCM2, and finally three times with Et2O, and dried in vacuo.

The final compound PSA-ALB-07 was obtained by mixing and subsequent cleavage from the resin for 2 hours using a mixture that included TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified using RP-HPLC.

The synthesis process described above is briefly depicted in Scheme 1.9.

Scheme 1.9: Combining Albumin Binder, DOTA Chelating Agent and PSA Precursor for PSA-ALB-07.

Scheme 1.9 (continued): Combination of the Albumin Binding Element, chelating agent DOTA and PSA precursor for PSA-ALB-07.

g) Synthesis of PSA-ALB-08

Relative to the amount of lysine-coated PSA precursor (9B), 4 equiv of Fmoc was used, as well as Alloc of protected L-lysine {(Fmoc-Lys(Alloc)-OH; Merck; catalog number 8521240005), 0.40 mmol, 452.50 g/mol, [ 181 mg]} which was activated with 3.96 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.396 mmol, 379.24 g/mol, [149 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (9B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (10B) was then washed three times with DMF1, and three times with DMF2. Selective removal of the Fmoc-protecting group from the compound (10B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (11B).

Relative to the amount of lysine- and aspartate-coated PSA precursor (11B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/ mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (11B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (12B) was then washed three times with DMF1, and three times with DMF2. Selective removal of the Fmoc-protecting group from the compound (12B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (15B).

Relative to the amount of lysine- and aspartate-coated PSA precursor (13B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/ mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (11B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (14B) was then washed three times with DMF1, and three times with DMF2. Selective removal of the Fmoc-protecting group from the compound (12B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (15B).

Relative to the amount of lysine- and aspartate-coated PSA precursor (15B), 3 equiv Fmoc, as well as tBu-protected D-aspartate {(Fmoc-D-Asp-OtBu; Merck; catalog number 8521440001), 0.30 mmol, 411.45 g/ mol, [123 mg]} was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/ mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution was added to the bis(tBu)-protected PSA precursor (11B) immobilized on resin and pre-swollen in DMF and stirred for 1 hour.

The resulting compound (16B) was then washed three times with DMF1, and three times with DMF2. Selective removal of the Fmoc-protecting group from the compound (14B) was carried out by washing with a mixture of DMX and piperidine in a ratio of 1:1 once for 2 minutes and then again for 5 minutes to obtain products (17B).

Relative to the amount of resin-coated product (17B), 4 eq tolyl-butyric acid (0.40 mmol) was used, which was activated with 3.96 eq HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [ 112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. The resulting compound (18B) was then washed three times with DMF1, three times with DMF2, three times with DCM2, three times with DCM1, and finally three times with DCM2.

Cleavage of Alloc-protecting groups from compound (18B) was carried out by reaction with 0.03 eq. morpholine {3.0 mmol, 87.12 g/mol, 0.999 g/ml, [262 μl]} in 3 ml anhydrous DCM The reaction was carried out in the dark for 2 hours using aluminum foil.

The resin was then washed three times with DCM1, three times with DCM2, three times with DMF1, and finally three times with DMF2. To remove residual palladium, the resin was washed an additional ten times with 1% DIPEA in DMF (300 µl DIPEA in 30 ml DMF2) and then another 10 times for 5 minutes with cupral solution {(Sigma; catalog number D3506-100G), 225.31 g/mol} in DMF2 at a concentration of 15 mg/ml (450 mg cupral in 30 ml DMF2). The resulting compound (19B) was then washed three times with DMF1 and three times with DMF2.

The conjugation of the chelating agent with compound (19B) immobilized on the resin was carried out with 3 equivalents of DOTA- tris (t-Bu)ether {([2-(4,7,10- tris (2-(t-butoxy)-2-oxoethyl) -1,4,7,10-tetraazacyclododecan-1-yl)acetic acid]; CheMatech; catalog number 137076-54-1), 0.30 mmol, 572.73 g/mol [171 mg]}. The chelating agent-binding building block was activated with 2.97 equiv HBTU {(Sigma; catalog number 12804-25G-F), 0.297 mmol, 379.24 g/mol, [112 mg]} in the presence of 4 equiv DIPEA {0.40 mmol, 129.24 g/mol, 0.742 g/ml, [70 µl]} in anhydrous DMF. Two minutes after the addition of DIPEA, the solution is added to the resin-immobilized and pre-swollen in DMF compound (17B). The addition of the DOTA chelating agent was continued for 2 hours with gentle agitation.

The resulting compound (20B) was then washed three times with DMF1, three times with DMF2, then three times with DCM1, and finally three times with DCM2, and finally three times with Et2O, and dried in vacuo.

The final compound PSA-ALB-07 was obtained by mixing and subsequent cleavage from the resin for 2 hours using a mixture that included TFA, TIPS and H2O in a ratio of 95:2.5:2.5. TFA was evaporated, the crude product was dissolved in ACN and water in a ratio of 1:1 and purified using RP-HPLC

The synthesis process described above is briefly depicted in Scheme 1.10.

Scheme 1.10: Combination of albumin-binding molecule, chelating agent, DOTA and PSA precursor for PSA-ALB-08.

Scheme 1.10 (continued): Combination of albumin-binding molecule, chelating agent, DOTA and PSA precursor for PSA-ALB-08.

1.1.3: INTRODUCING THE Tagged Atom ¹⁷⁷ Lu IN PSA LIGANDS AND THEIR EVALUATION IN VITRO

In vitro studies were performed using ¹⁷⁷ Lu-PSA-ALB-01/-03/-04/-05/-06/-07/-08. These included an initial assessment of labeling accuracy, determination of partition coefficients for n-octanol/PBS, and a study of serum protein binding. In addition, experiments to assess the uptake and internalization of compounds were carried out using a PSA-transfected PSA pos PC-3 PIP mouse line (positive control) and a mock-transfected PSA non-PC-3 flu mouse line (negative control).

a) PSA ligands and radionuclides

PSA ligands ¹⁷⁷ Lu-PSA-ALB-01/-03/-04/-05/-06/-07/-08 were synthesized as described above. A control compound (PSA-617) was obtained from Advanced Biochemical Compounds (ABX GmbH, Radeberg, Germany). The carrier-free ¹⁷⁷ Lu isotope in 0.05 M HCl was obtained from Isotope Technologies Garching (ITG GmbH, Germany).

b) Radioactive labeling

PSA-617 stock solution was prepared by diluting MilliQ in water to a final concentration of 1 mmol. ¹⁷⁷ Lu-PSA-ALB-01/-03/-04/-05/-06/-07/-08 was diluted in MilliQ/DMSO water to a final concentration of 1 mol. All compounds were labeled with ¹⁷⁷ Lu in a 1:5 mixture of sodium acetate (0.5 mol, pH 8) and HCl (0.05 mol, pH ~1) at pH 3.5–4.5. The compounds were labeled with ¹⁷⁷ Lu with a specific activity ranging from 5 to 50 MBq/nmol depending on the experimental conditions. The reaction mixture was incubated for 15 minutes at 95° C. followed by quality control using high performance liquid chromatography with a reverse phase C-18 column (XterraTM MS, C18, 5 μm, 150×4.6 mm; Waters). The mobile phase included MilliQ water with 0.1% trifluoroacetic acid (A) and acetonitrile (B) with a gradient of 95% A and 5% to 20% A and 80% B over 15 minutes at a flow rate of 1.0 ml/min. Radioactive ligands were diluted with MilliQ water containing Na-DTPA (50 μm (micromoles)) before being applied to the HPLC column.

c) Determination of the partition coefficient for n-Octanol/PBS

¹⁷⁷ Lu-PSA-ALB-01/-03/-04/-05/-06/-07/-08 and PSA-617 labeled ¹⁷⁷ Lu at a specific activity of 50 MBq/nmol. Then, a radioactive ligand (0.5 MBq; 10 pmol, 25 µl) was added to the reaction tube containing 1475 µl PBS pH 7.5 and 1500 µl n-octanol. The contents of the tubes were then mixed by vortexing followed by a centrifugation step to separate the phases. Finally, the radioactivity of a given volume of PBS and n-octanol was measured in a gamma counter (Perkin Elmer, Wallac Wizard 1480) to calculate distribution coefficients expressed in terms of the logarithm of the ratio of counts per minute (cpm - Count Per Minute) measured in the n-octanol phase. to the number of pulses per minute in the PBS phase.

d ) Ultrafiltration

Plasma binding of ¹⁷⁷ Lu-PSA-ALB-01/-03/-04/-05/-06/-07/-08 and ¹⁷⁷ Lu-PSA-617 was determined by ultrafiltration.

Therefore, compounds were labeled with ¹⁷⁷ Lu at a specific activity of 50 MBq/nmol and incubated with human plasma samples or PBS at room temperature. The free fraction and the plasma-bound fraction were separated using a centrifuge ultrafiltration apparatus (4104 filtration units [Millipore]; 30,000 Da nominal molecular weight limit, methylcellulose membranes for microparticles). The incubation solution was placed in an ultrafiltration apparatus and centrifuged at 2500 rpm for 40 minutes at 20°C. Collected samples from the filtrate and analyzed their radioactivity using a gamma counter. The amount of the compound bound to the plasma was determined as the fraction of radioactivity measured in the filtrate with respect to the corresponding initial solution (taken as 100%).

e) Analysis of the process of cellular internalization

The study of the processes of cell uptake and internalization was carried out with ¹⁷⁷ Lu-PSA-ALB-01/-03/-04/-05/-06/-07/-08 and the control compound ¹⁷⁷ Lu-PSA-617 using PSA-transfected PSA ^{pol +} PC-3 PIP cell line and mock-transfected PSA ^rep- PC-3 flu cell line, to study the specificity of new compounds.

Cells were grown in RPMI cell medium supplemented with 10% fetal calf serum, L-glutamine, antibiotics, and pyromucin (2 μg/ml) at 37°C and 5% CO2 (standard conditions). A standard cell culture was prepared twice a week using PBS/EDTA (2 mmol) to wash the cells and trypsin to separate the cells. Cells were transferred to 12-well plates (˜3×10 ⁵ cells in 2 ml RPMI medium per well) and allowed to adhere and grow overnight under standard conditions. Then the supernatant was removed and the cells were washed with PBS pH 7.4, after which RPMI medium without additives (975 μl/well) was added. Compounds were labeled with ¹⁷⁷ Lu at a specific activity of 5 MBq/nmol and diluted 1.5 MBq/ml in 0.05% bovine serum albumin (c)/0.9% NaCl solution to prevent sticking to plastic vials. Cells were incubated with 25 μL (~37.5 kBq per well) of PSA-labeled ligands under standard conditions for 2 h and 4 h, respectively. After incubation, the cells were washed three times with ice-cold PBS and the total uptake of radiolabeled ligands (PSA-bound fraction on the surface and internalized fraction) was determined. The fraction of internalized radioactive ligand was evaluated in cells that were washed with ice-cold PBS, followed by incubation with developing buffer for 10 minutes (0.05 M glycine developing buffer in 100 mmol NaCl, pH 2.8) and additionally washed again with ice-cold PBS. Cell samples were lysed by adding NaOH (1 M, 1 ml) to each well. Samples of cell suspensions were analyzed on a γ-counter (Perkin Elmer, Wallac Wizard 1480). After homogenizing the cell suspensions, the concentration of proteins in each sample was determined using the Micro BCA Protein Assay (Pierce, Therma Scientific). The results were expressed as a percentage of the total added radioactivity per 150 μg/ml of protein.

1.2 RESULTS

1.2.1 TAG EFFICIENCY

PSA-ALB-01 and -03 were successfully labeled with ¹⁷⁷ Lu with a specific activity of no more than 100 MBq/nmol and an excellent radioactive yield of >98%. PSA-ALB-04, -05, -06, -07 and -08 were labeled with ¹⁷⁷ Lu in preliminary tests with a specific activity of not more than 50 MBq/nmol and an excellent radioactive yield >97%.

The specific activity of the radioactive isotope used in the experiments (unless otherwise stated) was 50 MBq/nmol. The radiochemical purity of the compounds that were used for in vitro and in vivo studies was always >97% (Figure 1).

1.2.2 Partitioning coefficient for n-octanol/PBS

¹⁷⁷ Lu-PSA-ALB-01, -03, -04 and -06 showed the same distribution coefficients for n-octanol/PBS (LogD value), while coefficients for ¹⁷⁷ Lu-PSA-ALB-05, -07 and -08 indicated that these compounds are slightly more hydrophilic. In general, the results indicate that the introduction of an albumin-binding element reduces the hydrophilicity compared to the control compound ¹⁷⁷ Lu-PSA-617, however, all compounds are nevertheless hydrophilic with logD values > 2.7 (Figure 2).

1.2.3 ALBUMIN-BINDING PROPERTIES

Analysis of ¹⁷⁷ Lu-PSA-ALB-01, -03, -04, -05, -06 and -07 by ultrafiltration showed a high ability of the compounds to bind to plasma proteins (>94%), while they did not penetrate the filter during incubation with human plasma samples. The ability to easily pass through the filter of compounds was demonstrated when they were incubated in PBS without plasma proteins (Figure 3). The new synthesized compounds showed a more pronounced ability to bind to plasma proteins compared to the control compound ¹⁷⁷ Lu-PSA-617, which had an albumin-bound fraction of only about 44% (Figure 3)

1.2.4 INTERNALIZATION

Cellular uptake and internalization of PSA ligands ¹⁷⁷ Lu-PSA-ALB-01, 03, -04, -05, -06, -07 and -08 were studied and compared with the control compound ¹⁷⁷ Lu-PSA-617 in the PC-3 PIP cell line /flu (Figure 4). Uptake of all PC-3 compounds by PIP cells (PSApos+) was comparable to uptake of ¹⁷⁷ Lu-PSA-617 compound after 2 h or 4 h, respectively. Interestingly, the internalized fraction of PSA ligands was greater than that of ¹⁷⁷ Lu-PSA-617 at 2 h and 4 h, respectively. The rate of internalization of ¹⁷⁷ Lu-PSA-ALB-06 and 177 Lu-PSA-ALB-08 was again comparable to that of ¹⁷⁷ Lu-PSA-617. The capture of radioactive ligands of PC-3 flu cells (PSAtr-) was <0.5%, which proves the highly PSA-specific capture/internalization of all compounds.

Example 2 PSA Ligand Assay in vivo in mouse tumor models

The properties of ¹⁷⁷ Lu-PSA-ALB-01, -03, -04, -05, -06, -07 and -08 were studied in vivo . For this, immunodeficient Balb/c nude mice were injected with PSA pos PC-3 PIP and PSA non PC-3 flu cells. After intravenous (IV) administration of ligands, SPECT/CT examination and extensive biodistribution analysis were performed. Tumor cell uptake data, tumor cell/blood, tumor cell/kidney, and tumor cell/liver ratios for ¹⁷⁷ Lu-PSA-ALB-01-08 are shown in Figures 5 and 6.

2.1 MATERIALS AND METHODS

2.1.1 MOUSE TUMOR MODELS

Mice aged 5-6 weeks received in the laboratory of Charles River, Sulzfeld, Germany. Balb/c female athymic mice were injected subcutaneously in the right shoulder with PC-3 PIP cells (6 x 106 cells in 100 µl in Hank's balanced salt solution (HBSS) with Ca2+/Mg2+), and in the left shoulder with PC-3 flu cells ( 5 x 106 cells in 100 µl HBSS Ca2+/Mg2+). Two weeks later, when the tumors had reached a size of approximately 200-300 mm3, sufficient for biodistribution and imaging studies.

2.1.2 BIODISTRIBUTION ANALYSIS

Biodistribution was analyzed using PC-3 PIP/flu tumor-bearing mice treated with tumor cells two weeks prior to PSA ligand administration. Radioactive ligands were diluted with 0.9% NaCl and injected intravenously in a volume of 100-200 µl. Mice were euthanized at different times after injection (after administration) of radioactive ligands. Received samples of selected tissues and organs, weighed and measured the radioactivity using a gamma counter. The results were corrected for radioactive decay and expressed as a percentage of the amount of radioactivity introduced per gram of tissue sample (% IA/g).

2.1.3 SPECT/CT IMAGING

SPECT/CT examination was performed using a specially designed small animal SPECT/CT camera (NanoSPECT/CTTM, Mediso Medical Imaging Systems, Budapest, Hungary). PSA ligands were labeled at a specific activity of 5 MBq/nmol and diluted in saline containing 0.05% BSA. Scanning was performed 4 h, 24 h and 72 h after the administration of radiolabeled ligands (25 MBq, 1 nmol, 100 µl). Data were pre-processed using NanoSPECT/CTTM software, post-processing was performed using VivoQuant (version 3.0, inviCRO Imaging Services and Software, Boston USA). A post-reconstruction Gaussian filter (FWHM = 1 mm) was applied and the radioactivity scale was set on the images (minimum value = 0.095 Bq/voxel, maximum value 95 Bq/voxel).

2.1.4 TREATMENT OF MODEL MICE

Mice, statistically identical in body weight and tumor volume, were divided into 5 groups (Groups A - E, n=6) and on Day 0 of the study were administered vehicle only (saline containing BSA 0.05%, Group A), 177Lu -PSA-617 (Groups B and C) and 177Lu-PSA-ALB-06 (Groups G and E), respectively. (Table 2.1). Mice in Groups B and D received 2 MBq of radioactive ligand (1 nmol/mouse), while mice in Groups C and D received 5 MBq of radioactive ligand (1 nmol/mouse. Mice were observed, body weight and tumor volume were measured every other day within 12 weeks.Mice were euthanized when the pre-set endpoints of the experiment were reached or at the end of the study on Day 84. Relative body weight (RBW) was defined as the ratio [BWx/BW0], where BWx is the body weight in grams on a given day x, and BW0 is the body weight of the animals in grams at the beginning of the study, i.e. on Day 0. The size of the tumor was determined by measuring its longest dimension (L) and the dimension perpendicular to the first (W) using a digital caliper. V) was calculated using the equation V=0.5*(L*W2)].Relative tumor volume (RTV) was defined as the ratio [TVx/TV0], where TVx is the tumor volume in mm3 on a given day x and TV0 is the tumor volume in mm3 at the beginning of the study, i.e. on the day 0.

^a Radioactivity in syringe measured before and after administration to the appropriate animal.

^b There were no significant differences between the values measured for each group (p>0.05).

The efficacy of radionuclide therapy was expressed in terms of the tumor growth retardation index (TGDx), which was calculated as the time required to reach a tumor volume 10 times the baseline on Day 0. Tumor growth retardation index [TGDIx = TGDx(T)/TGDx(C )] was calculated as the ratio of treated mice TGDx (T) to control (C) for 2-fold (x=2, TGD2) and 5-fold (x=5, TGD5) increase in tumor volume. To assess undesirable side effects, the body weights of the animals on the day of euthanasia of the first control animal were compared. After euthanasia, the animals' kidneys, liver, and brain were removed and weighed. Weight ratios for organs (kidney/brain and liver/brain) were calculated using the organ weight measured on the day of death of the animals.

Data significance was analyzed, as disclosed in the study results section, using one-way analysis of variance (ANOVA) followed by multiple comparison using Tukey's test on GraphPad Prism software (version 7). A p<0.05 value was considered statistically significant. Survival analysis was performed on Kaplan-Meier curves and using logarithmic series tests (Mantel-Haengela).

2.2 RESULTS

2.2.1 BIODISTRIBUTION ¹⁷⁷ Lu-PSA-ALB-01 I ¹⁷⁷ Lu-PSA-ALB-03

The distribution of ¹⁷⁷ Lu-PSA-ALB-01 and ¹⁷⁷ Lu-PSA-ALB-03 in tissues was studied for 8 days. Compounds ¹⁷⁷ Lu-PSA-ALB-01 and ¹⁷⁷ Lu-PSA-ALB-03 showed similar tissue distribution profiles (Figure 5A).

The high level of radioactivity that can be observed in the bloodstream at early time points slowly but surely declines over time.

The uptake of both radioactive ligands of PSA ^sex+ PC-3 PIP by tumors increased until a plateau was reached and practically did not fall until the end of the study. Uptake of PC-3 flu by tumors was significantly lower than blood levels, suggesting a high specificity of PSA binding and uptake in vivo (Figure 5A). Biodistribution data for ¹⁷⁷ Lu-PSA-ALB-01 and -03 are presented in Tables 2.2 and 2.3 below.

2.2.2 BIODISTRIBUTION ¹⁷⁷ Lu-PSA-ALB-04 I ¹⁷⁷ Lu-PSA-ALB-05

The distribution of ¹⁷⁷ Lu-PSA-ALB-04 and ¹⁷⁷ Lu-PSA-ALB-05 in tissues was studied for 8 days (Figure 5B).

A high level of radioactivity in the blood of animals after the introduction of ¹⁷⁷ Lu-PSA-ALB-04 was observed at the initial stages of the study and remained significantly high. There was a large accumulation in PSA ^{sex +} PC-3 PIP tumors, which slowly decreased towards the end of the study. The activity accumulated in PSA ^otp -PC-3 flu tumors and other organs (non-targets) was significantly lower than the level in the blood, indicating a high PSA specificity of binding and uptake in vivo .

The high level of radioactivity in the bloodstream of animals treated with ¹⁷⁷ Lu-PSA-ALB-05 rapidly decreased and remained low until the end of the study. The highest level of capture of radioactive ligands was observed in PSA ^sex+ PC-3 PIP tumors in mice that were injected with ¹⁷⁷ Lu-PSA-ALB-05 followed by washing from tumor tissues. Uptake in PC-3 flu by tumors and other tissues was significantly lower than blood levels, suggesting high PSA-specific binding and uptake in vivo . Biodistribution data for ¹⁷⁷ Lu-PSA-ALB-04 and -05 are presented in Tables 2.4 and 2.5 below.

2.2.3 BIODISTRIBUTION ¹⁷⁷ Lu-PSA-ALB-06, ¹⁷⁷ Lu-PSA-ALB-07, ¹⁷⁷ Lu-PSA-ALB-08

The tissue distribution of 177Lu-PSA-ALB-06, -07 and -08 was analyzed no later than 3 days after administration (Figure 5B).

Radioactivity levels of all compounds in the blood rapidly decreased and were comparable throughout the study. The most pronounced accumulation in PSApos+ PC-3 PIP tumors was observed for the 177Lu-PSA-ALB-06 compound, which slowly and gradually decreased by the end of the study. Accumulation activity in PSAneg-PC-3 flu tumors and other organs (non-targets) was below blood levels, indicating the presence of PSA-specific binding and uptake in vivo for all tested compounds. Biodistribution data for 177Lu-PSA-ALB-06, -07 and -08 are presented in Tables 2.6, 2.7 and 2.8 below.

2.2.4 SPECT/CT Imaging

SPECT/CT images of PC-3 PIP/flu tumor bearing mice were obtained at various time points after administration of ¹⁷⁷ Lu-PSA-ALB-03 and 177 Lu-PSA-ALB-06. The exact injected radioactivity of ¹⁷⁷ Lu-PSA-ALB-03 and ¹⁷⁷ Lu-PSA-ALB-06 was 25 MBq and 23 MBq, respectively. The most favorable in vivo distribution pattern for ¹⁷⁷ Lu-PSA-ALB-03 and ¹⁷⁷ Lu-PSA-ALB-06 is shown in Figure 26.

2.2.5 TREATMENT OF MODEL MICE

Control animals (Group A) showed consistent tumor growth over time, which was comparable to the rate of tumor growth in mice treated with low-active ¹⁷⁷ Lu-PSA-617 (Group B: 2 MBq/mouse). The tumor growth retardation indices for the animals in Group B (TGDI ₂ =0.8, TGDI ₅ =1.4, Table 2.9) were, however, similar to those of the control animals, in which the TGDI was determined to be 1. The first control animal reached the endpoint on Day 16, while one animal from Group B had to be euthanized on Day 12 (Table 2.9). Treatment was effective in animals treated with high activity ¹⁷⁷ Lu-PSA-617 (Group B: 5 MBq/mouse) or low activity ¹⁷⁷ Lu-PSA-ALB-06 (Group D: 2 MBq/mouse). The TGDI ₂ and TGDI ₅ scores were identical for mice from both groups (Groups C and D) and therefore the mice were forced to euthanize at the same time intervals (Group C: Day 26 - Day 40; Group D: Day 28 - Day 44; data not shown). In mice treated with highly active ¹⁷⁷ Lu-PSA-ALB-06 (Group D: 5 MBq/mouse), tumor growth was most effectively inhibited. Four of the animals in Group G had tumors completely gone, with no recurrence until the end of the study on Day 84.

n/a ^a not defined because the animal was alive at the end of the study

Mice treated with high activity ¹⁷⁷ Lu-PSA-617 or low activity ¹⁷⁷ Lu-PSA-ALB-06 showed a significantly longer mean survival time (Group C: Day 32, Group D: Day 36, Table 2.9, Figure 27). At the end of the study on Day 84, four animals treated with highly active ¹⁷⁷ Lu-PSA-ALB-06 (Group D) were still alive and thus the median survival time was uncertain.

Example 3 Clinical Study of PSA Ligands

3.1: CASE 1:

The compound PSA-ALB-06, labeled with the therapeutic radioactive isotope Lutetium 177, was used in an individual therapeutic study in a patient suffering from moderately differentiated prostate adenocarcinoma with widespread metastases in both lobes of the liver, as well as disseminated osteoblastic metastases (in the pelvic region) and polytopic extensive damage to the lymph nodes. Biodistribution analysis and in vivo activity of radiolabeled PSA-ALB-06 was performed using SPECT-CT.

Visualization using SPECT-CT was performed at different time points, no later than 46 hours after injection (po). Long-term circulation of the PSA-ALB-06 compound in the blood and an improved bioavailability profile were established (Figure 7). Blood clearance was completed within the first hours, while non-specific uptake by healthy organs (in particular, the liver, salivary glands, kidneys) remained moderate throughout the study. SPECT-CT demonstrated almost complete specific uptake of the radiolabelled compound in tumor tissues (Figure 8).

These first human results confirm preclinical findings regarding an improved pharmacokinetic profile of compounds demonstrating potential for the treatment of PSA-positive tumors.

3.2 CASE 2:

The compound PSA-ALB-06, labeled with the therapeutic radioactive isotope Gallium-68, was used in an individual therapeutic study in a patient suffering from metastatic, castration-resistant prostate cancer, as diagnostic agents for PET-CT. Tumor tissues can be visualized with high specificity PET, while baseline radioactivity in healthy non-target organs remains moderate (Figure 9). The high image contrast increases over time, confirming the prolonged blood clearance of compounds and highly specific uptake by tumor cells.

Example 4 Efficacy Study of PSA Ligands in Combination with ⁴⁴ Sc for Use in PET-CT Diagnosis

4.1 BIODISTRIBUTION DATA ⁴⁴ Sc-PSA-ALB

⁴⁴ Sc were prepared in the Injector 2 device at the Paul Scherrer Institute as previously reported ²

Radiolabeling of the PSA-ALB-06 compound was carried out as previously described by the authors using the clinically established PSA-167 ⁵ ligand. Biodistribution analysis was performed on Balb/c female nude mice carrying PSA-positive PC-3 PIP tumor cells (in the right shoulder) and PSA-negative PC-3 flu tumor cells (in the left shoulder). For these purposes, mice were injected with tumor cells 12-14 days before the introduction of the radioactive ligand. Mice were euthanized and dissected 1 hour, 4 hours and 6 hours after injection (po). (Figure 10A, Table 4.1). ⁴⁴ Sc-PSA-ALB-06 was studied 6 h after administration, while data for ¹⁷⁷ Lu-PSA-ALB-06 are available for 24 h po. (Figure 10B).

4.2.3 PET/CT imaging in mice treated with ⁴⁴ Sc-PSA-ALB-06

PET/CT examinations were performed using a special small animal PET/CT camera (G8, Perkin Elmer, US) as previously described by the authors. ⁵ Images were acquired at 1 h, 4 h, and 20 h after administration of 5 MBq ⁴⁴ Sc-PSA-ALB-06. Figure 11 shows scans obtained using a single density scale. Additional images were obtained using an additional adjusted scale in order to visualize organs and tissues as much as possible. Figure 12 shows a scan 1 hour after administration, when the radioactive compound is mainly circulating in the blood and has not specifically accumulated in PSA-positive tumor tissues.

The Figure 13 shows the scan after 20 hours p / v obtained using an additional scale. Thanks to this scale, it is possible to obtain the most accurate image of the tumor at a time when the total radioactivity in the blood has already decreased.

4.3 CONCLUSION

The introduction of radioactive labels into the compound was successfully carried out using the radioactive isotope ⁴⁴ Sc with its specific activity of 5 MBq/nmol. Biodistribution analysis of the compound and PET-CT imaging data indicate that ⁴⁴ Sc-PSA-ALB-06 has the same properties as compound ¹⁷⁷ Lu-PSA-ALB-06, as previously established. Due to the high tumor uptake of ⁴⁴ Sc-PSA-ALB-0, it is believed that this radioactive ligand may be a useful tool for imaging even small tumor foci at late time points (>4 h.p.p.) when baseline activity has already declined. The clinical application of these discoveries is the most promising and should be studied at the next stage to confirm the proposed concept.

Example 5 Design and Preclinical Evaluation of NODAGA Functionalized Albumin PSA Binding Ligands

A long-circulating PSA-targeting agent suitable for stable copper complexing has been developed that can be used to contrast prostate cancer with PET-CT at distant time points. Thus, the chelating agent DOTA in PSA-ALB-06 was replaced by the chelating agent NODAGA to form PSA-ALB-89. PSA-ALB-89 and PSA-ALB-06 were labeled with ⁶⁴ Cu and studied for their radiolytic stability, ability to bind to serum albumin, and uptake by PSA-positive PC-3 PIP and PSA-negative PC-3 flu tumor cells. Biodistribution analysis and PET/CT imaging were performed on PC-3 PIP/flu tumor bearing mice.

The structural formula of PSA-ALB-89 is shown below:

5.1 MATERIALS AND METHODS

Solid phase synthesis of PSA ligand

The NODAGA-functionalized PSA ligand, designated PSA-ALB-89, was synthesized using a solid phase synthesis platform as previously described for the synthesis of the compound PSA-ALB-06 (see Example 1). The only difference concerned the conjugation of the chelating agent in the last step of the synthesis (Scheme 5.1). Conjugation was carried out with 3 equivalents of NODAGA- tris (t-Bu)ether [4-(4,7- bis (2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)- 5(tert-butoxy)-5-oxopentanoic acid], activated with 2.97 eq O-(benzotriazol-1-yl)- N,N,N ' ,N '-tetramethyluronium hexafluorophosphate (HBTU) in the presence of 4 eq N,N -diisopropylethylamine (DIPEA) in anhydrous N,N -dimethylformamide (DMF). The addition of the NODAGA chelating agent took place over 3 hours with slow stirring. The final product was cleaved from the resin and deprotected sequentially over 2 hours using a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H2O in a ratio of 95:2.5:2.5 (v/v).

Scheme 5.1: Synthesis of NODAGA-functionalized PSA ligand

5.2 RESULTS

Synthesis of PSA ligand. PSA-ALB-89 was synthesized in the solid phase using a support, similar to the synthesis of PSA-ALB-06 (Example 1). Instead of conjugating the DOTA chelating agent, the NODAGA chelating agent was used (Scheme 5.1). Using the described multi-step synthesis (17 steps), it was possible to obtain a highly purified compound (>98%) with a total yield of 8.7% after purification by semi-preparative HPLC.

Radioactive labeling, stability and properties in vitro ⁶⁴ Cu-labeled PSA ligands.PSA-ALB-89 and PSA-ALB-06 were labeled with radioactive⁶⁴Cu at a specific activity of not more than 50 MBq/nmol. Radioactive ligands showed a high degree of radiochemical purity (>98%) and the same delay time (~11 min).⁶⁴Cu-PSA-ALB-89 and⁶⁴Cu-PSA-ALB-06 were stable (>92%) over a period of at least 4 hours. Partition coefficient n-octanol/PBS (logD values) for⁶⁴Cu-PSA-ALB-89 (-2.3±0.7) were slightly but not significantly higher (p>0.05) than the logD values for⁶⁴Cu-PSA-ALB-06 (-3.1±0.1).

Albumin-binding properties. ⁶⁴ Cu-PSA-ALB-89 and ⁶⁴ Cu-PSA-ALB-06 showed similar plasma protein binding (>92%) when incubated in human plasma. Half-maximal binding (B ₅₀ ) of ⁶⁴ Cu-PSA-ALB-89 was achieved with a [BSA]--[radioactive ligand] ratio of 454. This indicates slightly stronger binding than was found for ⁶⁴ Cu-PSA-ALB-89, for which half-maximal binding was achieved at a ratio of [BSA]--[radioactive ligand] equal to 770 (Figure 14).

Cellular capture and internalization. Cellular uptake of ⁶⁴ Cu-PSA-ALB-89 PC-3 PIP by cells was ~46%, and the internalized fraction was ~14% after incubation for 2 h at 37°C. Cellular uptake increased slowly after 4 hours of incubation (~52%) while the internalized fraction remained unchanged (~14%). Similar values were determined for ⁶⁴ Cu-PSA-ALB-06 (Figure 15A). Uptake in PC-3 flu cells was below 0.5% for both radioactive ligands, indicating PSA-specific cellular uptake (Figure 15B).

Biodistribution analysis. The tissue distribution profile for ⁶⁴ Cu-PSA-ALB-89 was evaluated over 24 hours in tumor-bearing mice (Figure 16, Table 5.1). There was a rapid decline in activity in the bloodstream over time (<3.2% IA/g and <1.4% IA/g after 24 h po, respectively). Rapid and intense accumulation of ⁶⁴ Cu-PSA-ALB-89 in PC-3 PIP tumors was observed immediately after compound administration (25.9±3.41% IA/g after 1 h po) and slowly increased towards the end of the experiment (97.1±7.01% IA /g after 24 h p / v). Radioactivity accumulation in PC-3 flu tumor cells not expressing PSA was generally below blood levels. The pattern of uptake of ⁶⁴ Cu-PSA-ALB-89 by liver cells showed a level of radioactivity comparable to that in the bloodstream or even lower (Figure 17). The tumor/kidney distribution ratio increased over time, although the values after administration of compound ⁶⁴ Cu-PSA-ALB-89 were relatively low. The tumor/cell distribution ratio for ⁶⁴ Cu-PSA-ALB-89 was high. Tumor/muscle distribution ratios increased over time to 200±38.2 after 24 h p/v.

PET/CT examinations. PET/CT imaging was performed for 24 hours in PC-3 PIP/flu tumor-bearing mice at various time points after injection of ⁶⁴ Cu labeled radioactive ligand (Figure 17). ⁶⁴ Cu-PSA-ALB-89 was present in significant amounts in PSA-positive tumors (PC-3 PIP tumors), while there was no uptake in PSA-negative tumors (PC-3 flu tumor). It was shown by imaging that 16 hours post-injection, the tumor/kidney ratio for accumulated radioactive ligand was definitely >1 and increased over time. Baseline signals from other organs and tissues, due to total radioactivity in the blood, were visible on images obtained after 1 h po.

5.3 DISCUSSION

In the example described, long-circulating ^64Cu -labeled PSA ligands were synthesized in order to perform PET-CT even one day after radioligand administration. PSA-ALB-89 was synthesized as previously described for PSA-ALB-06, however, instead of the unifying chelating agent DOPA, the chelating agent NODAGA was used, as previously performed by the group of authors for other target agents.

PSA-ALB-89 was labeled with ⁶⁴ Cu at a high specific activity and radiochemical purity (50 MBq/nmol; >95%), which implies a high-quality synthesized ligand, as well as excellent radiochemical purity of ⁶⁴ Cu, which was obtained in-house at the Paul Scherrer Institute (PSI) in vitro, ⁶⁴ Cu-PSA-ALB-89 and ⁶⁴ Cu-PSA-ALB-06 both remained stable after incubation for several hours at room temperature with little degradation detectable 24 hours after administration. These results suggest that the NODAGA and DOTA chelating agents both form stable complexes with ⁶⁴ Cu in vitro.

The albumin-binding properties of ⁶⁴ Cu-PSA-ALB-89 were similar to those of ⁶⁴ Cu-PSA-ALB-06 when tested in vitro. PSA binding specificity did not change with the chelating agent used, as evidenced by the fact that ^64Cu -PSA-ALB-89 and ^64Cu -PSA-ALB-06 had similar cell binding parameters and internalized in vitro fractions.

Biodistribution profile data from well-established murine tumor models using PSA-positive and PSA-negative tumor cells showed that tumor uptake of ^64Cu -PSA-ALB-89 was significantly higher at all time points after administration, probably due to longer circulation in the blood. The maximum tumor uptake of ⁶⁴ Cu-PSA-ALB-89 was achieved only by the end of the study (after 24 h po). PET/CT images confirmed the favorable distribution profile of ⁶⁴ Cu-PSA-ALB-89 in tissues, taking into account increased uptake by tumor cells and accumulation in the liver. A slight accumulation of the drug in the liver is an important factor, since prostate cancer can metastasize to the liver, which in other cases may not be noticed due to non-specific radioactive accumulation.

5.4 CONCLUSION

In the present example, the DOTA chelating agent in PSA-ALB-06 was replaced with the NODAGA chelating agent to ensure stable ⁶⁴ Cu coordination for PET imaging. ⁶⁴ Cu-PSA-ALB-89 showed increased stability in vivo , which was expressed in increased accumulation of the compound by tumor cells without significant retention by liver tissues.

Example 6 Preparation and Evaluation of Additional DOTA-Functionalized PSA Binding Ligands

6.1 MATERIALS AND METHODS

Solid-phase synthesis of albumin-binding PSA ligands.

PSA ligands, designated as PSA-ALB-02, PSA-ALB-05, and PSA-ALB-07, respectively, were designed and synthesized on a solid phase synthesis platform. A urea-based PSA-binding pharmacopore, l-Glu-NH-CO-NH-l-Lys, was synthesized on 2-chlorotrityl resin (2-CT) in analogy to the method described by Eder et al . (2012). A linker site comprising 2-naphthyl-l-Ala and trans -cyclohexyl molecules was synthesized as described in Example 1. Such a resin-immobilized and bis( t -Bu)-protected precursor, l-Glu-NH-CO-NH- l-Lys-2-Nal-l-Ala-NH ₂ -Me-1,4- trans -CHX, referred to in the description as compound 1 - was used as the basis for the synthesis of three albumin-binding PSA ligands (Figure 18).

The next stages of the synthesis, which included conjugation of the structural element based on lysine and selective cleavage of Nα -Fmoc-protective groups, were carried out for all three compounds. Regarding the amount immobilized on the resin and bis( t -Bu)-protected precursor (0.3 mmol; compound ( 1 )), 4 eq of Nα -Fmoc- and N ε-Alloc-protected L-lysine (Fmoc-Lys(Alloc )-OH), which were activated with 3.96 eq of O- (benzotriazol-1-yl) -N , N , N ', N' -tetramethyluronium hexafluorophosphate (HBTU) in the presence of 4 eq of N , N -diisopropylethyleneamine (DIPEA) in N , N -dimethylformamide (DMF) and stirred for 1 hour. Then sequential selective removal of N α-Fmoc-protective groups was carried out with a mixture of DMF and piperidine in a ratio of 1:1 ( v / v ). The resulting precursor ( 2 ) was used for subsequent synthesis, which was specific for each particular compound.

PSA-ALB-02. PSA-ALB-02 was synthesized by combining an albumin-binding element with a resin-immobilized precursor (0.1 mmol; compound ( 2 )) using 4 equiv of 4-( p -iodophenyl)butyric acid activated with 3.96 equiv of HBTU in the presence of 4 equiv of DIPEA in DMF for 1 hour with slow stirring. Then, Nε-Alloc- protective groups were sequentially cleaved from compound (3) using 0.03 eq. hours in total darkness. To remove residual palladium, the resin was further washed with 1% DIPEA in DMF and then with a solution of sodium diethyldithiocarbamate in DMF (c=15 mg/ml). Finally, the chelating agent was conjugated to the resin- immobilized compound using 2 eq . 1,4,7,10-tetraazacyclo-dodecan-1-yl)acetic acid] activated with 1.98 eq HBTU in the presence of 4 eq DIPEA in DMF. Combining the DOTA chelating agent took place over 2 hours with slow stirring. The resulting compound ( 4 ) was washed with DMF, DCM and finally with Et ₂ O followed by drying in vacuo. The product was cleaved off the resin and deprotected sequentially over 2 hours using a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TPS) and H ₂ O in a ratio of 95:2.5:2.5 (v/v). TFA was evaporated, the crude product was dissolved in acetonitrile (ACN) and water in a ratio of 1:1 (v/v) and purified by high performance liquid chromatography in reverse phase (RP-HPLC) using a semi-preparative column (side information). The properties of PSA-ALB-02 were studied using analytical RP-HPLC (associated information) and laser desorption/ionization mass spectrometry (MALDI-MS) or sputter ionization mass spectrometry (ESI-MS), respectively.

The synthesis process described above is briefly depicted in Scheme 6.1.

Scheme 6.1: Conjugation of PSA precursor, albumin-binding element and chelating agent DOTA to form

PSA-ALB-05 and PSA-ALB-07. PSA-ALB-05 was synthesized by combining a D-aspartate building block with a resin-immobilized precursor (0.1 mmol; compound (2)) using 3 equiv of N-Fmoc- and Oβ-t-Bu-protected d- aspartate (Fmoc-d-Asp-O-t-Bu) activated with 2.97 equiv HBTU in the presence of 4 equiv DIPEA in DMF for 1 h with slow stirring. Selective cleavage of the N-Fmoc protecting groups from the resulting compound was carried out as described above. Similarly, the addition of one additional protecting group Fmoc-d-Asp-O-t-Bu and subsequent cleavage of N-Fmoc were repeated to form compound (5). In the next step, 4 equiv of 4-(p-iodophenyl)butyric acid was activated with 3.96 equiv of HBTU in the presence of 4 equiv of DIPEA in DMF and stirred for 1 h. Selective cleavage of the Nε-Alloc protecting group from product (6) proceeded as described above. Conjugation of the chelating agent with the resin-immobilized compound was carried out with 2 eq of DOTA-tris(tert-butyl) ether activated with 1.98 eq of HBTU in the presence of 4 eq of DIPEA in DMF for 2 h with slow stirring. The resulting compound (7) was washed with DMF, DCM, and finally with Et2O, followed by vacuum drying. The product was cleaved off the resin and deprotected sequentially over 2 hours using a mixture of TFA, TPS and H2O in a ratio of 95:2.5:2.5 (v/v). TFA was evaporated, the crude product was dissolved in acetonitrile (ACN) and water in a ratio of 1:1 (v/v) and purified by RP-HPLC (related information). The properties of PSA-ALB-05 were studied using analytical RP-HPLC (related information) and MALDI-MS or ESI-MS, respectively.

Synthesis and purification of PSA-ALB-07 was carried out by analogy with PSA-ALB-05 with one additional addition of the third protecting group Fmoc-d-Asp-O-t-Bu and subsequent elimination of N-Fmoc (8). In the next steps, 4-(p-iodophenyl)butyric acid (9) was conjugated, followed by selective cleavage of the Nε-Alloc protecting group and conjugation with DOTA-tris(t-Bu) ester (10). After cleavage from the resin, the compound was sequentially deprotected and purified/characterized as described for PSA-ALB-05 (related information). Synthesis of PSA-ALB-05 and PSA-ALB-07 is summarized in Scheme 6.2. The stability of each PSA ligand in the form of a lyophilized powder was tested by RP-HPLC and MALDI-MS after long-term storage (2 and 4 months, respectively) in a freezer (-18°C).

Scheme 6.2: Conjugation of PSA precursor, albumin-binding element and chelating agent DOTA to form (A) PSA-ALB-05 and (B) PSA-ALB-07 ^a .

Radiolabeling and Stability Studies

New PSA ligands (PSMA-ALB-02, PSA-ALB-05 and PSA-ALB-07, respectively) and PSA-617 (Advanced Biochemical Compounds, ABX GmbH, Radeberg, Germany) were dissolved in MilliQ water containing 10- 15% sodium acetate solution (0.5 M, pH 8) to prepare 1 mM stock solutions for subsequent radioactive labeling. PSA ligands were labeled with radioactive 177Lu (without the addition of 177LuCl3 carrier in 0.04 M HCl provided by Isotope Technologies Garching (ITG GmbH, Germany)) in a mixture of sodium acetate (0.5 M, pH 8) and HCl (0.05 M) at pH 4 and specific activity 5 -50 MBq/nmol. The reaction mixture was incubated for 10 minutes at 95°C. Quality control of radioactive ligands was carried out using RP-HPLC (supporting information). The solution with radioactive ligands was subsequently used for in vitro and in vivo experiments without additional purification steps.

The stability of the radioactive ligands over time was determined using RP-HPLC. PSA ligands were labeled with 177Lu (250 MBq) at a specific activity of 50 MBq/nmol both without and with the addition of l-ascorbic acid (0.5 M, 3 mg), followed by dilution in saline to a radioactivity concentration of 250 MBq/500 µl. The efficiency of radioactive labeling was determined immediately after obtaining the solutions (t = 0 h), and the integrity of the compounds was analyzed after incubation for various time intervals (t = 1 h, 4 h, and 24 h, respectively) at room temperature. The amount of the intact compound was calculated by integrating the chromatographic peak of the product with respect to the sum of all radioactive peaks of the decay products of unknown structure and traces of free 177Lu, which was taken as 100%.

Determination of n-octanol/PBS partition coefficients (LogD values).

Partition coefficients (logD values) for ¹⁷⁷ Lu-labeled radioactive ligands were determined using the shake flask method using liquid phase extraction followed by phase separation as previously described.

Briefly, PSA ligands were labeled with radioactive ¹⁷⁷ Lu at a specific activity of 50 MBq/nmol. A sample of the radiolabelled ligand was mixed with phosphate buffered saline (PBS) and n-octanol, followed by vigorous vortex mixing. After centrifugation to separate the phases, the activity in each layer was measured using a gamma counter (Perkin Elmer, Wallac Wizard 1480). For each compound, three experiments were performed with five parallel analyses.

Filtration analysis.

The ability of the radioactive ligands to bind to mouse and human plasma proteins was determined using ultrafiltration analysis as described previously (Example 1). 177Lu-labeled PSA ligands (50 MBq/nmol) were dissolved in mouse (Rockland, USA) and human (Stiftung Blutspede SRK Aargau-Solothurn, Switzerland), respectively, and incubated for 15 min at room temperature. In addition, radioactive ligands were dissolved in PBS (buffer solution without proteins) for the control experiment. Aliquots of the solutions were applied to an ultrafiltration column and centrifuged. The activity of the filtered sample was measured using a gamma counter and used to calculate the activity of bound plasma proteins (remaining on the filter membrane) expressed as a percentage of the total added activity. Three independent experiments were performed for each radioactive ligand (177Lu-PSA-ALB-02, 177Lu-PSA-ALB-05 and 177Lu-PSA-ALB-07, respectively), each experiment was repeated twice. An additional experiment was performed twice using 177Lu-PSA-617. Statistical analysis (one-way analysis of variance (ANOVA) with Bonferroni multiple comparisons to evaluate results) was performed using GraphPad Prism software version 7.

A p<0.05 value was considered statistically significant.

Cellular capture and internalization.

The total PSA-bound fraction on the cell surface, as well as the internalized fraction (denoted by the term cell uptake), as well as the separately internalized fraction of radioactive ligands, were determined at a specific activity of 5 MBq/nmol using PSA-positive PC-3 PIP and PSA-negative PC -3 flu cells as previously described (Example 1). The radiolabeled solution was diluted with saline containing 0.05% (w/v) bovine serum albumin (BSA) to prevent adhesion of the solution to laboratory tubes and vials. Further dilution of the radioactive ligand solution with cell culture medium resulted in a final concentration of BSA of 0.00125%, which had no effect on cellular uptake and internalization of radioactive ligands because was negligible. In parallel with each experiment with a new radioactive ligand, control experiments were performed with 177Lu-PSMA-617. Each experiment was carried out three times, with each ligand also being carried out three times.

Biodistribution analysis.

Biodistribution was studied 1 h, 4 h, 24 h, 48 h, 96 h, and 192 h after administration of radioactive ligands labeled with a specific activity of 5 MBq/nmol. The mass of the tumor at the time of the introduction of the radioactive ligand was 150±40 mg, which corresponds to an average volume of approximately 150 mm ³ . The corresponding radioactive ligand (5 MBq, 1 nmol, 100 µl) diluted in saline was injected into the lateral tail vein of mice. BSA (0.05%) was added to the saline solution to prevent absorption of the radioactive ligand to the walls of vials and syringes. Mice were euthanized at various times after administration of the compound (p/v), selected tissues and organs were removed, weighed and activity was measured using a gamma counter. For each time point, a group of 3-6 animals was used. In addition, blocking assays were performed by injecting 2-(phosphomethyl)-pentanedioic acid (2-PMPA, 500 nmol, 100 µl) diluted in saline. The 2-PMPA solution was administered 15 minutes prior to the administration of ¹⁷⁷ Lu-PSA-ALB-02, and the animals were euthanized 1 h and 4 h po, respectively. Results corrected for radioactive decay were expressed as a percentage of the total activity administered per gram of tissue weight (% IA/g). Area under the curve (AUC) was determined for all three albumin-binding PSA ligands and ¹⁷⁷ Lu-PSA-617 from non-radioactive decay-adjusted tumor, kidney, and blood biodistribution data obtained using GraphPad Prism Software, version 7 .

Statistical analysis was performed to compare areas under the curve (AUCs) obtained from the biodistribution data set using one-way analysis of variance (ANOVA) Bonferroni one-way analysis of variance with multiple comparisons to evaluate results using GraphPad Prism Software (version 7). A p value <0.05 was considered statistically significant.

SPECT/CT imaging studies.

SPECT/CT studies were performed 4 hours, 24 hours and 72 hours after the administration of the radioactive ligand. Animals were injected with the appropriate radioactive ligand (25 MBq, 1 nmol, 100 μL) diluted in saline containing 0.05% BSA via the lateral tail vein. In addition, SPECT/CT was performed 1 h, 4 h and 24 h after administration of 177Lu-PSA-ALB-02 in mice that received 2-PMPA (500 nmol, 100 µl) or PSA-ALB-02 (100 nmol, 100 µl) without a radioactive label, 15 minutes before the introduction of a radioactive ligand to block PSA. SPECT/CT studies were performed using a special small SPECT/CT camera (NanoSPECT/CTTM, Mediso Medical Imaging Systems, Budapest, Hungary). First, SPECT was performed for 45 minutes, then CT for 7.5 minutes. During the in vivo study, mice were euthanized with a mixture of isoflurane and oxygen. Data were pre-processed using NanoSPECT/CTTM software, post-processing was performed using VivoQuant (version 3.0, inviCRO Imaging Services and Software, Boston USA). A post-reconstruction Gaussian filter (FWHM = 1 mm) was applied and the radioactivity scale was set on the images (minimum value = 0.095 Bq/voxel, maximum value 95 Bq/voxel).

6.2 RESULTS

Synthesis of PSA ligands. PSA ligands with an albumin-binding element were synthesized using a platform for solid phase synthesis using a standard Fmoc(9-fluorenylmethyloxycarbonyl) protocol (Figure 19). The synthesis was started by immobilizing the C-terminus of the first amino acid on a 2-CT resin and continued in the C→N direction. At the last stage, the compound was cleaved from the resin, followed by complete removal of the protective groups, both processes took place in an acidic medium. Multi-stage synthesis of PSA-ALB-02 (17 stages), PSA-ALB-05 (20 stages) and PSA-ALB-07 (22 stages) made it possible to obtain compounds with a high degree of purity (>98%) and a total yield of 12.9-21.2% after purification with semi-preparative HPLC (Table 6.1). All three PSA ligands were stable for at least 4 months in the form of lyophilized powders stored at -18°C.

^a Mass spectrometry of unlabeled ligands, defined as [M+H]⁺.

^b Retention time of unlabeled ligand in analytical RP-HPLC. Analytical column (100 x 4.6 mm), used Chromolith RP-18e stationary phase with mobile phases containing 0.1% TFA in water (A) and ACN (B). For analytical runs, a linear gradient of solvent A (90-10% in 10 min) in solvent B at a flow rate of 1 ml/min was used.

^θ The purity of PSA-617 was determined based on the ABX GmbH certificate for this compound.

Radioactive labeling, stability and in vitro properties ¹⁷⁷ Lu-PSA ligands.

PSA-ALB-02, PSA-ALB-05 and PSA-ALB-07 were labeled with ¹⁷⁷ Lu with a specific activity of no more than 50 MBq/nmol. Radioactive ligands showed a high degree of radiochemical purity >98%. The addition of L-ascorbic acid resulted in ~97% of intact ¹⁷⁷ Lu-PSA-ALB-02, ~96% of ¹⁷⁷ Lu-PSA-ALB-05 and ~89% of intact ¹⁷⁷ Lu-PSA-ALB-07 after 24 h (Figure 20V). ¹⁷⁷ Lu-PSA-617 was less stable, yielding ~86% intact compound after 4 h, however, complete degradation (<2% intact compound) was observed after 24 h (Figure 20A). The presence of L-ascorbic acid prevented radiolysis completely, resulting in >98% yield of intact ¹⁷⁷ Lu-PSA-617 even after 24 h (Figure 20B). The n-octanol/PBS partition coefficient (logD value) for ¹⁷⁷ Lu-PSA-ALB-02 (-2.8±0.09) was the highest. The lowest logD value was obtained for ¹⁷⁷ Lu-PSA-617 (-4.4±0.15).

Study of cellular uptake and binding to albumin in vitro

The uptake of ¹⁷⁷ Lu-PSA-ALB-02, ¹⁷⁷ Lu-PSA-ALB-05 and ¹⁷⁷ Lu-PSA-ALB-07 PC-3 PIP cells ranged from 52-57%, while the internalized fraction ranged from 18 to 24% after a 2 hour incubation period at 37°C (Figure 21A). After 4 hours of incubation, cell uptake and internalization increased slightly to 60-63% and 20-26%, respectively. ¹⁷⁷ Lu-PSMA-617 showed similar cellular uptake (58%), however only 12% of the radioactive ligand was internalized after 4 hours of incubation. Uptake of PC-3 flu cells was below 0.5% for all albumin-binding radioactive ligands as well as for ¹⁷⁷ Lu-PSMA-617 (Figure 21B).

The results of ultrafiltration confirm the pronounced ability of ¹⁷⁷ Lu-PSA-ALB-02, ¹⁷⁷ Lu-PSA-ALB-05 and ¹⁷⁷ Lu-PSA-ALB-07 to bind to plasma proteins when incubated with mouse plasma (87±1.0%, 77±2 .1% and 64±2.1%, respectively) and human plasma (95±1.2%, 95±0.6 and 95±0.1%, respectively). The obtained values were significantly higher ( p <0.05) than for ¹⁷⁷ Lu-PSMA-617, which indicates a very low ability of this compound to bind to mouse plasma proteins (9.3±1.1%) and only insignificant binding to human plasma proteins (57± 2.3%). Control experiments that were carried out with PBS showed that the retention of radioactive ligands on the filter (<5%) was likely due to the absorption of the compounds on the filtration device (data not shown).

Biodistribution analysis.

The distribution of ¹⁷⁷ Lu-PSA-ALB-02, ¹⁷⁷ Lu-PSA-ALB-05, and ¹⁷⁷ Lu-PSA-ALB-07 in tissues was evaluated in tumor-bearing mice of the PC-3 PIP and PC-3 flu lines after injection into the right and left shoulder, respectively. The observation period was 192 hours (Figure 22).

The capture of all radioactive PC-3 PIP ligands by tumor cells had the same kinetic profile. ¹⁷⁷ Lu-PSA-ALB-02 rapidly accumulated in the tumor, after 4 h s/in the accumulation was 78.4±12.8% IA/g and remained at this level for 24 h s/v (76.4±2.49% IA/g) . All new compounds, in particular ¹⁷⁷ Lu-PSA-ALB-02, showed high levels of activity (18-21% IA/g), rapid clearance of radioactivity from the blood and rapid renal clearance. The maximum tumor uptake of ¹⁷⁷ Lu-PSA-617 was ~56% IA/g as early as 4 h po, by 192 h it decreased to approximately 20% IA/g. It was rapidly eluted from the bloodstream, reaching levels of <1% IA/g after 1 h, and was steadily leached by the kidneys: ~10% IA/g after 1 h po to <1% IA/g after 24 h p / w. Radioactivity levels in all other tissues were substantially below blood levels and gradually decreased over time. Tumor/blood, tumor/kidney and tumor/liver ratios were higher for all new compounds, especially for ¹⁷⁷ Lu-PSA-ALB-02. Due to rapid renal clearance, the tumor/surrounding intact tissue ratio for ¹⁷⁷ Lu-PSA-617 was high.

Additional studies were conducted in order to block PSA by administering 2-PMPA before injection of ¹⁷⁷ Lu-PSA-ALB-02. Uptake by tumor PC-3 PIP cells was reduced by 65% (17.6±3.24% IA/g) and 41% (46.0±7.29% IA/g) at 1 h and 4 h po, respectively, when compared to unblocked uptake at the same time points. The accumulated radioactivity in the kidneys decreased to 81% and 59% 1 hour and 4 hours, respectively, after the administration of the radioactive ligand. In all other organs and tissues, a slight and indistinct decrease in accumulated radioactivity was observed (data not shown).

To calculate the area under the curve (AUC), we used data on the accumulation of radioactive ligands in the bloodstream, tumors, kidneys and liver without correction for radioactive decay (Figure 23, Table 6.3).

For all new radioactive ligands, comparable AUCs were determined, reflecting the uptake of PIP by PC-3 tumor cells, which were almost two times greater than the AUC ( p < 0.05) calculated for ¹⁷⁷ Lu-PSMA-617. For all radioactive ligands, the tumor/blood, tumor/kidney, and tumor/liver distribution ratios were high. For ¹⁷⁷ Lu-PSMA-617, the high ratio of TBR: tumor/intact tissue accumulation and the large area under the curve were due to the rapid renal clearance of this radioactive ligand (Table 6.2).

SPECT/CT imaging studies. SPECT/CT images were obtained for mice inoculated with PC-3 PIP/flu tumors at 4 h, 24 h and 72 h after administration of the new radioactive ligands as well as ¹⁷⁷ Lu-PSA-617 (Figures 24 and 25).

The accumulation of all albumin-binding radioactive ligands in PC-3 PIP tumor xenografts was the same after 24 h ip. Renal uptake, especially for ¹⁷⁷ Lu-PSA-ALB-02, was low. Time-dependent SPECT/CT images obtained with ¹⁷⁷ Lu-PSA-ALB-02 revealed a decrease in tumor/surrounding intact tissue ratio over time. Compared to ¹⁷⁷ Lu-PSA-617, uptake of ¹⁷⁷ Lu-PSA-ALB-02 by tumor cells increased significantly throughout the study period, a similar accumulation occurred in the kidneys (Figure 25). No active accumulation was observed in PSA-negative PC-3 flu tumors.

Claims (111)

1. Compound of general formula (1a)

, where D is a chelating residue selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 2-(4,7-bis(carboxymethyl)-1, 4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA) or their derivatives, X is each independently selected from O, R ¹ and R ² are each independently H, F, Cl, Br, I, or C ₁ -C ₁₂ alkyl, Y is selected from the group consisting of a direct bond or C ₁ -C ₁₂ alkyl, R ³ , R ⁴ and R ⁵ are each independently selected from -CO ₂ H, the spacer has the formula (3a), (3b) or (3c)

, where m is an integer 1 or 2, n is an integer selected from 1, 2, 3, 4 or 5, o is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, the linker has the structural formula (6)

, where X is each independently O, Q is selected from C ₅ -C ₁₄ cycloalkyl, W is selected from -(CH ₂ ) _c -aryl, where c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and a, b are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or its pharmaceutically acceptable salts or radiolabeled complexes. 2. The compound according to claim 1, wherein said compound has the general formula (11)

, where D is a chelating residue selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 2-(4,7-bis(carboxymethyl)-1, 4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA) or their derivatives, X is each independently selected from O, R ¹ and R ² are each independently H, F, Cl, Br, I, or C ₁ -C ₁₂ alkyl, Y is selected from the group consisting of a direct bond or linear or branched C ₁ -C ₁₂ alkyl, R ³ , R ⁴ and R ⁵ are each independently selected from -CO ₂ H, the spacer has the formula (3a), (3b) or (3c)

, where m is an integer 1 or 2, n is an integer selected from 1, 2, 3, 4 or 5, o is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, the linker has the structural formula (6)

, where X is each independently O, Q is selected from C ₅ -C ₁₄ cycloalkyl, W is selected from -(CH ₂ ) _c -aryl, where c is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and a, b are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or its pharmaceutically acceptable salts or radiolabeled complexes. 3. Connection according to any one of paragraphs. 1, 2, where the chelating residue D is selected from DOTA, NODAGA, DO3AP, DO3AP ^PrA , or DO3AP ^ABn . 4. The connection according to any one of paragraphs. 1-3, where Y represents a linear or branched C ₁ -C ₁₀ alkyl, preferably a linear or branched C ₁ -C ₆ alkyl, more preferably a linear or branched C ₁ -C ₃ alkyl. 5. The compound according to claim 4, where Y is linear C ₁ -C ₃ alkyl. 6. A compound according to any one of the preceding claims, wherein R ¹ and R ² are each independently selected from H and halogen, preferably iodine or bromine, and C _1-6 alkyl, preferably C _1-3 alkyl, more preferably methyl. 7. The compound according to claim 6, where in the general formula (1a) the group

represents one of the structural formulas (2a), (2b) or (2c)

8. Connection according to any one of paragraphs. 2-7, where said compound has one of the general formulas (11.1)-(11.3)

9. Connection according to any one of paragraphs. 2-8, where said compound has one of the general formulas (12.1)-(12.4) or (13.1)-(13.4)

or its pharmaceutically acceptable salts or radiolabeled complexes, where D, spacer, linker, X, R ¹ -R ⁵ , a, b, m, n have the meanings defined in paragraph 2, and d is 4, more preferably D is a chelating residue selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 2-(4,7-bis(carboxymethyl)-1 ,4,7-triazononan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentadiic acid (DOTAGA) or their derivatives R ¹ and R ² are each independently selected from H, halogen, preferably iodine or bromine, or C _1-6 alkyl, preferably C _1-3 alkyl, even more preferably methyl, the linker has the general formula (6) as defined above, more preferably the linker has the general formula (6a)

, a, b are each independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, d is 4, m is an integer 1 or 2, and n is an integer selected from 0, 1, 2, 3, 4 or 5, more preferably a and b are each independently an integer selected from 0, 1, 2, 3, 4, 5 or 6. 10. A compound according to any one of the preceding claims, wherein Q is selected from C ₅ -C ₇ cycloalkyl. 11. A compound according to claim 10 wherein Q is cyclohexyl. 12. The compound according to any one of the preceding paragraphs, where W is selected from -(CH ₂ ) _c -naphthyl, -(CH ₂ ) _c -phenyl, -(CH ₂ ) _c -biphenyl, where c is an integer selected from 0 , 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 13. The compound according to claim 12, where W represents -(CH ₂ )-naphthyl. 14. A compound according to any one of the preceding claims, wherein the linker has the structural formula (6a)

15. A compound according to claim 14, wherein said compound has the general formula (1c)

or its pharmaceutically acceptable salts or radiolabeled complexes. 16. A compound according to claim 15, wherein said compound has the general formula (7a)

or its pharmaceutically acceptable salts or radiolabeled complexes. 17. A compound according to claim 16, wherein said compound has the structural formula (7a)(i), (7a)(ii) or (7a)(iii)

or pharmaceutically acceptable salts and radiolabeled complexes of compounds (7a)(i), (7a)(ii) or (7a)(iii). 18. A compound according to any one of the preceding claims, wherein said spacer has the formula (3b) or (3c)

, where m is an integer selected from 1 or 2 and n is an integer selected from 1, 2, 3, 4 or 5, preferably 1, 2 or 3;

, where o is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 19. A compound according to claim 18, wherein said compound has the structural formula (7b)(i), (7b)(ii) or (7b)(iii)

or pharmaceutically acceptable salts or radiolabeled complexes of compounds (7b)(i), (7b)(ii) or (7b)(iii). 20. The use of a compound according to any one of paragraphs. 1-19 to obtain radiolabeled complexes. 21. Radiolabeled complex containing a radionuclide and a compound according to any one of paragraphs. 1-19. 22. Radiolabeled complex according to claim 21, wherein the metal is selected from the group consisting of ⁹⁴ Tc, ^99m Tc, ⁹⁰ In, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ¹⁷⁷ Lu, ¹⁵¹ Tb, ¹⁸⁶ Re , ¹⁸⁸ Re, ⁶⁴ Cu, ⁶⁷ Cu, ⁵⁵ Co, ⁵⁷ Co, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ²²⁵ Ac, ²¹³ Bi, ²¹² Bi, ²¹² Pb, ²²⁷ Th, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁵² Gd, ¹⁵³ Gd, ¹⁵⁷ Gd or ¹⁶⁶ Dy. 23. Pharmaceutical composition with the ability to selectively bind to the membrane of the prostate-specific antigen (PSA), containing the compound according to any one of paragraphs. 1-19 or radiolabeled complex according to claims. 21, 22 in a safe and effective amount, and a pharmaceutically acceptable carrier and/or excipient. 24. A set containing a compound according to any one of paragraphs. 1-19, or a pharmaceutically acceptable salt thereof, or a radiolabeled complex, a radiolabeled complex according to PP. 21, 22 or a pharmaceutical composition according to claim 23, which have the ability to selectively bind to the membrane of the prostate-specific antigen (PSA). 25. The use of a compound according to any one of paragraphs. 1-19, labeled with a radioactive label of the complex according to paragraphs. 21, 22, a pharmaceutical composition according to claim 23 or a kit according to claim 24 as a drug and / or diagnostic agent for the detection of PSA-expressing target cells and tissues, as well as for the treatment and diagnosis of cancer. 26. The use of a compound according to any one of paragraphs. 1-19, labeled with a radioactive label of the complex according to paragraphs. 21, 22, a pharmaceutical composition according to claim 23 or a kit according to claim 24 for detecting the presence of cells and/or tissues expressing a prostate-specific membrane antigen (PSA). 27. The use of a compound according to any one of paragraphs. 1-19, labeled with a radioactive label of the complex according to paragraphs. 21, 22, a pharmaceutical composition according to claim 23 or a kit according to claim 24 for the diagnosis, treatment and/or prevention of prostate cancer, pancreatic cancer, kidney cancer or bladder cancer. 28. Application according to any one of paragraphs. 25-27, which includes: (a) administering to the patient said compound, radiolabeled complex or pharmaceutical composition, and (b) obtaining a radiographic image of said patient. 29. Application according to any one of paragraphs. 25-28, where radiological imaging includes positron emission tomography (PET) or single photon emission computed tomography (SPECT). 30. Application according to any one of paragraphs. 25-29, wherein said one or more cell or tissue includes (optionally cancerous) prostate cells or tissues, (optionally cancerous) spleen cells or tissues, or (optionally cancerous) kidney cells or tissues. 31. Application according to any one of paragraphs. 25-29, wherein the presence of PSA-expressing cells or tissues indicates the presence of prostate tumor cells, metastatic prostate tumor cells, kidney tumor cells, pancreatic tumor cells, bladder tumor cells, and combinations thereof. 32. An in vitro method for determining the presence of cells and / or tissues expressing a prostate-specific membrane antigen (PSA), including: (a) contacting said PSA-expressing cells and/or tissues with a compound according to any one of paragraphs. 1-19, labeled with a radioactive label complex according to paragraphs. 21, 22, a pharmaceutical composition according to claim 23 or a kit according to claim 24 and (b) using imaging techniques to detect said cells and/or tissues, optionally radiological imaging. 33. The method of claim 32, wherein the x-ray imaging comprises positron emission tomography (PET) or single photon emission computed tomography (SPECT). 34. The method of claim 32, wherein said one or more cells or tissues include (optionally cancerous) cells or tissues of the prostate, (optionally cancerous) cells or tissues of the spleen, or (optionally cancerous) kidney cells or tissues. 35. The method according to any one of paragraphs. 32-34, where the presence of PSA-expressing cells or tissues indicates the presence of prostate tumor cells, metastatic prostate tumor cells, kidney tumor cells, pancreatic tumor cells, bladder tumor cells, and combinations thereof. 36. Connection of any of the structural formulas

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