WO2025163029A1

WO2025163029A1 - Psma-targeting bimodal and heterobivalent fluorescent agents

Info

Publication number: WO2025163029A1
Application number: PCT/EP2025/052325
Authority: WO
Inventors: Francesco Blasi; Roberta Napolitano; Lorena Pizzuto; Federica CHIANALE
Original assignee: Bracco Imaging SpA
Current assignee: Bracco Imaging SpA
Priority date: 2024-01-31
Filing date: 2025-01-30
Publication date: 2025-08-07
Anticipated expiration: 2026-07-31

Abstract

The present invention generally relates to the field of fluorescent probes comprising a cyanine dye with near-infrared (NIR) emission labelled with a PSMA-targeting moiety and their use in the targeting and imaging of cancer, in particular PSMA-expressing cancers, such as prostate cancer and metastases thereof. The fluorescent probes of the invention also include a different targeting ligand or a prosthetic group that can be radiolabeled, such as a radioligand, forming hybrid tracers that can be respectively used as heterobivalent agents or as bimodal agents in nuclear imaging and Fluorescence Guided Surgery. The invention also relates to methods for preparing these compounds, to pharmaceutical compositions and kits incorporating them and to methods of use them as diagnostic agents in imaging or therapy of diseases, particularly those associated with PSMA-overexpression.

Description

PSMA-TARGETING BIMODAL AND HETEROBIVALENT FLUORESCENT AGENTS

FIELD OF THE INVENTION

BACKGROUND ART

Fluorophores (or dyes) are chemical entities that absorb photons of a specific wavelength upon light excitation and re-emit some of that energy, depending on quantum efficiency, usually at a longer wavelength. Particularly, cyanine dyes are fluorescent organic molecules characterized by a delocalized electron system that spans over a polymethine bridge and is confined between two nitrogen atoms. Given the favorable optical properties, low toxicity, and good solubility in aqueous media, cyanine dyes, in particular those emitting in the Near-InfraRed (NIR) region (700-900 nm), can be used as contrast agents for biomedical imaging, due to their higher penetration depth.

To date, multiple fluorescent conjugates, constituted by a targeting vector (antibodies, peptides, particles, and other small molecules) coupled to a Near Infrared Fluorescent (NIRF) dye, have been developed or are currently being explored and validated for localization and visualization of malignant lesions, interacting with the target cell-surface proteins or microenvironment of the cancer cells (Gioux S. et al., Mol Imaging 2010;9(5):237-255). Fluorescent contrast agents with low non-specific accumulation and high selectivity for the target tissue would be preferable for applications in living organisms.

To this purpose, many fluorescent labelled tracers have been investigated, comprising functionalized vectors able to link specific molecular targets with high affinity.

One of these targets is Prostate-Specific Membrane Antigen (PSMA)m a 750-residue type II transmembrane glycoprotein, with a short /V-terminal cytoplasmic tail, a single membrane-spanning helix and an extracellular part. It exists as a symmetric homodimer endowed with enzymatic activity and it is known to possess both /V-acetylated, o-linked acidic dipeptidase (NAALADase) and folate hydrolase (FOLH) activities. The substrate binding cavity lies deep within the PSMA structure and is formed with the contribution of three identified domains: the protease (coordinating two zinc atoms); apical; and C-terminal dimerization domains (Davis MI et al. Proc. Natl. Acad. Sci. USA 2005;102(17):5981-6). PSMA constitutively undergoes endocytosis from the plasma membrane, through clathrin-coated pits, while ligand-induced internalization has been characterized after binding of antibodies or antibody fragments. In benign prostatic cells, PSMA is localized to the cytoplasmic and apical side of the prostate epithelium (Jones W. et al., Cancers 2020; 12(6): 1367). As malignant transformation occurs, PSMA is transferred from the cytoplasm to the luminal surface of the prostatic ducts, where it presents a large extracellular domain to ligands. It has likely a transport function since its ligands are internalized through endocytosis.

PSMA is expressed in nearly all prostate cancers (PCa) with about 1000-fold increased expression in poorly differentiated, metastatic, and hormone-refractory carcinomas. It is abundantly expressed at all stages of prostate cancer, presented on the cell surface and not shed into the circulation. PSMA is also expressed by other cancers, such as bladder, pancreas, lung, and renal cell cancers, and at lower levels is physiologically expressed in tissues like healthy prostate, duodenum, kidney, salivary and lacrimal glands, neuroendocrine system, proximal renal tubules, liver and brain. The above-mentioned features make it valuable as target for molecular imaging applied to diagnosis, staging, follow up, surgery and therapy of malignant tumors (Kaewput et al., J. Clin. Med. 2022;ll:2738).

The classes of PSMA-targeting agents developed so far, for both imaging and/or therapeutic applications in patients with prostate cancer, include monoclonal antibodies and small chemical compounds. Some agents are already commercialized or under clinical development. In particular, ⁱⁿIn-capromab-pendetide (ProstaScint®; AYTU Bioscience Inc.) was the first commercialized anti-PSMA antibody approved by the US FDA in 1996.

However, the use of monoclonal antibodies entails many disadvantages, e.g. slow and inefficient tumor penetration and long delay between injection and imaging, leading to high accumulation in inflammatory tissue and substantial radiation exposure, and immunogenicity, precluding repeated administration for routine diagnostic procedures.

These problems could be circumvented with the use of small molecules, displaying faster tumor uptake and more rapid excretion (Jones W. et al., Cancers 2020; 12(6): 1367). Among small chemical compounds, the PSMA pharmacological inhibitors characterized so far comprise: phosphonate-based, e.g. (phosphonomethyl)pentanedioic acid (2-PMPA); urea- based, like /V-acetylaspartylglutamate (NAAG) analogs, where two amino acids (typically glutamate and lysine) are joined by a urea linkage; thiol-based; and hydroxamate derivatives. In the last few decades, several urea-based derivatives (e.g. EuK, EuE and other "EuX" groups) were developed as highly potent inhibitors for PSMA (EP3636635 Al).

The first inhibitors were primarily used as ⁶⁸Ga-labeled radiotracers, which showed a high tumor contrast. Following the approval by the US FDA of [⁶⁸Ga]Ga-PSMA-ll and [¹⁸F]F- DCFPyL for positron emission tomography (PET) imaging to identify suspected metastases or recurrence in patients with prostate cancer, [⁶⁸Ga]Ga-PSMA-ll (also known as HBED-CC, HBED, PSMA-HBED, or Prostamedix™) is now the most widely used radiopharmaceutical for PET-CT imaging of the prostate, able to detect even very small metastasis (Kaewput et al., J. Clin. Med. 2022;ll :2738). In many cases, prostate cancer surgery can be applied as a compromise between a complete oncological resection and the preservation of vital structures, improving the diagnostic accuracy of prostate cancer detection. In these case, accurate real-time identification of prostate cancer, such as that obtained using Near- Infra Red (NIR) fluorescence, might help to enhance the detection of tumor tissue during surgery and achieve complete oncological resections and prevent damage to vital structures.

In particular, it has been recently observed that many advantages could be obtained by developing flexible targeting platforms, e.g. diagnostic or therapeutic molecules incorporating multiple moieties enabling a simultaneous targeting of different markers or complex probes allowing a hybrid imaging, where a single tracer can be used for both pre-operative detection and intraoperative imaging.

One example of such hybrid probes is disclosed in W02020/074705, describing hybrid tracers, comprising linear cyanine scaffolds (fluorescent dyes) conjugated with both a PSMA- targeting ligand and a chelating moiety that can be radiolabeled, which can be used for imaging of a tumor after administration in a subject, for instance with a nuclear imaging technique like SPECT.

Ghosh S.C. et al, J. Med.Chem. 2012, 56(2):406-416 discloses a multimodal chelation platform using dual labeled compounds containing a NIR fluorescence agent like IRDye 800CW and conjugated with a radiometal chelating agent for multimodal (optical/nuclear) imaging.

Shallal H.M. et al, Bioconj. Chem 2014, 25(2): 393-405 discloses heterobivalent agents targeting PSMA and integrin-ovfh surface markers. In particular, the compound EUKL- cRGDfK-IRDye800 is provided as example of heterobivalent imaging agent, where the cyanine fluorescent dye is attached to one of the two targeting moieties.

Further PSMA-targeting compounds for use in imaging were reported in W02010/108125, which in one aspect of the invention discloses imaging agents bearing a PSMA-targeting moiety conjugated to a fluorescent dye moiety at one of their nitrogen atoms or at the phenyl ring and to a metal chelating moiety. Another broad class of hybrid tracers is disclosed in WO2023/222679 and represented by a complex comprising a dye group Z, a chelator residue A and a PSMA binding motif, optionally separated by a linker.

Moreover, WO2022/212958 discloses heterobivalent probes comprising one or more moieties targeting both PSMA and FAP-o proteins and an optical or radiolabeled functional group separated by bi-functional linkers forming a chemical bond with each group.

However, the repertoire of available dyes targeting PSMA expressing tumors is not yet extensive and diversified. Thus, despite the efforts made so far, there is still the urgent and unmet need to develop further efficient tracers for the diagnosis, staging and therapeutic monitoring of PSMA-expressing cancers, in particular PCa, to obtain better oncological outcomes following surgery resection of cancer.

The technical problem underlying the present invention can be seen as the provision of new fluorescent probes comprising a Cy7 cyanine conjugated in one site to a PSMA ligand and in a different site to a second targeting moiety or to a prosthetic group that can be radiolabeled, such as a cage for metal coordination (e.g., a radioligand), able to confer suitability for an additional imaging modality (e.g., nuclear medicine techniques) or for radiotherapy.

This technical problem is thus solved by the embodiments characterized in the claims and the description here below.

SUMMARY OF THE INVENTION

Generally, object of the present invention is to provide PSMA targeting fluorescent probes useful as contrast agents for imaging of tumor cells and tissues before and/or during surgery, adjuvating surgeons to obtain a better oncological outcome following the resection of cancer. Particularly, the present invention provides imaging agents represented by a flexible platform where a fluorescent cyanine dye is simultaneously labelled on one side to a PSMA binding moiety and on the other side to a moiety targeting a different biomarker (multitargeting approach) or to a chelating group able to host a radiolabel (hybrid probe approach).

Therefore, the heterobivalent compounds of the invention have the advantage to provide a concurrent targeting of different markers (e.g. PSMA and/or other surface proteins), enabling imaging of cancer cells that overexpress at least one of the two markers and/or enhancing the sensitivity and specificity of detection through the synergistic increase of binding affinity to the selected targets. The impact on diagnostic, staging and therapeutic monitoring of cancer is thus greater compared to probes targeting a single site.

On the other hand, the hybrid probes of the invention, combining a fluorescent dye and a chelating moiety that can include a radiolabel, may advantageously improve their field of application beyond the corresponding NIRF probes having a PSMA-targeting moiety only. In fact, these compounds can be used for instance in intraoperative visualization of PSMA- expressing cells or tissues and also in nuclear medicine as tracers and imaging agents for PSMA-expressing cancers, in particular prostate cancer. As an example, bearing both a fluorophore and a radiolabel, they may find simultaneous application in Fluorescence-Guided Surgery (FGS) and in preoperative nuclear imaging or radiotherapy of tumors.

In detail, among the several advantages that can be achieved by means of the present compounds, the following features can be highlighted for instance: high selectivity for the targets tissue and cellular uptake, low accumulation due to non-specific interaction with other tissues, high solubility in water, medium-moderate binding to albumin.

Thus, the present invention relates to PSMA-binding fluorescent probes and/or complexes thereof, or a pharmaceutical composition thereof, for imaging and/or treating and/or preventing PSMA-expressing cancer, in particular prostate cancer and/or metastases thereof in a patient in need thereof.

Preferably, the fluorescent probes of the invention are able to selectively link cells or tissues of tumors expressing PSMA, such as prostate cancer or a tumor selected from brain cancer, breast cancer, head and neck cancer, ovarian cancer, esophageal cancer, skin cancer, gastric cancer, pancreatic cancer, bladder cancer, oral cancer, lung cancer, renal cancer, uterine cancer, thyroid cancer, liver cancer, and colorectal cancer, including both primary tumors and regional and distant metastases.

A further aspect of the invention relates to such fluorescent probes for use as diagnostic agents, in particular for use in a method of optical imaging or nuclear imaging of a human or animal organ or tissue, wherein the imaging is a tomographic imaging of organs, intraoperative cancer identification, fluorescence-guided surgery, fluorescence life-time imaging, short-wave infrared imaging, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, open field surgery, laser guided surgery, position emission tomography (PET), single photon emission computed tomography (SPECT), scintigraphy, (intraoperative) gamma or beta-tracing/imaging, or a photoacoustic or sonofluorescence method.

Moreover, the invention relates to a manufacturing process for the preparation of the provided compounds and/or pharmaceutically acceptable salts thereof, and to their use in the preparation of a diagnostic agent.

According to a further aspect, the invention relates to a pharmaceutically acceptable composition comprising at least one compound of the invention, or a pharmaceutically acceptable salt thereof, in a mixture with one or more physiologically acceptable carriers or excipients. Said compositions are useful in particular as optical imaging agents to provide useful imaging of human or animal organs or tissues or as radio-imaging or radiotherapeutic agents.

In another aspect, the present invention refers to a method for the optical or nuclear imaging of a body organ, tissue or region by use of an optical or nuclear imaging technique that comprises the use of an effective dose of a compound of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, a first aspect of the invention relates to compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein each R¹ is independently a straight or branched Ci-io alkyl;

R² is a group -SO3H or a group -CONH-Y wherein Y is a straight or branched C1-10 alkyl substituted with at least two hydroxyl groups;

R³ is selected from a group -SO3H; a group -CONH-Y, wherein Y is a straight or branched Ci-10 alkyl substituted with at least two hydroxyl groups; and a group -CONH-L2-T2, wherein L2 is a bond or a bifunctional linker and T2 is selected from a moiety targeting a carbonic anhydrase enzyme (CA) or an integrin receptor and a chelating moiety;

R⁴ is a straight or branched C1-10 alkyl-SOsH or a straight or branched C1-10 alkyl-CO- L2-T2 , wherein L2 is a bond or a bifunctional linker and T2 is selected from a moiety targeting a carbonic anhydrase enzyme (CA) or an integrin receptor and a chelating moiety;

Li is a straight or branched Ci-io alkyl-CO-;

Ti is a PSMA-targeting moiety of formula (II) wherein X is a radical of an amino acid, such as lysine, glutamic acid, optionally further substituted with a group selected form 3-(2-furyl)-alanine, 2-(2'-propynyl)-alanine and 3-(2-naphtyl)-alanine-tranexamic acid; provided that at least one of R³ or R⁴ comprises the group -L2-T2.

Preferably Li is a group -(CH2)s-CO-.

In a preferred embodiment Ti is a group having the structure (Ila), corresponding to the residue Glu-urea-L

In another preferred embodiment Ti is a group having the structure (lib), corresponding to the residue Glu-urea-Lys-3-(2-naphtyl)-alanine-tranexamic acid:

Preferably the invention relates to a compound of formula (la) wherein R¹, R³ and R⁴ are as defined above. Preferably the linker L2 is a bond (i.e. it is absent). Alternatively in a preferred embodiment L2 is group -NH-(CH2)_P-CO- or a diradical of one or more moieties selected from the group consisting of an amino acid, such as for instance glycine, alanine, 0-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid and the like; a peptide comprising from 2 to 10 amino acids in L or D configuration; 4-aminomethylbenzoic acid; cysteic acid; a polyethylene glycol, such as a group of formula -NH-(O-CH2-CH2)_P-, -NH-(CH2-CH2-O-)_P- CH2-CH2-, -N H-(O-CH2-CH₂)_P-CO-, or -NH-(CH2-CH2-O-)_P-CH₂-CH2-CO- or derivatives thereof; amino-polyethylene glycol-carboxylic acid; diaminobutyric acid; and diaminopropionic acid; or it is a group -L3-L4- wherein L3 is a diradical of a diamine, such as for instance an amino-polyethylene glycol amine of formula -NH-(O-CH2-CH2)_P-NH- or N H-(CH2-CH2-O)_P-NH- or a diradical of ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexanediamine and the like; and l_4 is a diradical of a dicarboxylic acid, such as for instance succinic acid, glutaric acid, suberic acid, adipic acid and the like; wherein p is an integer comprised between 1 and 20.

In another preferred embodiment the group T2 is a moiety targeting a protein selected from a carbonic anhydrase (CA) enzyme, an integrin receptor, fibroblast activation protein alpha (FAP-o), epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and a receptor for folic acid.

Preferably T2 is a moiety targeting a carbonic anhydrase (CA) enzyme. More preferably, it is represented by the following moiety CA-AAZ:

In another preferred embodiment T2 is an integrin receptor and comprises an Arg-Gly- Asp (RGD) binding motif. More preferably it comprises a cyclo-(Arg-Gly-Asp-D-Phe-Lys) peptide, also named c(RGDfK) binding motif.

In further preferred embodiment the group T2 is a macrocyclic or linear chelating group selected from 1,4,7, 10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), 2- [l,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA), l,4,7,10-tetraazacyclododecane-l,4,7-triacetic acid (DO3A), 1,4,7- triazacyclononanetriacetic acid (NOTA), 1,4,7-triazacyclononane-l-glutaric acid-4, 7-acetic acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane phosphinic acid (TRAP), l,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6- methylperhydro-l,4-diazepine (AAZTA), S-acetylmercaptoacetyltriglycine (MAG3) and S- acetylmercaptoacetyltriserine (MAS3).

In another aspect the invention provides for a complex comprising a compound of formula (I) as defined above, wherein T2 is a chelating moiety, and a radioactive or nonradioactive cation (e.g. as chelated radionuclide).

The present invention also relates to methods for preparing the compounds of formula (I) and the complexes thereof by means of synthetic transformations steps.

The invention further comprises compounds of formula (I) for use as fluorescent agents for the detection of tumor margins in fluorescence-guided surgery.

Preferably, the invention's fluorescent probes can selectively bind tumor cells or tissues expressing PSMA. In particular, they are able to bind prostate cancer or other tumors including brain cancer, breast cancer, head and neck cancer, ovarian cancer, esophageal cancer, skin cancer, gastric cancer, pancreatic cancer, bladder cancer, oral cancer, lung cancer, renal cancer, uterine cancer, thyroid cancer, liver cancer and colorectal cancer or diseases associated to cancer-related angiogenesis. In addition, the fluorescent probes of the invention are able to target metastatic spreads of the above-mentioned cancers in tissues and organs different from the primary source. Furthermore, the fluorescent probes of the invention are able to target pre-neoplastic lesions and dysplasia in different tissues and organs.

Description of the Figures

Figure 1 shows the binding curves of representative compounds of the invention to PSMA-expressing LNCaP cells.

Figure 2 shows the binding curves of representative compounds of the invention to CA- IX-expressing HT-29 cells.

Figure 3 shows the cellular uptake of representative compounds of the invention over time in LNCaP cells on ice (endocytosis blocked) and at 37 °C (internalization-permissive temperature). The graphs also report the uptake of the PSMA-only targeting Reference 1 and of the unconjugated dye (IRDye800CW).

Figure 4 shows the cellular uptake of representative compounds of the invention over time in HT-29 cells on ice (endocytosis blocked) and at 37 °C (internalization-permissive temperature). The graphs also report the uptake of the CA-IX-only targeting References 2 and 3 and of the unconjugated dye (IRDye800CW).

Figure 5 shows the cellular uptake of representative compound 3 of the invention over time in HT-29 cells on ice (endocytosis blocked) and at 37 °C (internalization-permissive temperature). The graphs also report the uptake of the integrin-only targeting Reference 4 and of the unconjugated dye (IRDye800CW).

Definitions

In the present description, and unless otherwise provided, the following terms and phrases as used herein are intended to have the following meanings.

The expression "straight or branched Ci-Cio alkyl" refers to an aliphatic hydrocarbon radical group, which may be a straight or branched chain, having from 1 to 10 carbon atoms in the chain. Similarly, "Ci-Ce alkyl" comprises within its meaning a linear or branched chain comprising from 1 to 6 carbon atoms. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, pentyl, hexyl and the like. Unless otherwise specified, the straight or branched alkyl is a monovalent radical group. In some cases, it may be a "bivalent" or "multivalent" radical group, wherein two or more hydrogen atoms are removed from the above hydrocarbon radical group and substituted, e.g. methylene, ethylene, iso-propylene groups and the like. In such cases, the expression "straight or branched Ci-Cx alkylene" can be used, where "x" is a number from 1 to 10.

The term "hydroxyalkyl" refers to any of the corresponding alkyl chain wherein one or more hydrogen atoms are replaced by hydroxyl groups.

The term "protecting group" (Pg) designates a protective group adapted for preserving the function of the group to which it is bound. Specifically, protective groups are used to preserve amino, hydroxyl or carboxyl functions. Appropriate protective groups may include, for example, benzyl, carbonyl, such as formyl, 9-fluoromethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc), isopropyloxycarbonyl or allyloxycarbonyl (Alloc), phtalimide, alkyl, e.g. methyl, tert-butyl or triphenylmethyl, sulfonyl, acetyl groups, such as trifluoroacetyl, benzyl esters, allyl, or other substituents commonly used for protection of such functions, which are well known to the person skilled in the art (see, for instance, the general reference T.W. Green and P.G.M. Wuts, Protective Groups in Organic Synthesis, Wiley, N.Y. 2007, 4^th Ed., Ch. 5).

Moreover, the invention also relates to the precursors or intermediates compounds suitable for the preparation of a desired compound of formula (I) or salts thereof. In such intermediates any functional group, such as a carboxylic acid or carboxamide, can be protected with an appropriate protecting group (Pg) as defined above, preferably with alkyl or ester groups. If necessary, also hydroxyl groups of Y groups can be protected with an appropriate protecting group (Pg) during the preparation of the compounds of formula (I), forming for instance acetoxy, alkoxy or ester groups.

The expression "coupling reagent" refers to a reagent used for instance in the formation of an amide bond between a carboxyl moiety and an amino moiety. The reaction may consist of two consecutive steps: activation of the carboxyl moiety and then acylation of the amino group with the activated carboxylic acid. Non limiting examples of such coupling agents are selected from the group consisting of: carbodiimides, such as N,N'-diisopropylcarbodiimide (DIC), N,N’-dicyclohexylcarbodiimide (DCC), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and l-ethyl-3- (3-dimethylaminopropyl)carbodiimide (WSC); phosphonium reagents, such as (benzotriazol- l-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-l-yloxy- tripyrrolidino-phosphonium hexafluorophosphate (PyAOP), [ethyl cyano(hydroxyimino)acetato-O2]tri-l-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP) and 3- (diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one (DEPBT); and aminium/uronium- imonium reagents, such as N,N,N',N'-tetramethyl-O-(benzotriazol-l-yl)uronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-O-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU), N,N,N',N '-tetramethyl-O-(7-aza benzotriazol- l-yl)uronium hexafluorophosphate (HATU), O-(lH-6-chlorobenzotriazole- 1-yl)- 1,1, 3,3- tetramethyluronium hexafluorophosphate (HCTU), l-[l-(cyano-2-ethoxy-2-oxoethylidene- aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU), 2-(2,5- dioxopyrrolidin-l-yl)-l,l,3,3-tetramethylisouronium tetrafluoroborate (TSTU), N,N,N’,N’- tetramethyl-O-(N-succinimidyl)uronium hexafluorophosphate (HSTU) and fluoro-N,N,N',N'- tetramethylformamidinium hexafluorophosphate (TFFH) or other compounds well known to the person skilled in the art.

The expression "activated carboxylic acid" refers to a derivative of a carboxyl group that is more susceptible to nucleophilic attack than a free carboxyl group; suitable derivatives may include for instance acid anhydrides, thioesters, acyl halides, NHS ester, sulfo NHS esters, tetrafluoro ester, nitrophenol ester and pentafluorophenol ester.

The terms "moiety" or "residue" are herewith intended to define the residual portion of a given molecule once properly attached or conjugated, either directly or through a suitable linker, to the rest of the molecule.

A "chelating moiety" or "chelating group" comprises i) a macrocyclic ring structure with 8 to 20 atoms of which 2 or more are heteroatoms selected from oxygen and nitrogen atoms; ii) an acyclic, open chain chelating structure with 8 to 20 main chain atoms of which 2 or more are heteroatoms selected from oxygen and nitrogen atoms; or iii) a branched chelating structure containing a quaternary carbon atom.

The term "imaging agent" refers to a detectable entity that can be used in in vitro, ex vivo or in vivo visualization or detection of a biological element including cells, biological fluids and biological tissues originating from a live mammal patient, and preferably, human patient, as well as human body organ, regions or tissues, when the said detectable entity is used in association with a suitable diagnostic imaging technique.

Targeting moiety Ti CPSMA binding motif)

According to the invention, the targeting moiety (Ti) is a molecule that binds with particular selectivity to the PSMA biological target and facilitates the accumulation of the contrast agent in a specific tissue or part of the body expressing PSMA protein, thus allowing the detection and imaging of a cancer, in particular prostate cancer. Generally, it is represented by a natural or synthetic molecule for use in biological systems.

Such specific binding can be achieved through a ligand, such as for instance a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or fragment thereof or an aptamer, interacting with a specific biological target expressed on the surface of the tissues or cells of interest.

Preferably such targeting moiety is represented by a small molecule. Among the known PSMA pharmacological inhibitors characterized so far, it can be mentioned the class of phosphonate-based derivatives, such as for instance (phosphonomethyl)pentanedioic acid (2- PMPA); urea-based derivatives, like N-acetylaspartylglutamate (NAAG) analogs, in which two amino acids (glutamate (E) and/or lysine (K)) are joined through their -NH2 groups by a urea linkage; thiol-based derivatives; and hydroxamate derivatives.

In a preferred embodiment, this targeting moiety is represented by the vector glutamic acid-urea-lysine (EuK) or other PSMA binding vectors of formula "EuX" as described in EP3636635 Al, namely glutamic acid linked to another amino acid or similar via a bridging urea. More preferably, the group Ti corresponds to the ligand glutamic acid-urea-lysine (EuK).

In some cases, the "EuX" binding motifs have been functionalized with several spacers, in order to increase their biological activity. In particular, the introduction of an apolar chain was shown to improve the interaction of the targeting motifs with the hydrophobic pocket of the enzyme: one example is disclosed for instance in Benesova et al., J.Nucl.Med. 2015;56:914-920 which describes the use of peptidomimetic glutamate-urea-lysine-3-(2- naphtyl)-alanine-tranexamic acid. Other derivatives are represented for instance by EuFA (glutamic acid-urea-3-(2-furyl)-alanine), EuPG (glutamic acid-urea-2-(2'-propynyl)-alanine), EuE (glutamic acid-urea-glutamic acid), or other urea-based peptidomimetics. All such PSMA binding motif can be used in alternative embodiments of the invention.

Targeting or chelating moiety (T2)

According to some preferred embodiments of the invention, the group T2 may be a targeting moiety, that is a molecule that binds with particular selectivity to a biological target and facilitates the accumulation of the contrast agent in a specific tissue or part of the body. Generally, it is represented by a natural or synthetic molecule for use in biological systems.

Suitable biological targets for the compounds of the invention can be for instance a carbonic anhydrase (CA) enzyme, such as CAIX, CAII or CAXII; an integrin receptor, such as av03, av05, av06 or os[3i integrin receptors; fibroblast activation protein alpha (FAP-o); an epithelial growth factor (EGF) receptor, such as EGFR or HER.2; a vascular endothelial growth factor (VEGF) receptor, such as VEGFR1 or VEGFR2; a receptor for the folic acid, such as FR- alpha; a mucin glycoprotein, such as MUC1; a glucose transporter, such as GLUT-1; a sodiumhydrogen antiporter, such as NHE1; a carcinoembryonic glycoprotein, such as the carcinoembryonic antigen (CEA); a chemokine receptor, such as the chemokine receptor type 4 (CXCR4); a cell adhesion molecule, such as ICAM, EPCAM, VCAM, E-Selectin, P-Selectin; the hepatocyte growth factor HGFR (c-met); a receptor for the transferrin; a ephrin receptor, such as EPHA2; a glycoprotein binding hialuronic acid, such as CD44 or a bombesin receptor, such as BB1, BB2, BB3.

Preferably the moiety T2 is a group able to bind a target selected from a carbonic anhydrase (CA) enzyme, an integrin receptor, fibroblast activation protein alpha (FAP-o), epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and a receptor for folic acid.

As an example, an integrin receptor targeting moieties is represented by a linear or cyclic peptide comprising the sequence Arg-Gly-Asp (RGD). This tripeptide has high binding specificity for the receptor, being recognized as ligand by the family of the integrin receptors located in the cell membrane. In fact, it has been identified in some extracellular matrix glycoproteins, such as fibronectin or vitronectin, which exploit this RGD motif to mediate cell adhesion. Linear and cyclic peptides and peptidomimetics containing the sequence Arg-Gly- Asp (RGD), such as for instance cRGD, cRGDfK, cRGDyK, cRGDfC, RGD-4C, RGD-2C, AH111585, NC100692, RGD-K5 (Kapp et al., Sci Rep, 2017, 7:3905), or analogues and derivatives thereof, are well-known examples of binding motif targeting cancer tissues on which cell membrane integrins are up-regulated compared to healthy tissues.

In another embodiment, the compounds of the invention can be conjugated to other small molecules, peptides, proteins or antibodies, such as for instance monoclonal antibodies already used for therapy. Small molecules containing the drug acetazolamide, such as for instance ligands 4a, 5a, 6a, 7a and 8a (Wichert et al., Nat Chem 2015, 7: 241-249), or analogues and derivatives thereof, are examples of small molecules targeting the enzyme CAIX. Linear and cyclic peptides and peptidomimetics, such as peptide GE11 (described in Li et al., FASEB J 2005, 19: 1978-85) and/or peptide LI (described in Williams et al., Chem Biol Drug Des 2018, 91 :605-619), or analogues and derivatives thereof, are examples of peptides targeting the epithelial growth factor receptor (EGFR). Among the proteins, derivatives of the epithelial growth factor (EGF) are examples of small protein targeting the epithelial growth factor receptor (EGFR). Among the antibodies, panitumumab and cetuximab are examples of monoclonal antibodies targeting the epithelial growth factor receptor (EGFR).

According to other preferred embodiments of the invention, the moiety T2 is a chelating group which is linked to the compounds of formula (I) optionally through a linker L2. Preferably, the chelating moiety is selected from 1,4,7, 10-tetraazacyclododecane- N,N',N",N"'-tetraacetic acid (DOTA), 2-[l,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA), l,4,7,10-tetraazacyclododecane-l,4,7-triacetic acid (DO3A), 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4,7-triazacyclononane-l-glutaric acid-4, 7-acetic acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7- triazacyclononane phosphinic acid (TRAP), l,4-bis(carboxymethyl)-6- [bis(carboxymethyl)]amino-6-methylperhydro-l,4-diazepine (AAZTA), S- acetylmercaptoacetyltriglycine (MAG3) and S-acetyl mercaptoacetyltriserine (MAS3).

In this case the compounds of formula (I) can be obtained by covalent binding of a carboxyl group of the chelating group to the rest of the molecule via an ester or amide bond, preferably an amide bond, optionally through attachment to the linker L2.

In another aspect, the compounds of the invention bearing such chelating moieties (T2) may form complexes when the chelating group is complexed with a radiolabel.

The term "radiolabel" or "radionuclide" as used herein refers to a radioactive isotope readily forming a radioactive or non-radioactive cation, for instance selected from a Ga, Cu, Lu, Y, Ac, In, Pb, Bi and Tc cation, or a cation that binds to ¹⁸F, preferably Al.

Preferred examples of such radiolabels are selected from the group consisting of ⁶⁸Ga, ⁶⁷Ga, ⁶⁴Cu, ⁶⁷Cu, ¹⁷⁷Lu, ⁹⁰Y, ²²⁵Ac, In, ^99mTc, ²¹²Pb, ²⁰³Pb, ²¹²Bi and ²¹³Bi. Alternatively, the chelating moiety can be linked to a cationic molecule that binds to ¹⁸F, such as a cation of Al or Sc (e.g. ¹⁸F-[AIF]²⁺).

Depending on the radioactive isotope, a radiolabel may emit y-radiation or a- or [3- particles when it decays. For instance, In and ^99mTc are y-emitters (emitting y-radiation); ²²⁵Ac and ²¹³Bi are o-emitters; ⁶⁸Ga, ⁹⁰Y and ¹⁷⁷Lu are ^-emitters. Moreover, some of the above radiolabels, for instance the 0-emitter radionuclides, are known for their application in targeted radionuclide therapy (e.g. ¹⁷⁷Lu).

Linker 1_2

According to the invention, L2 is a bifunctional linker, optionally present, that separates the targeting moiety T2 from the dye.

The presence of a linker is particularly useful for some embodiments where the ligand and the dye risk adversely interacting with each other. Moreover, the presence of the linker may be advantageous when the dye is relatively large and may interfere with the binding of the targeting moiety to the target site.

The linker can be either flexible (e.g., including linear alkyl chains) or rigid (e.g., including amino acids with aryl groups) so that the dye is oriented away from the target. The linker can also modify pharmacokinetic and metabolism of the conjugates of formula (I) used as imaging agents in a living organism.

Hydrophilic linkers may reduce the interaction with plasma proteins, reduce blood circulation time and facilitate excretion. For example, if the linker is a polyethyleneglycol (PEG) moiety, the pharmacokinetics and blood clearance rates of the imaging agent in vivo may be altered. In such embodiments, the linker can improve the clearance of the imaging agent from background tissue (/.e., muscle, blood) thus giving a better diagnostic image due to high target-to-background contrast. Moreover, the introduction of a particular hydrophilic linker may shift the elimination of the contrast agent from hepatic to renal, thus reducing overall body retention.

Preferably the linker L2 is a bond (i.e. it is absent).

In another preferred embodiment, the linker L2, when present, is a group -NH-(CH2)_P- CO- or a diradical of one or more moieties selected from the group consisting of an amino acid, such as for instance glycine, alanine, 0-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid and the like; a peptide comprising from 2 to 10 amino acids in L or D configuration; 4-aminomethylbenzoic acid; cysteic acid; a polyethylene glycol such as a group of formula -NH-(O-CH2-CH₂)p-, -NH-(CH2-CH2-O-)_P-CH2-CH2-,

NH-(O-CH2-CH2)_P-CO-, -NH-(CH2-CH2-O-)_P-CH2-CH2-CO- or derivatives thereof; aminopolyethylene glycol-carboxylic acid; diaminobutyric acid; and diaminopropionic acid; or it is a group -L3-L4- wherein L3 is a diradical of a diamine, such as for instance amino-polyethylene glycol amine of formula -NH-(O-CH2-CH2)_P-NH- or -NH-(CH2-CH2-O)_P-NH- or a diradical of ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexanediamine and the like; and l_4 is a diradical of a dicarboxylic acid, such as for instance succinic acid, glutaric acid, suberic acid, adipic acid and the like; wherein p is an integer comprised between 1 and 20.

More preferably, L2 is selected from a group -NH-(CH2)_P-CO-; a polyethylene glycol of formula -NH-(O-CH2-CH2)_P-CO-; and a diradical of from one to five amino acids, wherein p is an integer comprised between 1 and 20. The compounds of the above formula (I) may have one or more asymmetric carbon atoms, otherwise referred to as chiral carbon atoms, and may thus give rise to diastereomers and optical isomers. Unless otherwise provided, the present invention further includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof.

The present invention further relates to compounds of the above formula (I) in which the sulfonyl groups may be in the form of a negatively charged ion or a pharmaceutically acceptable salt.

Detailed description of the embodiments

In a preferred embodiment, the invention relates to a compound of formula (I) wherein R² is a group -SO3H.

In another preferred embodiment the invention relates to a compound of formula (I) wherein R² is a group -CONH-Y, wherein Y is selected from the group consisting of

More preferably, the invention relates to a compound of formula (I) wherein R² is a group -CONH-Y and Y is a group of formula (ii) as defined above. Preferably, the group (ii) has the following stereochemical configuration, obtained by using a D-glucamine in the preparation of the compounds:

In another preferred embodiment the invention relates to a compound of formula (I) wherein Ti is a PSMA-targeting moiety of formula (II), as defined above. More preferably, Ti is a group having the structure (Ila): Even more preferably the invention relates to a compound of formula (la) wherein R¹, R³ and R⁴ are as defined above.

In another preferred embodiment both R² and R3 are a group -SO3H, and R⁴ is a straight or branched C1-6 alkyl-CO-l_2-T2 as in the following formula (lb) wherein R¹, Li, L2, Ti and T2 are as defined above.

Alternatively, in another preferred embodiment R² and R³ are a group-CONH-Y wherein

Y is a straight or branched C1-10 alkyl substituted with at least two hydroxyl groups and R⁴ is a straight or branched C1-6 alkyl-CO-l_2-T2, as per formula (Ic): wherein Y, R¹, Li, L2, Ti and T2 are as defined above.

In a further preferred embodiment R² is a group -SO3H, R⁴ is a straight or branched Ci-

6 alkyl-SOsH and R³ is a group -CONH-L2-T2 as per formula (Id) wherein R¹, Li, L2, Ti and T2 are as defined above.

Especially preferred and representative of the invention are the compounds of formula

(I) listed in Table I. The present invention is also directed to methods for synthesizing the compounds of formula (I) prepared as illustrated in the following description.

The compounds of the invention are tracers that are useful as imaging agents in the detection of tumors in both humans and animals. Preferably the imaged subject is a human.

Accordingly, the invention provides the compounds of formula (I) as defined above, for use as fluorescent probes for the detection and demarcation of a tumor tissue during diagnostic, interventional imaging and intraoperative procedures, in particular wherein said tumor is a tumor showing an increased or variable expression of PSMA. In a preferred embodiment, said detection and demarcation of the tumor tissue is carried out under NIR radiation.

The fluorescent probes of the invention are able to identify in vivo a diseased tissue in a subject in need thereof. This can be accomplished by administering a compound of formula (I) as defined above and irradiating in vivo a body of the subject in need containing the diseased tissue with light having at least one excitation wavelength in the NIR range from about 650 nm to about 850 nm. Fluorescence emitted from said administered compound which is specifically bound to the diseased tissue in the body part in response to the at least one excitation wavelength is directly viewed to determine location and/or surface area of the diseased tissue in the subject.

In detail, the present invention provides compounds of formula (I), in particular compounds wherein T2 is a targeting moiety, as fluorescent probes for biomedical optical imaging applications in mammal. Preferably said fluorescent probes are for use in a method of optical imaging of a human or animal organ or tissue, wherein the imaging is selected from a tomographic imaging of organs, intraoperative cancer identification, fluorescence-guided surgery, fluorescence life-time imaging, short-wave infrared imaging, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, open field surgery, laser guided surgery and a photoacoustic or sonofluorescence method.

In a preferred embodiment said fluorescent probes are imaging agents for use in a method for detecting the possible presence of a disease in a subject comprising the steps of:

- administering to a subject in need of diagnosis an amount of a compound of formula (I) as defined above for a time and under conditions that allow for binding said compound to targeted cells;

- measuring a signal from said compound in a biological sample;

- comparing said signal with at least one control data set comprising signals from the compound of formula (I) contacted with a biological sample that does not comprise the target cell type, for indicating the possible presence of a disease.

The present invention further provides a compound of formula (I) as imaging agent for use in a method of imaging tissues and cells comprising the steps of:

- contacting the tissues or cells with a compound of formula (I) as defined above; - irradiating the tissues or cells at a wavelength absorbed by the imaging agent;

- detecting a near-infrared emission using a fluorescent camera.

In particular, in a preferred embodiment the invention provides a method for performing imaging guided surgery on a subject comprising the steps of:

- administering a composition comprising a compound of formula (I) as defined above under conditions and for a time sufficient for the compound to accumulate at a given surgical site;

- illuminating and visualizing said compound using near-infrared light;

- performing surgical resection of the areas that fluorescence upon excitation by the nearinfrared light and/or back-table fluorescence guided imaging.

The invention further relates to a compound of formula (I) as imaging agent for use in the method described above.

In another aspect, the invention provides compounds of formula (I) as defined above, when T2 is a chelating moiety, for use as radio-imaging agents or as radiotherapeutics for the treatment of proliferating cells. For instance, the radio-imaging may comprise imaging of radioactive decay and fluorescence imaging. As an example, the imaging of radioactive decay may be performed before a surgery and fluorescence imaging may be carried out during surgery to better define the margin of tumors and guide the tumor resection.

In particular the compounds of the invention are useful as agents for the imaging or treatment of PSMA expressing cancer cells, more specifically of PSMA-expressing prostate cancer cells.

In the preferred embodiments wherein T2 is a chelating moiety, the invention further provides compounds of formula (I) or radiolabeled complexes thereof as agents for use in an imaging method selected from position emission tomography (PET), single photon emission computed tomography (SPECT), scintigraphy and gamma- or beta-diagnostic imaging. Preferred imaging methods are position emission tomography (PET) or single photon emission computed tomography (SPECT).

Preferably the compounds of the invention bearing a chelating moiety form complexes with a radionuclide selected from the group consisting of ⁶⁸Ga, ⁶⁷Ga, ⁶⁴Cu, ⁶⁷Cu, ¹⁷⁷Lu, ⁹⁰Y, ²²⁵Ac, In, ^99mTc, ²¹²Pb, ²⁰³Pb, ²¹²Bi and ²¹³Bi or are represented by a group Al¹⁸Fs or Sc¹⁸Fs. More preferably, the radionuclide is selected from ⁶⁸Ga, ¹⁷⁷Lu, In, ^99mTc and ²¹²Bi or is represented by a group Al¹⁸Fs or Sc¹⁸Fs.

Radio-imaging or therapy may be carried out in a manner known to the skilled person, for instance by injecting a suitable amount of an imaging composition comprising a compound of formula (I) to provide adequate imaging and scanning with a suitable imaging or scanning instrument, such as a tomograph or gamma camera.

Accordingly, the invention further provides a method for nuclear imaging on a subject comprising the steps of: - administering to a patient a composition comprising a complex of a compound of formula (I), wherein T2 is a chelating moiety, with a radionuclide;

- exposing a region of the patient to a scanning device; and

- obtaining an image of the region of the patient.

Moreover, the invention relates to a complex of the compound of formula (I), wherein T2 is a chelating moiety, for use as a medicament, preferably for use in radiotherapy.

In a further embodiment the invention relates to a complex of the compound of formula (I), wherein T2 is a chelating moiety, for use in the treatment of a disease associated with an overexpression of PSMA.

A further aspect of this invention relates to a pharmaceutical composition comprising a fluorescent probe of formula (I) as defined above, or a complex comprising a compound of formula (I) wherein T2 is a chelating moiety and a radionuclide, or a salt thereof, and one or more pharmaceutically acceptable adjuvants, excipients, carriers or diluents.

Another aspect of this invention relates to a diagnostic kit comprising at least one compound of formula (I) as defined above or a complex comprising a compound of formula (I) wherein T2 is a chelating moiety and a radionuclide, or a pharmaceutical composition thereof. In addition, the kit can contain additional adjuvants for implementing a biomedical optical imaging application. These adjuvants are, for example, suitable buffers, vessels, detection reagents or directions for use. The kit preferably contains all materials for an intravenous administration of the compounds of the invention.

An effective amount of a compound of the invention may be administered by different routes prior to the imaging procedure, based on the disease to be treated and the location of the suspected disease to be diagnosed. For instance, it can be administered to the organ or tissue to be imaged by a topical route, e.g. transdermally, an enteral route, e.g. orally, or a parenteral route, e.g. intradermally, subcutaneously, intramuscularly, intraperitoneally or intravenously. In some embodiments the compounds of the invention can be administered by topically spraying or nebulizing pharmaceutical compositions comprising them and/or specifically formulated for that use.

The compositions are administered in doses effective to achieve the desired optical image of a tumor, tissue or organ, which can vary widely, depending for instance on the compound used, the tissue subjected to the imaging procedure and the imaging equipment being used. The exact concentration of the imaging agents in a pharmaceutical composition is dependent upon the experimental conditions and the desired results, but typically may range between 1 pM to 0.1 mM/kg body weight. The optimal concentration is determined by systematic variation until satisfactory results with minimal background fluorescence are obtained. Once administered, the imaging agents of the invention are exposed to a light source, or other form of energy, which can pass through a tissue layer. Preferably the radiation wavelength or waveband matches the excitation wavelength or waveband of the photosensitizing agent and has low absorption by the non-target cells and the rest of the subject, including blood proteins. Typically, the optical signal is detectable either by observation or instrumentally, and its response is related to the fluorescence or light intensity, distribution and lifetime.

In the preferred embodiments wherein T2 is a chelating moiety the concentration is indicated as activity dosage, i.e. the amount of radioactivity administered to a patient. Preferably said activity dosage is determined taking into account several factors like therapeutic progress and/or adverse effects observed for the patient, as known by the expert in the art. As an example, a preferred activity dosage may be at least 100 kBq/kg body weight.

The preparation of the compounds of formula (I), as such or in the form of pharmaceutically acceptable salts, represents a further aspect of the invention. The compounds of the invention can be prepared for instance according to the methods described in the experimental part. A general teaching about the preparation of cyanine scaffold can be found in Mujumdar R.B. et al., Bioconjugate Chem. 1993; 4(2): 105-111, which relates to the synthesis and labelling of sulfoindocyanine dyes.

In some cases, due to the presence of different functional moieties such as carboxylic acid or amide groups in the cyanines of the present invention, the use of protecting groups may be necessary to direct the reactions on the desired functional group. Generally, special attention is required when manipulating the cyanines at the strong pH and temperature conditions necessary to remove the protecting groups, since the stability of the polymethine scaffold can be compromised in some cases, with severe degradation of the dyes.

EXPERIMENTAL PART

The invention and its particular embodiments described in the following part are only exemplary and not to be regarded as a limitation of the present invention: they show how the present invention can be carried out and are meant to be illustrative without limiting the scope of the invention.

Materials and Equipment

All commercially available reagents used in the synthesis were obtained from Sigma Aldrich and TCI and used without further purification. Other known starting materials were prepared according to the procedures described in the literature: e.g., the moiety Glu-urea-Lys (EuK) was synthesized as described in Maresca K.P. et al., J. Med. Chem. 2009;52(2): 347-357; the ligand C8-AAZ was prepared according to Mahalingam, S. M. et al. Bioconjugate Chem. 2018, 29, 3320-3331; the ligand "4a" was prepared according to Wichert M. et al., Nature Chemistry, 2015, 7, 241-249; DOTA amine 10-[2-[(2-Aminoethyl)amino]-2-oxoethyl]-l,4,7,10- tetraazacyclododecane-l,4,7-triacetic acid was synthetized according to the procedures described in Barge A. et al., Org. Biomol. Chem. 2008, 6, 1176-1184. Moreover, the vector c(RGDfK) was obtained from Bachem, while compounds IRDye800CW-COOH and IRDye800CW-NHS were obtained from Li-Cor. All the reactions were monitored by HPLC (Agilent mod. 1100/1200) and HPLC-MS (Agilent mod. 1260, Quadrupole LC/MS Mod. 6120) equipped with an absorption detector set at different wavelengths (Column: YMC-Triart Phenyl, 250 x 4.6 mm I S-5 pm I 12 nm), eluents: 0.1% ammonium acetate and acetonitrile.

Flash chromatographic purifications were performed on an automated purification system (CombiFlash® Rf+, Teledyne ISCO), using pre-packed KP-Sil cartridge or silica C18 cartridges (Biotage® SNAP or SFAR, Teledyne RediSep Gold® C18Aq), generally eluting with methanol/ethyl acetate or water/acetonitrile gradient, respectively.

Human prostate carcinoma LNCaP cells (CRL-1740, ATCC) were cultured in RPMI medium (Euroclone) supplemented with 10% HyClone Fetal Clone III (Euroclone), 2 mM L- glutamine (Sigma-Aldrich), 100 ZU/mL penicillin, 0.1 mg/mL streptomycin, 0.25 pg/mL amphotericin B (Antibiotic-Antimycotic solution, Life Technologies) and cells were grown at 37 °C in humidified atmosphere enriched with 5% CO2. DPBS without MgCh and CaCI? (Sigma- Aldrich) was used for cell rinsing. Human colon adenocarcinoma HT-29 cells (ATCC) were cultured in McCoy's 5A medium (Sigma-Aldrich), supplemented with 10% HyClone Fetal Clone III (Euroclone), 2 mM L-glutamine (Sigma-Aldrich), 100 lU/mL penicillin, 0.1 mg/mL streptomycin, 0.25 pg/mL amphotericin B (Antibiotic-Antimycotic solution, Life Technologies).

Samples from cellular experiments were analyzed with the flow cytometer Accuri™ C6 (BD Biosciences) according to the following general parameters: threshold for event detection 2000000 in FSC-H; gate on living cells based on physical parameters (reasonable FSC-A, low SSC-A, doublets exclusion); at least 10000 valid events for each sample (/.e. inside the established gate); Flow rate: "medium"; events / pL < 1000; excitation: 640 nm laser; collection in the FL4 channel (780/60 nm filter) for each sample.

List of abbreviations

AC2O Acetic anhydride

ACOH Acetic Acid

K2CO3 Potassium Carbonate

KOAc Potassium Acetate

DMF /V,/V-Dimethylformamide

DIPEA Diisopropylethylamine

C8-AAZ 8-amino-/V-(5-sulfamoyl-l,3,4-thiadiazol-2-yl)octanamide

EuK Glutamic acid-urea-lysine

EuE Glutamic acid-urea-glutamic acid

HCOOH Formic Acid

HPLC High performance liquid chromatography

RT Room temperature

CV Column volume

DMSO Dimethyl sulfoxide DPBS Dulbecco's phosphate buffered saline

FSC Forward Scatter

SSC Side Scatter

CV% Coefficient of Variation

NMM /V-methylmorpholine

HATU l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

HBTU O-Benzotriazol-l-yl-ZV^/V'/V'-tetramethyluronium hexafluorophosphate

TBTU 2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethylaminium tetrafluoroborate

TFA Trifluoroacetic acid

TSTU O-(/V-Succinimidyl)-l,l,3,3-tetramethyluronium tetrafluoroborate

HCI Hydrochloric acid

N2 Nitrogen

NaOH Sodium hydroxide

The biological results obtained for the representative compounds of the invention and described here below were compared with the results obtained with the corresponding cyanine dye (e.g. IRDye800 CW, obtained from Li-Cor) conjugated with a single targeting moiety, Ti or T2. The structures of said conjugates are reported in the following Table II as References 1 to 4 and were prepared starting from IRDye800CW- NHS ester following procedures analogous to that described in the following Examples 1, 3 and 4:

Example 1: Preparation of Intermediate (4)

Preparation of Intermediate (2): To a solution of l-(5-carboxypentyl)-2,3,3-trimethyl-5-sulfo-3H-indole (Intermediate (1), 503.3 mg, 1.424 mmol) in DMF (5 mL), a solution of EuK(tBu)s (833.29 mg, 1.708 mmol) in DMF (5 mL) was added. Then, DIPEA (745 pL, 4.272 mmol) and HBTU (1.080 g, 2.848 mmol) were added and the red solution was stirred for 3 hours at room temperature in N2 atmosphere. Then, solvent was evaporated under reduced pressure, the residue was dissolved in 1/1 water/acetonitrile (5 mL) and purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 30 g) with an automated flash chromatographic system eluting with a water- acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile in 20 CV). Fractions containing the desired pure Intermediate (2) (eluted with 50% acetonitrile) were concentrated under reduced pressure to afford it as a red solid (824.24 mg, 1.001 mmol,

Yield: 70%). [M + H]⁺ 824.1.

Preparation of Intermediate (3):

In a dried round bottom flask, Intermediate (2) (772 mg, 0.938 mmol) and Intermediate (1) (331 mg, 0.938 mmol) were suspended in acetic anhydride (30 mL) and acetic acid (15 mL). The mixture was heated at 55 °C, until the two powders were fully solubilized. Then, 2-chloro- 3-(hydroxymethylene)-l-cyclohexene-l-carboxaldehyde (198 mg, 1.13 mmol) was added to the mixture. Potassium acetate (184 mg, 1.876 mmol) was added and the mixture was heated at 100 °C for 1 hour. Solvents were concentrated under reduced pressure (up to 5 mL) and cold diethyl ether (50 mL) was added obtaining a green suspension. The solid was filtered, dissolved in water and the pH was adjusted to 3 with 0.5 N HCI. The green mixture was let under stirring, in order to hydrolyze the by-products (anhydride formed on free carboxylic acid moiety). After 1 hour the pH was adjusted to 7 with 0.5 N NaOH.

The product was purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 60 g) with an automated flash chromatographic system eluting with water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 20 CV). Fractions containing the desired pure product (eluted with 30% acetonitrile) were concentrated under reduced pressure and dried under vacuum at 40 °C, recovering pure Intermediate (3) as a green solid (393 mg, 0.299 mmol, Yield: 32%). [M + H]⁺ 1312.4. To a solution of Intermediate (3) (388 mg, 0.296 mmol) in dry DMSO (30 mL), 4- hydroxybenzene sodium sulfonate (581 mg, 2.96 mmol) and anhydrous potassium carbonate (409 mg, 2.96 mmol) were added. The mixture was stirred at 40 °C for 3 hours. A cold solution of 1 :2 ethyl acetate : diethyl ether (450 mL) was added to the green mixture, the solid was filtered, dissolved in water and the pH was adjusted to 4 with 0.5 N HCI. The crude product was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 60 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 20 CV). Fractions containing the desired pure product (eluted with 20% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering pure Intermediate (4) as a green solid (304, 0.210 mmol, Yield: 71%). [M+H]⁺ 1450.1.

Example 2: Synthesis of Compound 1

In a 50 mL conical-bottom glass centrifuge, Intermediate (4) (30 mg, 0.0208 mmol) was dissolved at room temperature in anhydrous DMF (3 mL), obtaining a dark green dispersion. Then, NMM (5.3 pL, 0.048 mmol) and TSTU (9.4 mg, 0.0312 mmol) were added obtaining complete dissolution. The dark green solution was stirred at RT and in the dark for one hour. Ice cold ethyl acetate (25 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After 30 minutes, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green precipitate was washed with ethyl acetate and dried under N2 flow. It was dissolved in dry DMF (1.5 mL) directly in the conical-bottom glass centrifuge tube. A solution of C8-AAZ (13 mg, 0.0406 mmol) in anhydrous DMF (1 mL) was dropped, followed by a solution of DIPEA (27 pL, 0.156 mmol) in anhydrous DMF (0.5 mL). The solution was stirred overnight at room temperature under N2 atmosphere. Ice cold diethyl ether (25 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After two hours, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow.

Crude intermediate (5) was dissolved at room temperature in 95:5 TFA : water (1 mL) directly in the conical-bottom glass centrifuge, obtaining a brown solution. After 30 minutes, cold diethyl ether (25 mL) was added at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark green precipitate. After two hours, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow. The solid was dissolved in water and the pH was adjusted to 4 with 0.5 N NaOH. The crude product was purified by flash chromatography on a prepacked C18 silica column (Biotage® SNAP ULTRA 12 g) with a 0.1% HCOOH/acetonitrile gradient (stepped gradient from 100% HCOOH buffer to 100% acetonitrile over 30 CV). Fractions containing the desired pure product (eluted with 25% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering pure Compound 1 as a green solid (13.8 mg, 0.0087 mmol, Yield: 42%). [M+H]⁺ 1585.2.

Example 3: Synthesis of Compound 2

In a 50 mL conical-bottom glass centrifuge, Intermediate (4) (20 mg, 0.0138 mmol) was dissolved at room temperature in anhydrous DMF (2 mL), obtaining a dark green dispersion. Then, NMM (3.5 pL, 0.0317 mmol) and TSTU (6.2 mg, 0.0207 mmol) were added obtaining complete dissolution. The dark green solution was stirred at RT and in the dark for 1.5 hour.

Ice cold ethyl acetate (45 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After 30 minutes, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was washed with ethyl acetate and dried under N2 flow. The crude product was dissolved in dry DMF (1 mL) directly in the conical-bottom glass centrifuge tube. A solution of "4a" ligand, prepared as described in Wichert M. et al, Nat. Chem. 2015; 7(3): 241-249, (14.9 mg, 0.0241 mmol) in anhydrous DMF (1 mL) was dropped, followed by a solution of DIPEA (12 |j L, 0.069 mmol) in anhydrous DMF (0.5 mL). The solution was stirred overnight at room temperature under N2 atmosphere. Ice cold diethyl ether (25 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After two hours, the tube was centrifuged (T : 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow.

In a 50 mL conical-bottom glass centrifuge, the crude Intermediate (6) was dissolved at room temperature in 95:5 TFA : water (1 mL), obtaining a brown solution. After 30 minutes, cold diethyl ether (25mL) was added to the brown mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark green precipitate. After two hours, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow. The crude solid was dissolved in water and the pH was adjusted to 8.5 with 0.5 N NaOH. It was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 12 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 17 CV). Fractions containing the desired pure product (eluted with 10% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering Compound 2 as a green solid (14.6 mg, 0.0073 mmol, Yield: 53%). [M+H]⁺ 1883.2.

Example 4: Synthesis of Compound 3

In a 50 mL conical-bottom glass centrifuge, Intermediate (4) (20 mg, 0.0138 mmol) was dissolved at room temperature in anhydrous DMF (2 mL), obtaining a dark green dispersion. Then, NMM (3.5 pL, 0.0317 mmol) and TSTU (6.2 mg, 0.0207 mmol) were added obtaining complete dissolution. The dark green solution was stirred at RT and in the dark for 1.5 hours.

Cold ethyl acetate (25 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After 30 minutes, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was washed with ethyl acetate and dried under N2 flow. It was dissolved in dry DMF (1 mL) directly in the conical-bottom glass centrifuge tube. A solution of c(RGDfK)

(13.8 mg, 0.0169 mmol) in anhydrous DMF (1 mL) was dropped, followed by a solution of DIPEA (12 pL, 0.069 mmol) in anhydrous DMF (0.5 mL). The solution was stirred overnight at room temperature under N2 atmosphere. Cold diethyl ether (25 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After two hours, the tube was centrifuged (T:

5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow.

In a 50 mL conical-bottom glass centrifuge, crude Intermediate (7) was dissolved at room temperature in 95:5 TFA : water (5 mL), obtaining a brown solution. After one hour, cold diethyl ether (25 mL) was added to the brown mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark green precipitate. After two hours, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow. It was dissolved in water and the pH was adjusted to 8.5 with 0.5 N NaOH. The crude product was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 30 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 19 CV). Fractions containing the desired pure product (eluted with 10% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering 15.2 mg of Compound 3 as a green solid (15.2 mg, 0.008 mmol, Yield: 58%). [M+H]⁺ 1868.2.

Example 5: Synthesis of Compound 4

In a round-bottom flask, Intermediate (4) (30 mg, 0.0201 mmol) was dissolved in DMF (6 mL). HATU (15.7 mg, 0.0413 mmol), DIPEA (11 pL, 0.063 mmol) and l-(5-amino-3-aza-2- oxopentyl)-4,7, lO-tris(tertbutyloxycarbonylmethyl)- 1,4,7, 10-tetraazacyclododecane (16 mg, 0.026 mmol) were added and the green solution was stirred for 2 hours at room temperature in N2 atmosphere. Cold diethyl ether (15 mL) was added to the green mixture, the solid was filtered and recovered from the filter with MeOH. The solvent was concentrated under reduced pressure recovering crude Intermediate (8).

In a round bottom flask, crude Intermediate (8) was dissolved at room temperature in 95:5 TFA : water, obtaining a brown solution. After one hour cold diethyl ether (25mL) was added to the brown mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark green precipitate. After two hours, the solid was filtered and recovered from the filter with water. The pH was adjusted to 3 with 0.5 N NaOH and it was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 12 g) with a 0.1% HCOOH/acetonitrile gradient (stepped gradient from 95% HCOOH buffer to 100% acetonitrile over 20 CV). Fractions containing the desired pure product (eluted with 20% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering Compound 4 as a green solid (27.7 mg, 0.0162 mmol, Yield: 81%). [M+H]⁺ 1712.5.

Example 6: Synthesis of Compound 4a

Compound 4 (11.6 mg, 0.00678 mmol) was dissolved in H2O (6 mL). A solution of In(NOs)3 (850 pL of a 3.4 mg/mL solution) was added and the pH adjusted to 4.6 with 0.1 N NaOH. The green solution was stirred for 2 hours at room temperature, maintaining the pH at 4.6 with 0.1 N NaOH. Then it was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 12 g) with a water/acetonitrile gradient (stepped gradient from 0% to 100% acetonitrile over 20 CV). Fractions containing the desired pure product (eluted with 15% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering Compound 4a as a green solid (7 mg, 0.0038 mmol, Yield: 56%). [M + H]⁺ 1822.7.

Example 7: Preparation of Intermediate 11

Preparation of Intermediate 10:

In a dried round-bottom flask, Intermediate (2) (608 mg, 0.739 mmol) and Intermediate (9) (251 mg, 0.739 mmol) were suspended in acetic anhydride (30 mL) and acetic acid (15 mL). The mixture was heated at 55 °C, until the two powders were fully solubilized. Then, 2-chloro- 3-(hydroxymethylene)-l-cyclohexene-l-carboxaldehyde (153 mg, 0.887 mmol) was added to the mixture. Potassium acetate (145 mg, 1.478 mmol) was added and the mixture was heated at 100 °C for 1 hour. Solvents were concentrated under reduced pressure (up to 5 mL) and cold diethyl ether (50 mL) was added obtaining a green suspension. The solid was filtered, dissolved in water and purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 60 g) with an automated flash chromatographic system eluting with water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 65 CV). Fractions containing the desired pure product (eluted with 30% acetonitrile) were concentrated under reduced pressure and dried under vacuum at 40 °C, recovering pure Intermediate (10) as a green solid (219 mg, 0.169 mmol, Yield 23%). [M+H]⁺ 1298.3.

Preparation of Intermediate 11:

To a solution of Intermediate (10) (209 mg, 0.161 mmol) in dry DMSO (20 mL), 4- hydroxybenzene sodium sulfonate (316 mg, 1.612 mmol) and anhydrous potassium carbonate (223 mg, 1.612 mmol) were added. The mixture was stirred at 40 °C for 1 hour. A cold solution of 1 :2 ethyl acetate : diethyl ether (200 mL) was added to the green mixture, the solid was filtered, dissolved in water and the pH was adjusted to 7.8 with 0.5 N HCI. The crude product was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 30 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 45 CV). Fractions containing the desired pure product (eluted with 20% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering pure Intermediate (11) as a green solid (136, 0.094 mmol, Yield 58%). [M+H]⁺ 1436.7.

Example 8: Synthesis of Compound 5

In a 50 mL conical-bottom glass centrifuge, Intermediate (11) (20 mg, 0.0134 mmol) was dissolved at room temperature in anhydrous DMF (2 mL), obtaining a dark green dispersion. Then, NMM (3.4 pL, 0.0308 mmol) and TSTU (6.0 mg, 0.0201 mmol) were added obtaining complete dissolution. The dark green solution was stirred at RT and in the dark for one hour. Ice cold ethyl acetate (40 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After 2 hours, the tube was centrifuged (T: 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green precipitate was washed with ethyl acetate and dried under N2 flow. It was dissolved in dry DMF (1 mL) directly in the conical-bottom glass centrifuge tube. A solution of C8-AAZ (5.4 mg, 0.0167 mmol) in anhydrous DMF (1 mL) was dropped, followed by a solution of DIPEA (11.7 pL, 0.067 mmol) in anhydrous DMF (0.5 mL). The solution was stirred overnight at room temperature under N2 atmosphere for 2 days. Ice cold diethyl ether (25 mL) was added to the mixture at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark precipitate. After two hours, the tube was centrifuged (T : 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under In flow. The crude Intermediate (12) was dissolved at room temperature in 95:5 TFA : water (1 mL) directly in the conical-bottom glass centrifuge, obtaining a brown solution. After 30 minutes, cold diethyl ether (25 mL) was added at 0 °C (ice bath) under vigorous stirring, observing the formation of a dark green precipitate. After two hours, the tube was centrifuged (T : 5 °C, 4010 RPM, 10 minutes): the pale-yellow solvent was decanted and discarded, whereas the green solid was dried under N2 flow. The solid was dissolved in water and the pH was adjusted to 4 with 0.5 N NaOH. The crude product was purified by flash chromatography on a prepacked C18 silica column (Biotage® SNAP ULTRA 12 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 25 CV). Fractions containing the desired pure product (eluted with 20% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering pure Compound 5 as a green solid (8.9 mg, 0.0056 mmol, Yield: 42%). [M + H]⁺ 1572.3.

Example 9: Synthesis of Compound 6

In a dried round bottom flask Intermediate (13) (1 g, 4.18 mmol) was dissolved in sulfolane (10 mL); N-(4-Bromobutyl)phtalimide (3.53 g, 12.55 mmol) was added and the mixture was stirred at 110 °C for 24 hours. Cold ethyl acetate (50 mL) was added obtaining a suspension. The solid was filtered, dissolved in water and purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 120 g) with an automated flash chromatographic system eluting with water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 10 CV). Fractions containing the desired pure product (eluted with 50% acetonitrile) were concentrated under reduced pressure and dried under vacuum at 40 °C, recovering pure Intermediate (14) as a pale brown solid (960 mg, 2.18 mmol, Yield: 52%). [M + H]⁺ 441.5.

Preparation of Intermediate (15):

In a dried-round bottom flask, Intermediate (1) (233 mg, 0.659 mmol) and Intermediate (14) (290 mg, 0.659 mmol) were suspended in acetic anhydride (20 mL) and acetic acid (10 mL). The mixture was heated at 55 °C, until the two powders were fully solubilized. Then, 2-chloro- 3-(hydroxymethylene)-l-cyclohexene-l-carboxaldehyde (136 mg, 0.791 mmol) was added to the mixture. Potassium acetate (90 mg, 0.923 mmol) was added and the mixture was heated at 100 °C for 2 hours. Solvents were concentrated under reduced pressure (up to 5 mL) and cold diethyl ether (50 mL) was added obtaining a green suspension. The solid was filtered, dissolved in water and the pH was adjusted to 3 with 0.5 N HCI. The green mixture was let under stirring, in order to hydrolyze the by-product (anhydride formed on free carboxylic acid moiety). After 1 hour the pH was adjusted to 7 with 0.5 N NaOH.

The product was purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 60 g) with an automated flash chromatographic system eluting with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 35 CV). Fractions containing the desired pure product (eluted with 30% acetonitrile) were concentrated under reduced pressure and dried under vacuum at 40 °C, recovering pure Intermediate (15) as a green solid (238 mg, 0.256 mmol, Yield: 39%). [M+H]⁺ 930.8.

Preparation of Intermediate (16)

To a solution of Intermediate (15) (51 mg, 0.0547 mmol) in dry DMSO (5 mL), 4- hydroxybenzene sodium sulfonate (107 mg, 0.547 mmol) and anhydrous potassium carbonate (76 mg, 0.547 mmol) were added. The mixture was stirred at 40 °C for 16 hours. A cold solution of 1 :2 ethyl acetate : diethyl ether (200 mL) was added to the green mixture, the solid was filtered, dissolved in 0.1% HCOOH and the pH was adjusted to 2 with 0.5 N HCI. The crude product was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 30 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 20 CV). Fractions containing the desired pure product (eluted with 25% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering pure Intermediate (16) as a green solid (40 mg, 0.0374 mmol, Yield 68%). [M+H]⁺ 1068.5.

To a solution of Intermediate (16) (40 mg, 0.0375 mmol) in DMF (25 mL), a solution of EuK(tBu)s (22 mg, 0.045 mmol) in DMF (25 mL) was added. Then, DIPEA (19 pL, 0.1126 mmol) and TBTU (24 mg, 0.0751 mmol) were added and the green solution was stirred for 2 hours at room temperature in N2 atmosphere. Then, solvent was evaporated under reduced pressure and crude Intermediate (17) was dissolved in a 33% methylamine solution in absolute ethanol (11 mL). The red solution was stirred for 2 hours at room temperature in N2 atmosphere. Then glacial acetic acid (13 pL, 0.225 mmol) was added at 0 °C. The solvent was evaporated under reduced pressure, the residue was dissolved in water (5 mL) and purified on a pre-packed silica C18 column (BIOTAGE® SNAP ULTRA 30 g) with an automated flash chromatographic system eluting with a water-acetonitrile gradient (stepped gradient from 100% H2O to 100% acetonitrile in 20 CV). Fractions containing pure Intermediate (18) (eluted with 35% acetonitrile) were concentrated under reduced pressure to afford it as a green solid (14.8 mg, 0.0105 mmol, Yield: 28%). [M+H]⁺ 1408.1.

Synthesis of Compound 6

In a round-bottom flask, Intermediate (18) (28.4 mg, 0.0202 mmol) was dissolved in DMF (15 mL). TBTU (13 mg, 0.0404 mmol), DIPEA (11 pL, 0.0606 mmol) and 2-[l,4,7,10- Tetraazacyclododecane-4,7,10-tris(t-butyl acetate)]-pentanedioic acid-l-t-butyl ester DOTAGA-tetra (t-Bu ester) (17 mg, 0.024 mmol) were added and the green solution was stirred for 1 hour at room temperature in N2 atmosphere. Solvent was concentrated under reduced pressure recovering crude Intermediate (19).

In a round bottom flask, crude Intermediate (19) was dissolved at room temperature in 95:5 TFA : water (10 mL), obtaining a brown solution. After six hours solvents were concentrated under reduced pressure and the crude was dissolved in water and purified by flash chromatography on pre-packed C18 silica column (Biotage® SNAP ULTRA 30 g) with a water/acetonitrile gradient (stepped gradient from 100% water to 100% acetonitrile over 30 CV). Fractions containing the desired pure product (eluted with 20% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering Compound 6 as a green solid (24 mg, 0.014 mmol, Yield: 70%). [M+H]⁺ 1698.1.

Example 10: Synthesis of Compound 6a

Compound 6 (13 mg, 0.00766 mmol) was dissolved in H2O (4 mL). A solution of In(NOs)3 (850 pL of a 3.4 mg/mL solution) was added and the pH was adjusted to 4.6 with 0.1 N NaOH. The green solution was stirred for 2 hours at room temperature, maintaining the pH at 4.6 with 0.1 N NaOH. Then, it was purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP ULTRA 12 g) with a water/acetonitrile gradient (stepped gradient from 0% to 100% acetonitrile over 20 CV). Fractions containing the desired pure product (eluted with 20% acetonitrile) were combined, concentrated under reduced pressure and freeze-dried recovering Compound 6a as a green solid (7.6 mg, 0.0042 mmol, Yield: 55%). [M + H]⁺ 1810.7.

Example 11: Binding affinity to PSMA-expressing LNCaP cells

LNCaP prostate cells were selected as in vitro model to assess the specific binding of compounds of the invention to PSMA, after internal validation of their PSMA expression in its physiological environment at the cell-surface. Indeed, by means of cytometry, LNCaP cells were demonstrated to constitutively express high levels of PSMA, estimated in about 130,000 PSMA molecules per cell by mean of the DAKO QUIFIKIT (Agilent).

All probes were incubated in increasing concentrations (range 0-4 pM, except for compound 5 incubated in the range 0-2 pM) with LNCaP cells on ice. Cell-associated fluorescence was collected by flow cytometry analysis and the binding constant at equilibrium was calculated by mathematical fitting.

In more detail, LNCaP cells were detached with StemPro® Accutase® Cell Dissociation Reagent (Life Technologies), collected in DPBS and counted. At least 2-10⁵ cells were placed in 1.5 mL tubes on ice and resuspended in 100 pL of cold FACS buffer (eBioscience™ Flow Cytometry Staining Buffer, Invitrogen) containing the Test Articles (representative compounds of the invention), two-fold serially diluted in the range stated above. The unstained sample, incubated with FACS buffer, was used to record the basal autofluorescence of cells. After incubation (2 hours on ice in the dark), cells were rinsed 3 times in cold FACS buffer by centrifugation (5 min, 4 °C, 350 RCF), discarding the supernatant. Cell pellets were then resuspended in 100 pL of cold FACS buffer (or in a suitable volume in order to obtain no more than 1000 recorded events per pL during FACS analysis) and analysed with the flow cytometer Accuri™ C6 (BD Biosciences).

The mean cell-associated fluorescence was plotted against concentration and the dissociation constant at equilibrium (KD) was inferred by mathematical fitting with GraphPad Prism v.9 software (One site, Total). In detail, the equation used was the following: where

Y is the mean cell-associated fluorescence;

X is the concentration of the compound;

Bmax is the maximum specific binding in the same units as Y;

KD is the equilibrium dissociation constant, in the same units as X;

NS is the slope of nonspecific binding in Y units divided by X units; background is the amount of nonspecific binding with no added compound.

All compounds were analyzed in replicate independent experiments. The calculated KD are summarized in Table III for some representative compounds of the invention together with the conjugate EuK-IRDye800 CW used as reference, while corresponding binding curves are shown in Figure 1.

Table III - Binding constant of equilibrium (KD) to LNCaP cells of representative compounds of formula (I).

This assay confirmed that the bi-functional compounds of the invention maintain a remarkable affinity to PSMA (in particular to PSMA-positive LNCaP cells), in the same nanomolar range of the EuK-IRDye800CW reference compound and with a KD lower than about 100 nM. Surprisingly, the second functionalization other than the PSMA targeting moiety did not impair the ability of the compounds of formula (I) to target PSMA with high affinity. The affinity and targeting specificity to PSMA was confirmed also for the representative bimodal Compound 4a, which is functionalized with a chelating moiety labeling the [ In] radiotracer, demonstrating that it can be a valid candidate as contrast agent for applications in pre-operative nuclear imaging, intraoperative radioguidance and fluorescence guided surgery of PSMA lesions with a single administration only.

Example 12: Binding affinity to CA-IX

Affinity to CA-IX-expressinq HT-29 cells

HT-29 cells were selected as in vitro model to assess the binding to CA-IX, physiologically expressed at the cell surface, of PSMA/CA-IX heterobivalent compounds. By mean of flow cytometry (DAKO QIFIKIT, primary anti-CA-IX antibody MAB2188 R&D Systems), HT-29 cells were estimated to express about 150000 CA-IX molecules per cell. All compounds were incubated at increasing concentrations (Compound 1: range 0 - 10 pM, Compound 2: range 0 - 20 pM; Compound 5: range 0 - 5 pM) with HT-29 cells on ice, and the binding constant at equilibrium was calculated by mathematical fitting after flow cytometry analysis, as described above. The estimated KD is reported in Table IV, together with the conjugate C8- AAZ-IRDye800CW used as Reference Compound (Ref.2), uniquely targeting CA-IX. Representative binding curves are shown in Figure 2.

Table IV - Binding constant of equilibrium (KD) to HT-29 cells of representative compounds of formula (I).

As shown above, the PSMA/CA-IX bi-functional compounds of the invention maintain a remarkable affinity to CA-IX, in the same nanomolar range of the corresponding reference compound bearing the CA-IX binding motif only: in fact, the estimated KD of the above compounds still indicate that the ability to specifically bind the CA-IX target is retained and not impaired by the presence of the PSMA-binding motif.

Therefore, the affinity to the metabolic (/.e. hypoxia) CA-IX biomarker can provide further advantages to the probes of the invention with respect to fluorescent probes targeting PSMA only, in particular with respect to imaging of PSMA prostate carcinomas which benefit of such additional targeting for tumor homing.

Affinity to isolated CA enzyme

The affinity to the catalytic site of isolated carbonic anhydrase enzymes was performed to further confirm the binding affinity to CA-IX for the heterobivalent probes of the invention bearing a CA-IX targeting moiety. Considering that the CA-IX targeting moieties present in the compounds of the invention are expected to generally target all the isoforms of the CA family, the test was conducted using the recombinant bovine CA-II enzyme (bCAII, recombinant, expressed in E. Coli, Sigma-Aldrich), which has a catalytic site with high degree of homology to that of the CA-IX isoform (J Enzyme Inhib Med Chem 2013; 28(2) : 267-277). The affinity was calculated through a fluorescence-based method based on competition with 5-(Dimethylamino)-l-naphthalenesulfonamide (dansylamide, DNSA, Sigma-Aldrich 218898, reconstituted in 20 mM Tris pH 8). Briefly, in a 96-well plate format, scalar concentrations of probes (Compound 1 : range 0 - 9.5 pM; Compound 2: range 0 - 22.5 pM; Compound 5: range 0 - 20 pM) were incubated with 0.25 pM of bovine CA-II and 5 pM of DNSA in 20 mM Tris, pH 8. DNSA fluorescence emission at 462 nm from each well was recorded after excitation at 285 nm (Trp wavelength of excitation) and consequent selective excitation of DNSA bound to bCAII by FRET. Fractional saturation of bCAII was experimentally determined through normalization of fluorescence data between 1 (no competing compounds) and 0 (minimal fluorescence signal), according to the equation below: r^Llobs ^{r L}min r = - 1 F ‘I-‘max — ¹ F ‘I-‘min where r is fractional saturation, FLobs is the recorded fluorescence intensity, FLmin is the fluorescence intensity recorded in samples with DNSA only, and FLmax is fluorescence recorded in absence of competitor.

The KD of CA-binding compounds (KD comp) was then derived by mathematical fitting of experimental data according to the equation: where r is fractional saturation (calculated from experimental data as described above), [DNSA] is DNSA nanomolar concentration, KD_DNSA is the dissociation constant at equilibrium of DNSA (previously experimentally estimated = 169.6 ± 7.4 nM), and [comp] is the nanomolar concentration of the CA-IX targeting compound under analysis. Mathematical fitting was performed using Microsoft Excel Solver.

The obtained KD to bovine CA-II is summarized in Table V together with the corresponding conjugates of IRDye800CW with the respective CA-IX targeting moiety, used as Reference Compounds (/.e. Ref. 2 for Compound 1 and 5 and Ref. 3 for Compound 2).

Table V - Binding constant of equilibrium (KD) to bCA-II enzyme of representative compounds of formula (I). KD values are expressed in nM (mean of KD resulting in replicate independent experiments, with SD). All the representative heterobivalent probes shown above displayed a KD to bCA-II very close (same nanomolar range) to that of the monovalent Reference compounds bearing the corresponding CA-IX binding motif only, indicating that the double functionalization did not affect the ability to interact with the Carbonic Anhydrase binding site. Surprisingly, the affinity to bCA-II of Compound 5 was improved compared to both Compound 1 and Reference 2, even if bearing the same CA targeting moiety.

Example 13: Binding affinity to RGD-binding integrins

Affinity to RGD-binding integrins of the PSMA/integrin bivalent probes of the invention was evaluated by estimation of the KD of representative Compound 3 for the purified dimer av06, a well-recognized tumor biomarker mostly expressed by transformed epithelial cells (Liu et al., Am J Nucl Med Mol Imaging 2014; 4(4): 333-345) by mean of a fluorescence-based binding assay in plate.

Briefly, Black MaxiSorp™ 96-well plates were coated overnight at 4 °C with of integrin av06, 2 pg/mL in Coating Buffer (20 mM Tris HCI pH 7.4; 150 mM NaCI; 1 mM MnCh; 0.5 mM MgCh; 2 mM CaCh), 0.1 mL per well. After coating, plates were washed and blocked (coating buffer/BSA 3%, 0.3 mL/well, 2 hours RT). Wells were then washed three times with 0.3 mL coating buffer and filled with 0.1 mL of two-fold serially diluted Compound 3 in sample buffer (/.e., coating buffer/BSA 1%) in a concentration range from 1.25 to 6.10- 10^-4 pM, two replicates per each condition. After incubation 1 hour at RT, wells were washed three times in coating buffer (0.3 mL/well) and finally filled with 0.1 mL/well of coating buffer. As controls, Compound 3 was also incubated in uncoated (blocked) wells, to estimate non-specific binding at each concentration; moreover, both coated and uncoated wells incubated in sample buffer alone, to calculate blank. Fluorescence was read at the Spark® multimode microplate reader (Tecan) with following parameters: 775 ± 15 nm and 815 ± 20 nm for excitation and emission, respectively. The assay was performed two times.

The results of the estimated KD for av06 of representative Compound 3 are shown in the Table VI below and compared to the corresponding conjugate of IRDye800CW with c(RGDfK), Reference 4.

Table VI - Binding constant of equilibrium (KD) to OvPe integrin of a representative compound compared to the corresponding integrin-monovalent reference compound. KD values are expressed in nM (mean of KD resulting in replicate independent experiments, with SD). The assay confirmed that the integrin/PSMA-heterobifunctional Compound 3 of the invention also maintained a remarkable affinity to target with high affinity an RGD-binding integrin, such as av06, and that this ability is surprisingly not impaired by the presence of the PSMA-binding motif. Thus, the affinity to this stromal biomarker (RGD-binding integrins) represents a further advantage to the probes of the invention with respect to fluorescent probes targeting PSMA only, in particular with respect to imaging of PSMA prostate carcinomas which benefit of such additional targeting for tumor homing.

Example 14: Cellular uptake to PSMA-expressing cells

Cellular uptake experiments were performed using flow cytometry analysis to assess if cellular internalization of bi-functional (either heterobivalent or bimodal) PSMA-targeting probes occurs in LNCaP PSMA-expressing cells.

Time-dependent cellular uptake of compounds 1, 2, 3, 4a, and 5 together with their corresponding reference compound (Ref. 1) and the unconjugated dye IRDye800CW was evaluated. Cell incubation at 37 °C (endocytosis permissive temperature) vs on ice (endocytosis blocked) was performed, to differentiate cell-associated fluorescence deriving from internalized compounds from the one due to cell surface-associated ones.

The assay was performed according to the general procedure below. The same assay was used to assess CA-IX or integrin mediate cellular uptake using HT-29 cells and reference compounds 2 and 3, respectively. Cells, grown in wells of 24-well plates, were treated with multifunctional probes using the working concentration of 1 pM, in serum-free medium containing 25 mM HEPES pH 7.4. In addition, cell incubation with both the corresponding single-targeting reference compounds and the unconjugated dye was performed. Some wells were finally left untreated to assess the basal autofluorescence of samples. Every treatment was performed in independent replicates. After treatment (1, 2, 4 hours, at 37 °C or on ice), cells were rinsed 3 times with cold DPBS and detached from the plate with StemPro® Accutase® Cell Dissociation Reagent, at 37 °C. After few minutes, the plates were placed on ice and cold DPBS was added to each well to dilute and inactivate the dissociation reagent. The cell suspension of each well was collected and transferred to 1.5 mL tubes, on ice. Cells were then centrifuged (5 minutes, 350 RCF, 4 °C), the supernatant was discarded, and the cell pellet was resuspended in cold FACS buffer, in a suitable volume in order to obtain no more than about 1000 recorded events per pL during FACS analysis.

Samples were analyzed with the flow cytometer Accuri™ C6 (BD Biosciences) using the 640 nm excitation laser and the emission 780/60 nm filter. At least 12000 valid events were collected for each sample and the resulting mean fluorescence was considered the fluorescence intensity value of the sample. For each sample, the normalized cell-associated fluorescence was calculated according to the following equation:

Normalized FL ^^treated -untreated) I '^untreated

Compounds 2 and 3 and Ref. 1 displayed the same time-dependent cellular uptake at the endocytosis-permissive temperature of 37 °C, which increased over time (1 - 2 - 4 hours cell treatment), with a minor cell-associated fluorescence recorded after cell incubation on ice, which resulted roughly constant at all time points. This result indicates that the predominant quote of cell-associated fluorescence can be ascribed to internalized cell surface-associated PSMA-targeting probes.

Cell-associated fluorescence recorded after cell incubation with Compound 1 was higher at all time points compared to the one recorded with Compound 2, 3 and Ref. 1, even though all compounds display similar KD to LNCaP cells. The cell-associated fluorescence after cell treatment with the unconjugated dye IRDye800CW at 37 °C was largely lower compared to cell treatment with all the tested heterobivalent probes, representing the basal targetindependent cellular uptake, with undetectable levels of unconjugated dye remaining at the cell surface. Results are represented in Figure 3, panel A.

The cellular uptake of Compound 5 was tested in a separated experiment, together with Ref.l and the unconjugated dye IRDye800CW as well. Similarly to the other tested compounds, time-dependent internalization of Compound 5 was observed, with cell-associated fluorescence values higher than in the case of cell treatment with Ref.l, both at 37 °C and on ice. Moreover, the comparatively very low cell-associated fluorescence recorded after cell treatment with the unconjugated IRDye800CW indicated the negligible quote of non-specific cellular uptake. Results are represented in Figure 3, panel B.

Finally, the LNCaP uptake profile of the bimodal Compound 4a resulted superimposable to the one obtained, in the same test, with Ref. 1, indicating that the chemical modification for indium coordination did not alter the PSMA-targeting properties of the compound. The result is shown in Figure 3, panel C.

Example 15: Cellular uptake to CA-IX expressing cells

The same logic stated in the Example above was applied to PSMA/CA-IX heterobivalent probes, to assess cellular internalization after incubation with CA-IX-expressing HT-29 cells. The cellular uptake of Compounds 1, 2 and 5 was similar to that of the corresponding CA-IX- targeting monovalent References, in terms both of time-dependent cellular uptake at 37 °C and of cell-surface associated residual fluorescence recorded after cell incubation on ice. The non-specific uptake, represented by the cell-associated fluorescence recorded after cell treatment with the unconjugated dye IRDye800CW was largely minor in every test. Results are shown in Figure 4.

Example 16: Integrin mediated cellular uptake of PSMA/integrin bivalent probes (Compound 3)

The integrin-mediated cellular uptake was assessed using HT-29 cells, which do not express PSMA and predominantly express the RGD-binding dimer avfJs, together with lower levels of av06 and a negligible number of ovfh (estimated in about 30000, 10000, and 200 avfJs, av06, and avfh receptors per cell, respectively, through flow cytometric analysis with the DAKO QIFIKIT). The time-dependent cellular uptake observed after cell treatment at 37 °C was similar for the representative PSMA/integrin heterobivalent Compound 3 and the integrin monovalent Ref. 4, with undetectable levels of cell surface binding for both compounds. As in the Examples above, the non-specific cellular uptake, represented by cell-associated fluorescence after incubation with the unconjugated dye IRDye800CW, was a minor quote. The result is shown in Figure 5.

Example 17: Binding Affinity (K_a) to Human Serum Albumin (HSA)

The binding affinity of the probes of the invention for human serum albumin (HSA) was determined to assess the influence of structural features on their albumin binding properties and get insights into possible differences in the expected bioavailability of the probes.

It was performed by UV-VIS spectrophotometry after either ultrafiltration (for Compounds 1, 2, 3 and 4a) or by peak shift (Compound 5). Briefly, for the centrifugation-based assay, HSA (A9511, Sigma-Aldrich) was prepared in a 0.5 M stock solution in PBS, used to obtain a series of dilutions in PBS (0, 9.52-10 ⁷, 4.76- 10 ⁶, 9.52-10 ⁶, 1.90-10 ⁵, 3.81-10 ⁵, 5.71-10 ⁵, 9.52-10- ⁵, 1.43-10^-4, 1.90-10^-4, 2.86-10^-4, 3.81-10^-4 M), in presence of 4.76 pM of each bi-functional probe (prepared from a 50 pM stock solution in PBS), in a total volume of 0.525 mL. The samples were centrifuged (10000 g for 30 min at 25 °C) in a Microcon device (10 KDa MW cut off, Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane) and the absorbance measurements of the filtrates were obtained with the Specord200 Plus (Analytik Jena) spectrophotometer at the maximum absorbance wavelength of the fluorophore. When the peak shift protocol was applied, HSA was diluted in PBS from the 0.5 M stock solution, in a final volume of 1 mL, in scalar concentrations (0, 1- 10 ⁶, 5-10^-6, 1-10 ⁵, 2-10^-5, 4-10^-5, 6-10’ ⁵, 8- 10^-5, 1-10⁴, 2-10^-4, 3-10^-4 M) and in presence of 2.5 pM B28710 (prepared from the 50 pM stock in PBS). Absorbance of solutions was measured at the maximum absorbance of the shifted peak (791 nm). Data analysis was performed fitting the raw data with the following formula, using Microsoft Excel Solver: where:

AA/b = Absorbance measured (b = 1 cm) KRL = KA calculated by fitting

AE ■ Rt was calculated by fitting

[L] = Albumin concentration

AA/b was obtained subtracting the absorbance of the control "0 HSA sample" to the absorbance of each other samples (AAX = Asampiei ^— Asamplex) .

The results of the estimated association constants at equilibrium (KA) of representative probes of the invention are summarized in Table VII.

Table VII - Estimated association constant of equilibrium (KA) of representative Compounds 1 -5

Overall, all probes displayed a medium-to-moderate affinity to human albumin, comparable to the affinity of the corresponding cyanine IRDye800CW.

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Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein each R¹ is independently a straight or branched Ci-io alkyl;

Li is a straight or branched C1-10 alkyl-CO-;

Ti is a PSMA-targeting moiety of formula (II) wherein X is a radical of an amino acid, such as lysine, glutamic acid, optionally further substituted with a group selected from 3-(2-furyl)-alanine, 2-(2'-propynyl)-alanine and 3-(2-naphtyl)-alanine-tranexamic acid; provided that at least one of R³ or R⁴ comprises the group -L2-T2.

2. The compound of formula (I) according to claim 1 wherein Ti is a PSMA-targeting moiety of formula (Ila):

3. The compound of formula (I) according to claim 1 wherein T2 is a moiety targeting carbonic anhydrase IX (CA-IX).

4. The compound of formula (I) according to claim 3 wherein T2 is a moiety C8-AAZ

5. The compound of formula (I) according to claim 1 wherein T2 is a moiety targeting an integrin receptor and comprising an Arg-Gly-Asp (RGD) binding motif.

6. The compound of formula (I) according to claim 5 wherein T2 comprises a c(RGDfK) binding motif.

7. The compound of formula (I) according to claim 1 wherein T2 is a chelating moiety selected from l,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), 2- [l,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid]-pentanedioic acid (DOTAGA), l,4,7,10-tetraazacyclododecane-l,4,7-triacetic acid (DO3A), 1,4,7- triazacyclononanetriacetic acid (NOTA), 1,4,7-triazacyclononane-l-glutaric acid-4, 7- acetic acid (NODAGA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7- triazacyclononane phosphinic acid (TRAP), l,4-bis(carboxymethyl)-6- [bis(carboxymethyl)]amino-6-methylperhydro-l,4-diazepine (AAZTA), S- acetylmercaptoacetyltriglycine (MAG3) and S-acetylmercaptoacetyltriserine (MAS3).

8. A complex comprising a compound of formula (I) according to claim 1, wherein T2 is a chelating moiety, and a radioactive or non-radioactive cation, wherein the cation is selected from a Ga, Cu, Lu, Y, Ac, In, Pb, Bi and Tc cation or a cation that binds to ¹⁸F, preferably Al.

9. A complex according to claim 8 wherein the radioactive or non-radioactive cation is selected from ⁶⁸Ga, ⁶⁷Ga, ⁶⁴Cu, ⁶⁷Cu, ¹⁷⁷Lu, ⁹⁰Y, ²²⁵Ac, In, ^99mTc, ²¹²Pb, ²⁰³Pb, ²¹²Bi, or is represented by a group Al¹⁸Fs or Sc¹⁸Fs.

10. A compound as defined in claim 1, for use as fluorescent probe for biomedical optical imaging applications in mammals.

11. A compound according to claim 10 wherein the imaging is selected from a tomographic imaging of organs, intraoperative cancer identification, fluorescence-guided surgery, fluorescence life-time imaging, short-wave infrared imaging, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, open field surgery, laser guided surgery and a photoacoustic or sonofluorescence method.

12. A complex as defined in claims 8 or 9 for use as agents for imaging applications or treatment of PSMA expressing cancer cells.

13. A complex according to claim 12 wherein the imaging application is selected from position emission tomography (PET), single photon emission computed tomography (SPECT), scintigraphy and gamma- or beta-diagnostic imaging.

14. A pharmaceutical composition comprising a compound of formula (I) as defined in claim 1 or a complex as defined in claim 8 and at least one pharmaceutically acceptable adjuvants, excipients, carriers or diluents.

15. Diagnostic kit comprising at least one compound as defined in any of the preceding claims 1 to 9 together with additional adjuvants thereof for implementing the imaging applications as defined in claims 10 to 13.