PSMA-targeting fluorescent probes

Abstract

The present invention relates to the field of optical imaging. More particularly, it relates to fluorescent probes able to efficiently target Prostate-Specific Membrane Antigen (PSMA) and comprising heptamethine cyanine dyes with near-infrared (NIR) emission. The invention also relates to methods for preparing these compounds, to pharmaceutical compositions and kits incorporating them and to methods of use them as optical diagnostic agents in imaging or therapy of diseases, particularly prostate cancer.

Description

[0001] PSMA-TARGETING FLUORESCENT PROBES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of optical imaging. More particularly, it relates to fluorescent probes able to efficiently target Prostate-Specific Membrane Antigen (PSMA) and comprising heptamethine cyanine dyes with near-infrared (NIR) emission. The invention also relates to methods for preparing these compounds, to pharmaceutical compositions and kits incorporating them and to methods of use them as optical diagnostic agents in imaging or therapy of diseases, particularly prostate cancer.

[0004] BACKGROUND ART

[0005] Prostate-Specific Membrane Antigen (PSMA)

[0006] PSMA is a 750-residue type II transmembrane glycoprotein (about 84 KDa), with a short N-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 N-acetylated, a-linked acidic dipeptidase (NAALADase) and folate hydrolase (FOLH) activities, hydrolyzing respectively the y-peptide bonds between N-acetylaspartate and glutamate in the abundant neuropeptide N-acetylaspartylglutamate (NAAG) and the y- glutamyl linkages in pteroylpolyglutamate. 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.

[0007] PSMA is expressed in nearly all prostate cancers with increased expression in poorly differentiated, metastatic, and hormone-refractory carcinomas, with expression levels about 1000-fold higher compared to the physiologic levels found in other tissues such as kidney, small intestine, or brain. It is abundantly expressed at all stages of prostate cancer, presented on the cell surface and not shed into the circulation.

[0008] PSMA is also expressed by other cancers, due to an overexpression on cancer-related neovascular structures, such as the bladder, pancreas, lung, and renal cell cancers. Physiological expression of PSMA has been demonstrated, at lower levels, in other tissues including healthy prostate, duodenum, kidney, salivary and lacrimal glands, neuroendocrine system, proximal renal tubules, liver and brain. The above-mentioned features and pattern of expression make it valuable as target for molecular imaging applied to diagnosis, staging, follow up, surgery and therapy (Kaewput et al., J. Clin. Med. 2022; ll:2738). Therefore, PSMA targeting using specific tools could open new important fields to improve the conventional therapies and the early diagnosis and prognosis of malignant tumors.

[0009] The classes of PSMA-targeting agents developed so far, for both imaging (e.g. PET imaging) and / or therapeutic applications in patients with prostate cancer, include monoclonal antibodies and small chemical compounds. Some agents belonging to these classes of compounds are already commercialized or currently under clinical development. In particular,111In-capromab-pendetide (ProstaScint®; AYTU Bioscience Inc.) was the first commercialized anti-PSMA antibody approved by the US FDA in 1996 to target PSMA.

[0010] However, although monoclonal antibodies are ligands of choice for most tumor targeting applications, their use entails many disadvantages: in fact, they are characterized by slow and inefficient tumor penetration and long delay between injection and imaging, due to their long half-lives, which leads to high accumulation in inflammatory tissue and substantial radiation exposure. Additionally, they may be immunogenic, precluding repeated administration for routine diagnostic procedures.

[0011] These problems could be circumvented with the use of small molecules, which show similarly accurate localization of prostate cancer lesions but faster tumor uptake and more rapid excretion, thus reducing radiation exposure and allowing physicians to obtain diagnostic information much more quickly (Jones W. et a / ., Cancers 2020; 12(6): 1367).

[0012] Among small chemical compounds, the PSMA pharmacological inhibitors characterized so far comprise: phosphonate-based, such as for instance (phosphonomethyl)pentanedioic acid (2-PMPA); urea-based, 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; and hydroxamate derivatives. In the last few decades, several urea- based derivatives (e.g. EuK, EuE and other "EuX" groups) have been developed as highly potent inhibitors for PSMA (EP3636635 Al).

[0013] In many cases, the EuK and EuE 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-naphthyl)-alanine-tranexamic acid.

[0014] The first inhibitors were primarily used as68Ga-labeled radiotracers, the development of which was considered a breakthrough due to their excellent characteristics, such as 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 ProstamedixTM) 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; 11 :2738).

[0015] 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. However, prostate surgery fully relies on white light endoscopy, not allowing a sensitive and specific visual identification of cancer extent and presence, locally and regionally. In fact, this limited intraoperative ability to differentiate cancerous from noncancerous tissue explains the high incidence (30-40%) of recurrence in patients with locally advanced cancers. Accurate real-time identification of prostate cancer during surgery, such as that obtained using Near-InfraRed (NIR) fluorescence, might help to enhance the detection of tumor tissue during surgery and achieve complete oncological resections and prevent damage to vital structures.

[0016] Fluorescent labeling

[0017] 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.

[0018] The current clinically available fluorescent probes, such as Indocyanine green (ICG), have restricted utility for imaging the tumor tissue, since they distribute in tissues by a combination of passive diffusion and enhanced permeability and retention (EPR) effect. In general, this first generation of fluorescent contrast agents requires a large mass dose (> 1 mg / kg) for proper tumor visualization. Although ICG could be applicable for certain indications, visualization is hampered due to non-specific binding and lack of tumor specificity (Galerna HA et al., Eur J Surg Oncol. 2022;48(4):810-821). To date, multiple fluorescent conjugates, constituted by a targeting vector (antibodies, peptides, particles, and other small molecules) coupled to a fluorescent NIR 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). Their in vivo behavior may be strongly affected by the biological properties of the fluorescent moiety. For example, small structural modifications of cyanine Cy5 strongly modulate the accumulation in tumor and off-target tissues of the relative bioconjugates (Bunschoten A. et al., Bioconjugate Chem. 2016;27: 1253-1258). Fluorescent contrast agents with low non-specific accumulation and high selectivity for the target tissue would be preferable for applications in living organisms.

[0019] One example of cyanine dyes conjugated with low molecular weight moieties targeting PSMA is described in WO2017 / 044584 (On Target Laboratories) which discloses among others the compound OTL-78 currently under clinical development, comprising the NIR dye S0456 conjugated in meso position to the PSMA targeting moiety EuE, through linkers formed by 8- aminooctanoic acid and Phe-Tyr dipeptide.

[0020] However, the conjugation at the central position may suffer from higher lability of the central substituent, that could be removed in vivo by reaction with endogenous nucleophiles ( / .e., it is susceptible of in vivo nucleophilic substitution), causing the loss of the targeting unit and leading to a potential in vivo degradation and de-activation of the probe. This drawback has been partially resolved by replacing the central C-0 bond with a C-C bond, much more stable in vivo, but in this case the water solubility of the dye tends to dramatically decrease due to a higher tendency of aggregation.

[0021] Other examples of fluorescent probes, characterized by conjugation at position 1 or 3 of the indole, are disclosed in patent applications WO2017 / 184383 (Intuitive Surgical Operations) and W02009 / 026177 (Purdue Research Foundation) disclosing different PSMA binding conjugates, in both cases comprising PSMA binding ligands linked to known NIR sulfocyanine dyes.

[0022] Another broad class of PSMA targeting compounds, also including few examples of conjugates with fluorescent dyes, is disclosed in W02010 / 108125 (J. Hopkins).

[0023] Unfortunately, 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 efficient fluorescent probes targeting PSMA with high affinity and specificity in order to be used in diagnosis and / or therapy.

[0024] With respect to the above-mentioned probes, the compounds of the present invention are characterized by Cy7 cyanines with a different functionalization pattern and / or conjugation to a PSMA-targeting moiety at an alternative position of the cyanine scaffold. Such probes have surprisingly shown a high affinity for the target PSMA and optimal cell internalization and tumor imaging efficacy results, besides a higher stability during their preparation.

[0025] SUMMARY OF THE INVENTION

[0026] Generally, object of the present invention is to provide PSMA targeting fluorescent probes useful as contrast agents for optical imaging and aimed at solving the above- mentioned issues. Particularly, the present invention provides fluorescent probes with optimal properties for different molecular imaging applications, being able to interact with PSMA expressing cells, to accumulate in pathological cells and tissues and to specifically display a fluorescent signal in correspondence to pathological tissues with high signal-to-noise ratio and improved imaging efficacy at low mass doses.

[0027] The preferred sites of conjugation to the cyanine dyes disclosed in the art are represented by the positions 1 or 3 of the indolenine groups for Cy7 cyanines, both linear or with cyclohexenyl central ring, for instance by conjugation at an alkyl chain functionalized with a carboxylic acid or ester. In some cases, Cy7 cyanines with cyclohexenyl central ring can be also conjugated at the central (meso) position of the heptamethine scaffold, typically by attachment to a phenyl or phenoxy ring that can be functionalized in para with a carboxy, carboxamido or ester group, optionally by interposition of a linker. These cyanine compounds generally have a symmetrical structure, bearing -SO3H, alkyl or alkyl-SO3H groups in the other positions.

[0028] Conversely, the fluorescent probes of the invention comprise asymmetric dyes and are characterized by a heptamethine cyanine dye conjugated to a PSMA-targeting moiety at a different position of the cyanine scaffold, namely at position 5 of at least one of the indolenine rings, affording strong binding results to the target and unexpectedly displaying an optimal in vitro and in vivo behaviour, thus being particularly suitable for molecular imaging.

[0029] 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 target tissue and cellular uptake, low accumulation due to non-specific interaction with other tissues, high solubility in water, low binding to albumin.

[0030] 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 of a human or animal organ or tissue, wherein the imaging is a tomographic imaging of organs, monitoring of organ functions including angiography, urinary tract imaging, bile duct imaging, nerve imaging, 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, or a photoacoustic or sonofluorescence method.

[0031] 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.

[0032] 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. In another aspect, the present invention refers to a method for the optical imaging of a body organ, tissue or region by use of an optical imaging technique that comprises the use of an effective dose of a compound of the invention.

[0033] DESCRIPTION OF THE INVENTION

[0034] Accordingly, a first aspect of the invention relates to compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein each R1is independently a straight or branched C1-C10 alkyl substituted by a group - SO3H;

[0035] R2is selected from -SO3H, a group -CONH-Y wherein Y is a straight or branched C1-C10 alkyl substituted with at least two hydroxyl groups, and a group of formula (II)

[0036] R3is selected from hydrogen and a group phenyl or -O-phenyl, optionally substituted with -SO3H;

[0037] L is a bond or a linker;

[0038] T is a PSMA-targeting moiety of formula (III): wherein X is an amino acid or a derivative thereof; and n is an integer equal to 0 or 1.

[0039] Preferably, X is an amino acid group selected from lysine, glutamic acid and the like or a derivative thereof selected from 3-(2-furyl)-alanine, 2-(2'-propynyl)-alanine and the like.

[0040] More preferably, X is lysine or glutamic acid. Even more preferably it is lysine.

[0041] Preferably, L is a group of formula -NH-(CH2)P-CO- or a diradical of one or more moieties selected from the group consisting of an amino acid or a derivative thereof; a peptide comprising from 2 to 10 amino acids in L or D configuration; 4-aminomethylbenzoic acid; cysteic acid; a polyethylene glycol or a derivative thereof; amino-polyethylene glycolcarboxylic acid; trans-4-(aminomethyl)-cyclohexanecarboxylic acid (TXA, tranexamic acid); diaminobutyric acid; diaminopropionic acid, and combinations thereof; or it is a group -L1-L2- wherein -Li- is a diradical of a diamine and -L2- is a diradical of a dicarboxylic acid; wherein p is an integer comprised between 1 and 20.

[0042] More preferably, the linker L 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 or an amino acid derivative, such as for instance 3-(2-naphthyl)-alanine; 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- or - NH- (O-CH2-CH2)P-CO- or derivatives thereof; amino-polyethylene glycol-carboxylic acid; trans-4-(aminomethyl)-cyclohexanecarboxylic acid (TXA, tranexamic acid); diaminobutyric acid; diaminopropionic acid, and combinations thereof; or it is a group -L1-L2- wherein Li is a diradical of a diamine, for instance selected from an amino-polyethylene glycol amine of formula -NH-(O-CH2-CH2)P-NH-, ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexanediamine and the like, and L2 is a diradical of a dicarboxylic acid, for instance selected from succinic acid, glutaric acid, suberic acid, adipic acid and the like; wherein p is an integer comprised between 1 and 20.

[0043] More preferably, L is selected from a group of formula -NH-(CH2)P-CO-; a polyethylene glycol of formula -NH-(O-CH2-CH2)P-CO-; and a diradical comprising from one to five amino acids, wherein p is an integer comprised between 1 and 20.

[0044] In another preferred embodiment L is a combination of tranexamic acid and an amino acid derivative, such as for instance the group of formula (L3): corresponding to the tranexamic acid-(3-(2-naphtyl)-alanine) linker described for instance in Benesova et al., J Nucl Med 2015; 56: 914-920.The present invention also relates to methods for preparing the compounds of formula (I) by means of synthetic transformation steps.

[0045] The invention further comprises compounds of formula (I) for use as fluorescent agents for the detection of tumor margins in fluorescence-guided surgery. Description of the Figures

[0046] Figure 1 shows the binding curves of the representative compounds 1-4 of the invention to PSMA-positive LNCaP cells and PSMA-negative PC-3 cells.

[0047] Figure 2 shows the cellular uptake of compounds 1-4 (black bars) over time in LNCaP on ice (cell endocytosis blocked) and at 37 °C (internalization-permissive temperature). For each compound, the corresponding uptake of the unconjugated dye is reported (striped bars).

[0048] Figure 3 shows the cellular uptake of representative compounds 1-4 in presence or absence of a competing excess of 2-PMPA.

[0049] Figure 4 shows the tumor imaging efficacy, in terms of tumor-to-background ratio (TBR), of representative compounds 1-4 of the invention in in vivo experiments on LNCaP tumor bearing animals.

[0050] Definitions

[0051] In the present description, and unless otherwise provided, the following terms and phrases as used herein are intended to have the following meanings.

[0052] The term "diradical" refers to a chemical group wherein the hydrogen atoms at two terminal portions of the molecule are removed to form a bond.

[0053] 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. For instance, "Ci-Cs alkyl" comprises within its meaning a linear or branched chain comprising from 1 to 8 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-Cio alkylene" can be used.

[0054] The term "hydroxyalkyl" refers to any of the corresponding alkyl chain wherein one or more hydrogen atoms are replaced by hydroxyl groups.

[0055] 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), alkyl, e.g. 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, 4thEd., Ch. 5).

[0056] 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.

[0057] 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- 1 -pyrrolid inyl phosphonium hexafluorophosphate

[0058] (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-azabenzotriazol-l-yl)uronium hexafluorophosphate (HATU), O-(lH-6-chlorobenzotriazole-l-yl)-l,l,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.

[0059] 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 and sulfo NHS esters. 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.

[0060] 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.

[0061] Preferably, the fluorescent probes of the invention are able to 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.

[0062] Targeting moiety CT)

[0063] According to the invention, a targeting moiety (T) 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.

[0064] 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.

[0065] 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.

[0066] 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. For example, it can be represented 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.

[0067] Linker (L)

[0068] According to the invention, L is a linker, optionally present, that separates the PSMA targeting ligand from the dye.

[0069] The presence of a linker is particularly useful for some embodiments where the ligand (e.g., an urea-based peptidomimetic inhibitor) 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.

[0070] 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.

[0071] 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.

[0072] Therefore, in one preferred embodiment, the linker, 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, -alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid and the like or an amino acid derivative, such as for instance 3- (2-naphthyl)-alanine; 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- or -NH-(O-CH2-CH2)P-CO- or derivatives thereof; amino-polyethylene glycol-carboxylic acid; trans-4-faminomethvl)cvclohexanecarboxylic acid (tranexamic acid); diaminobutyric acid; and diaminopropionic acid, and combinations thereof, or it is a group - L1-L2- wherein Li is a diradical of a diamine, such as for instance amino-polyethylene glycol amine of formula -NH-(O-CH2-CH2)P-NH- or a diradical of ethylenediamine, propylenediamine, putrescine, spermidine, spermine or hexanediamine and the like, and L2 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.

[0073] More preferably, L 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.

[0074] In another preferred embodiment L is a combination of tranexamic acid and an amino acid derivative, such as the tranexamic acid-(3-(2-naphtyl)-alanine) linker described for instance in Benesova et al., J Nucl Med 2015; 56: 914-920.

[0075] 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.

[0076] The present invention further relates to compounds of the above formula (I) in which the sulfonyl groups of R1, R2and / or R3may be in the form of a negatively charged ion or a pharmaceutically acceptable salt.

[0077] Detailed description of the embodiments

[0078] In a preferred embodiment, the invention relates to a compound of formula (I) wherein R2is a group -SO3H.

[0079] In another preferred embodiment the invention relates to a compound of formula (I) wherein R2is a group -CONH-Y, wherein Y is selected from the group consisting of

[0080] More preferably, the invention relates to a compound of formula (I) wherein R2is 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:

[0081] In another preferred embodiment the invention relates to a compound of formula (I) wherein R2is a group (III), as defined above. More preferably, R2is a group of the following formula (Illa), wherein L is defined above:

[0082] (Illa).

[0083] Preferably, L is a bond or a group -NH-(CH2)s-CO-.

[0084] More preferably, L is represented by the formula (L3)

[0085] In another preferred embodiment both R1are a group -(CH2)4-SO3H.

[0086] In one preferred embodiment n is 1 and R3is a group -O-phenyl substituted with -SO3H, as represented by the following formula (la) wherein R1, R2, L and T are as defined above.

[0087] In another preferred embodiment n is 0 and R3is hydrogen, as represented by the following formula (lb)

[0088] wherein R1, R2, L and T are as defined above.

[0089] A more preferred embodiment of the invention relates to a compound represented by the following formula (Ic)

[0090] Especially preferred and representative of the invention are the compounds of formula

[0091] (I) listed in Table I.

[0092]

[0093] The present invention is also directed to methods for synthesizing the compounds of formula (I) prepared as illustrated in the following description .

[0094] The compounds of the invention are useful as imaging agents in the detection of tumors in both humans and animals. 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 a n increased or variable expression of PSMA. Preferably the imaged subject is a human . The invention also provides a compound of formula (I) for use as fluorescent probe as defined above, wherein the detection and demarcation of the tumor tissue is carried out under NIR radiation.

[0095] 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.

[0096] The 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.

[0097] In detail, the present invention also provides a compound of formula (I) as imaging agent for use in a method for detecting the possible presence of a disease in a subject comprising the steps of:

[0098] - 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 PSMA expressing cells;

[0099] - measuring a signal from said compound in a biological sample;

[0100] - 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.

[0101] 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:

[0102] - contacting the tissues or cells with a compound of formula (I) as defined above;

[0103] - irradiating the tissues or cells at a wavelength absorbed by the imaging agent;

[0104] - detecting a near-infrared emission using a fluorescent camera.

[0105] In particular, in a preferred embodiment the invention provides a method for performing imaging guided surgery on a subject comprising the steps of:

[0106] - 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;

[0107] - performing surgical resection of the areas that fluorescence upon excitation by the nearinfrared light and / or back-table fluorescence guided imaging.

[0108] Moreover, the invention relates to a compound of formula (I) as imaging agent for use in the method described above.

[0109] A further aspect of this invention relates to a pharmaceutical composition comprising a fluorescent probe of formula (I) as defined above, or a salt thereof, and one or more pharmaceutically acceptable adjuvants, excipients, carriers or diluents.

[0110] Another aspect of this invention relates to a diagnostic kit comprising at least one compound of formula (I) as defined above 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.

[0111] 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.

[0112] 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. 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.

[0113] 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 labeling of sulfoindocyanine dyes.

[0114] 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.

[0115] Contrary to what was expected, the compounds of the invention were found very stable at basic pH ( / .e., at about pH 11-12) and none or negligible degradation has been observed during the removal of the protecting groups.

[0116] EXPERIMENTAL PART

[0117] 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.

[0118] Materials and Equipment

[0119] 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 moiety glutamate-urea-lysine-3-(2-naphthyl)-alanine-tranexamic acid was prepared by solid-phase peptide chemistry following the procedure described in Benesova et al., Journal of Nuclear Medicine;56(6):914-920.

[0120] All the reactions were monitored by HPLC-UV (Agilent mod. 1100) and HPLC-UV-MS (Agilent mod. 1260, MS detector single quadrupole Mod. 6120) equipped with an absorption detector set at different wavelengths or a DAD detector (Column : YMC-Triart Phenyl, 250 x 4.6 mm I S-5 pm I 12 nm, eluents: 0.1% ammonium acetate or 10 mM ammonium formate and acetonitrile).

[0121] 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.

[0122] The absorption spectra were recorded with a UV-VIS spectrophotometer (Lambda 365, Perkin Elmer), with acquisition range of 500 - 1000 nm. The emission spectra and the absolute quantum yield were carried out on the FluoroLog-3 1IHR-320 spectrofluorometer with 450W Xenon Light source and equipped with an F-3018 integrating sphere accessory (Horiba Jobin Yvon). Detection was performed by photomultiplier tubes (PMT-NIR, R5509) cooled detector.

[0123] Human prostate carcinoma LNCaP cells (CRL-1740, ATCC) and human prostate adenocarcinoma PC-3 cells (CRL-1435, ATCC) used for cellular experiments were cultured in RPMI medium (Euroclone) and Ham's F-12K nutrient mixture (Euroclone) respectively, 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) and cells were grown at 37 °C in humidified atmosphere enriched with 5% CO2. DPBS without MgCh and CaC (Sigma-Aldrich) was used for cell rinsing.

[0124] Samples from cellular experiments were analyzed with the flow cytometer AccuriTM 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 (i.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.

[0125] To evaluate the in vivo efficacy of the compounds of the invention, a LNCaP prostate cancer, orthotopically induced, was used as an animal model due to the well-known PSMA overexpression on prostate cancer cells. LNCaP cells were collected and washed twice with PBS. One million cells was re-suspended in 20 pL serum-free medium and injected in the left ventral prostate lobe of each 7 weeks old male mouse in general anesthesia. Tumor development was followed at least twice a week, from 14 days after cells implant, by Magnetic Resonance Imaging (MRI) acquisitions of anatomical images. The MRI acquisition protocol was performed with the following steps: a) Localizer sequence to verify the correct positioning of the animal; b) Optimization adjustment protocol (baseline frequency, 1stand 2ndorder shimming, radiofrequency pulse); c) T2-weighted spin echo sequence (over axial, coronal and sagittal geometry) to acquire a full reference set of images for geometry definition of next sequences; d) T2-weighted spin echo image at high resolution (TR = 2500 ms, TE = 30.15 ms, rare factor = 8, FOV = 35 mm x 35 mm, matrix size = 256 x 256, number of slices = 25, slice thickness 0.8 mm, number of averages = 4).

[0126] List of abbreviations

[0127] DMF / V, / V-Dimethylformamide

[0128] EuK Glutamic acid-urea-lysine EuE Glutamic acid-urea-glutamic acid

[0129] HPLC High performance liquid chromatography

[0130] RT Room temperature

[0131] CV Column volume

[0132] DMSO Dimethyl sulfoxide

[0133] DPBS Dulbecco's phosphate buffered saline

[0134] FSC Forward Scatter

[0135] SSC Side Scatter

[0136] CV% Coefficient of Variation

[0137] NMM / V-methylmorpholine

[0138] HATU 1- [ Bis(d i methyla mi no) methylene] -1H-1, 2, 3-triazolo[4,5-b] pyridinium 3-oxid hexafluorophosphate

[0139] TSTU O-( / V-Succinimidyl)-l,l,3,3-tetramethyluronium tetrafluoroborate

[0140] Example 1: Synthesis of compound 1

[0141] To a suspension of 4-hydrazinobenzenesulfonic acid (2.09 g, 0.011 mol) in glacial acetic acid (10 mL) 3-methylbutan-2-one (1.5 mL, 0.014 mol) and sodium acetate (1.64 g, 0.020 mol) were added. After stirring for 4.5 hours at 110 °C the orange solution was cooled to RT and precipitated in cold diisopropyl ether (100 mL). The solid was filtered under suction and dissolved with methanol together with SiO?. Methanol was removed and the slurry in silica gel was loaded onto a silica gel column eluting with 30% methanol in ethyl acetate, giving intermediate 10 as pale brown solid (2.46 g).

[0142] Yield: 94%

[0143] HPLC purity: 90% 270 nm

[0144] MS: [M + H]+240.1

[0145] A mixture of the intermediate 10 (1.32 g, 5.51 mmol) and 1,4-butanesultone (0.84 mL, 7.58 mmol) in sulfolane (2 mL) was heated at 120 °C for 24 hours. Then, the mixture was cooled down to RT and cold ethyl acetate (50 mL) was added, stirring for 1 hour in an ice bath. Then, the precipitate was filtered under suction and washed twice with cold ethyl acetate (2 x 50 mL). The crude product was dissolved in methanol, dried under vacuum and purified by flash chromatography on a pre-packed KP-Sil cartridge (Biotage® SNAP, 60 g) eluting with 50% methanol / ethyl acetate. Intermediate 11 was recovered as a pink solid (0.573 g).

[0146] Yield: 28%

[0147] HPLC purity: 98.3% at 270 nm

[0148] MS: [M + H]+376.0

[0149] Preparation of the intermediate compound (13)

[0150] To a suspension of 4-hydrazinobenzoic acid (10 g, 0.053 mol) in glacial acetic acid (150 mL) 3-methylbutan-2-one (146 mL, 0.130 mol) and sodium acetate (11 g, 0.130 mol) were added. After stirring for 3 hours at 135 °C the brown solution was cooled to RT, concentrated under reduced pressure and suspended in 30 mL 9 : 1 water / methanol mixture. The solid (intermediate 12) was filtered under suction and washed with other 40 mL of 9: 1 water / methanol, then dissolved in 200 mL sat. NaHCOs solution and extracted with dichloromethane (3 x 125 mL). The organic layer was concentrated under reduced pressure and the orange solid was oven-dried for 18 hours at 45 °C, obtaining 9.4 g of a pale pink solid.

[0151] Yield: 87%

[0152] HPLC purity: 98% at 270 nm

[0153] MS: [M + H]+204.8

[0154] A mixture of intermediate 12 (4.01 g, 19.7 mmol) and 1,4-butanesultone (2.4 mL, 23.5 mmol) in butyronitrile (4 mL) was heated at 120 °C for 54 hours. The mixture was cooled down to RT and cold acetone (100 mL) was added, stirring for 1 hour in an ice bath. Then, the precipitate was filtered under suction and washed twice with cold ethyl acetate (2 x 50 mL). The crude product was dissolved in 0.1% ammonium acetate aqueous solution and purified by flash chromatography on a pre-packed C18 silica column (Biotage® SNAP, 60 g), eluting the desired product with water, and removing by-products with acetonitrile. Desalting was performed on the same silica C18 column, loading the product dissolved in sodium acetate solution and washing with 0.05% formic acid aqueous solution (2 CV) and then water (2 CV). Fractions containing the desired product were concentrated under reduced pressure and freeze-dried, obtaining 4.8 g of a pink solid corresponding to intermediate 13. Yield: 72%

[0155] HPLC purity: 98% at 270 nm

[0156] MS: [M + H]+342.1

[0157] Intermediate 11 (500 mg, 1.331 mmol), intermediate 13 (451.74 mg, 1.331 mmol) and potassium acetate (261.25 mg, 2.662 mmol) were dissolved in a 8:2 acetic anhydride I glacial acetic acid mixture (18 mL) at 55 °C. Then, 2-chlorocyclohex-l-ene-l,3-dicarbaldehyde (287.22 mg, 1.664 mmol) was added and the mixture was heated at 100 °C in the dark for 2.5 hours. The mixture was cooled down to RT and solvents were removed under reduced pressure. The viscous residue was partially dissolved in methanol (2 mL) and precipitated in cold diethyl ether (150 mL) after stirring for 1 hour in an ice bath. The precipitate was filtered under suction, dissolved in water, and purified by flash chromatography on a pre-packed C18 silica column (Biotage® SFAR 60 g) with a water-acetonitrile gradient (the cyanine was eluted with 15% acetonitrile). Intermediate compound 14 was freeze-dried, obtaining 328 mg of a green solid.

[0158] Yield: 29%

[0159] HPLC purity: 98.2% at 790 nm; 96% at 254 nm

[0160] MS: [M + H]+851.2

[0161] Intermediate 14 (328 mg, 0.385 mmol) was dissolved in DMSO (10 mL) and dropped into a suspension of sodium 4-hydroxybenzenesulfonate (755.76 mg, 3.85 mmol) and potassium carbonate (532.52 mg, 3.85 mmol) in DMSO (5 mL). The mixture was stirred in the dark at RT overnight. Then, the mixture was precipitated in cold ethyl acetate (225 mL), stirring for 1 hour in an ice bath. The precipitate was filtered under suction, dissolved in water, quickly acidified at pH 7.5 with diluted HCI and purified by flash chromatography on a pre- packed C18 silica column (Biotage® SFAR 60 g) with a water / acetonitrile gradient (the cyanine was eluted with 10% acetonitrile). Fractions containing the pure product were concentrated under reduced pressure and freeze-dried obtaining 242.75 mg of a green solid corresponding to intermediate compound 15.

[0162] Yield: 63.7% HPLC purity: 99.1% at 790 nm; 97.4% at 254 nm

[0163] MS: [M + H]+989.1

[0164] Intermediate 15 (40 mg, 0.0371 mmol) was dissolved in anhydrous DMF (7 mL), then HATU (17.67 mg, 0.0464 mmol), DIPEA (12.9 pL, 0.0742 mmol) and intermediate 16, EuK(tBu)s,

[0165] (21.76 mg, 0.0445 mmol) were added. The dark green solution was stirred in the dark for 1 hour at RT, then cold diethyl ether (50 mL) was added and the mixture was stirred in an ice bath for 30 minutes. The precipitate (intermediate 17) was filtered under suction and directly dissolved in TFA (1 mL). The brown solution was stirred in the dark for 30 minutes at RT, then cold diethyl ether (50 mL) was added and the mixture was stirred in an ice bath for 30 minutes. The precipitate was filtered under suction, dissolved in water and the pH immediately adjusted to 8.5 with 1 N NaOH. The solution was loaded on a pre-packed C18 silica column (Biotage® SFAR 30 g) and purified by flash chromatography with a water-acetonitrile gradient (the cyanine was eluted with 10% acetonitrile). Fractions containing the pure product were concentrated under reduced pressure and freeze-dried, obtaining 32.86 mg of a green solid. Yield: 68%

[0166] HPLC purity: 99.6% at 780 nm; 98.7% at 254 nm

[0167] MS: [M + 2H]2+645.7, [M-2H]2’ 643.8

[0168] Example 2: Synthesis of compound 2

[0169] Preparation of the peptidomimetic cjlutamate-urea-lvsine-3-(2-naphthyl)-alanine-

[0170] Intermediate 18a was obtained by well known procedures of solid-phase peptide chemistry, following the procedure described in Benesova et al., Journal of Nuclear Medicine;56(6) :914-920. The intermediate was cleaved from the resin, deprotected and purified by RP-HPLC / UV-MS. Preparative HPLC purifications were performed on a Waters HPLC system equipped with a MS Detector and photodiode array detector. Runs were performed using an Atlantis dCis OBD Prep Column, lOOA, 5 pm, 19 mm X 150 mm column. The final compound was analyzed using a UPLC-H-Class system equipped with Acquity QDa MS Detector and TUV detector, with a UPLC Kinetex® 1.7mm F5 100 A 100x2.1 mm column . Solvent A consisted of 0.01% TFA in water and solvent B consisted of 0.01% TFA in acetonitrile. Fractions containing the desired pure product were combined, evaporated under reduced pressure and freeze-dried, recovering 55 mg of a white solid.

[0171] MS: [M + H]+656.4

[0172] Synthesis of Compound 2

[0173] In a 50 mL centrifuge tube, intermediate 15 (18 mg, 0.0182 mmol) was dissolved in anhydrous DMF (2 mL), then NMM (4.4 pL, 0.040 mmol) and TSTU (8.22 mg, 0.0273 mmol) were added. The dark green solution was stirred for 1 hour at RT. Cold ethyl acetate (25 mL) was added and the suspension was centrifuged for 10 min at 4010 rpm and 5 °C. Then, the organic layer was decanted, the solid was suspended twice in ethyl acetate (10 mL) and centrifuged again. The crude solid was finally dried under nitrogen atmosphere, dissolved in 10 mL of DMF / DMSO mixture and added to a solution of glutamate-urea-lysine-3-(2- naphthyl)-alanine-tranexamic acid (intermediate 18: 14 mg, 0.0182 mmol) and DIPEA (7.92 pL, 0.0455 mmol) in anhydrous DMF (1 mL). The solution was stirred in the dark at RT overnight. Cold ethyl acetate (25 mL) was then added and the precipitate was filtered under suction. The crude material was dissolved in water and acetonitrile (pH adjusted to 7.5) and purified twice by flash chromatography on a pre-packed C18 silica column (Biotage® SFAR 30 g) with 0.1% ammonium acetate / acetonitrile gradient (the cyanine was eluted with 20% acetonitrile). The pure product was dissolved in water and desalted on a pre-packed C18 silica column (Biotage® SFAR 30 g) with 0.1% formic acid (5 CV), washed with water (5 CV) and eluted with a water-acetonitrile gradient (the cyanine was eluted with 20% acetonitrile). The pure product was freeze-dried obtaining 7.04 mg of a green solid.

[0174] Yield: 24%

[0175] HPLC purity: 98.7% at 780 nm; 98.0% at 254 nm

[0176] MS: [M + 2H]2+813.7

[0177] Example 3: Synthesis of compound 3

[0178] Preparation of the ligand EuK-C6 (intermediate 20)

[0179]

[0180] EuK(t-Bu)3 (intermediate 16: 75mg, 0.154 mmol) was dissolved in anhydrous DMF (5 mL), then HATU (88 mg, 0.23 mmol), NMM (34 pL, 0.308 mmol) and / V-(tert-butoxycarbonyl)- 6-amino hexanoic acid (39 mg, 0.169 mmol) were added. The yellow solution was stirred for 1 hour at RT, then tert-butyl methyl ether (20 mL) was added and the organic phase was washed twice with water (40 mL). The organic phase was concentrated under reduce pressure and directly dissolved in TFA (3 mL). The solution was stirred for 3 hours at RT, then cold tert-butyl methyl ether (50 mL) was added and the mixture was stirred in an ice bath for 30 minutes. The white precipitate was filtered under suction, dissolved in water / acetonitrile and purified by flash chromatography on a pre-packed C18 silica column (Biotage® SFAR 12 g) with a water / acetonitrile gradient (the product was eluted with 10% acetonitrile). Fractions containing pure intermediate compound 20 were concentrated under reduced pressure and freeze-dried, obtaining 44 mg of a white solid.

[0181] Yield: 52% HPLC purity: 90% at 220 nm

[0182] MS: [M + H]+433.5. Synthesis of compound 3

[0183]

[0184] In a 50 mL centrifuge tube, intermediate 15 (23.6 mg, 0.0327 mmol) was dissolved in anhydrous DMF (3mL), then NMM (7.9 pL, 0.0719 mmol) and TSTU (14.77 mg, 0.049 mmol) were added. The dark green solution was stirred for 1 hour at RT. Cold ethyl acetate (25 mL) was added and the suspension was centrifuged for 10 min at 4010 rpm and 5 °C. Then, the organic layer was decanted, the solid was suspended twice in ethyl acetate (10 mL) and centrifuged again. The crude solid was finally dried under nitrogen atmosphere, dissolved in 2.5 mL of DMSO and added to a solution of intermediate 20 (C6-EuK: 15.55 mg, 0.036 mmol) and DIPEA (39.87 pL, 0.2289 mmol) in DMSO (2 mL). The solution was stirred in the dark at RT overnight. Cold ethyl acetate (25 mL) was then added and the precipitate was filtered under suction. The crude material was dissolved in water (pH adjusted to 7.5) and purified twice by flash chromatography on a pre-packed Cis silica column (Biotage® SFAR 30 g) with water / acetonitrile gradient (the cyanine was eluted with 20% acetonitrile). The pure product was freeze-dried obtaining 22.87 mg of a green solid. Yield: 49.8%

[0185] HPLC purity: 99.1% at 780 nm; 97.6% at 254 nm

[0186] MS: [M + 2H]2+702.3

[0187] Example 4: Synthesis of compound 4

[0188] Preparation of intermediate compound (22)

[0189] To a solution of intermediate 13 (500 mg, 1.47 mmol) in methanol (8 mL), cone. H2SO4 (0.820 mL) was added and the solution was refluxed for 2 hours. The mixture was cooled down to RT and concentrated under reduced pressure, diluted with water (pH adjusted to 2 with 1 M NaOH ) and purified by flash chromatography on a pre-packed C18 silica column (Biotage® SFAR 60 g) with a water / acetonitrile gradient (the desired compound was eluted with 15% acetonitrile). After freeze-drying, intermediate 21 was obtained as a pale brown solid (468 mg).

[0190] Yield: 88%

[0191] HPLC purity: 99.3% at 270 nm

[0192] MS: [M + H]+354.1

[0193] Intermediate 21 (424.6 mg, 1.201 mmol), intermediate 13 (407.7 mg, 1.331 mmol) and potassium acetate (200.4 mg, 2.042 mmol) were dissolved in a 2: 1 acetic anhydride / glacial acetic acid mixture (30 mL) at 50 °C. Then, 2-chlorocyclohex-l-ene-l,3- dicarbaldehyde (251.67 mg, 1.441 mmol) was added and the mixture was heated at 100 °C in the dark for 1 hour. The mixture was cooled down to RT and solvents were removed under reduced pressure. The viscous residue was partially dissolved in methanol (2 mL) and precipitated in cold diethyl ether (200 mL) after stirring for 1 hour in an ice bath. The precipitate was filtered under suction, dissolved in water and purified by flash chromatography on a pre-packed Cis silica column (Biotage® SFAR 60 g) with a water / acetonitrile gradient (the cyanine was eluted with 20% acetonitrile). Intermediate compound 22 was freeze-dried, obtaining 362.9 mg of a green solid.

[0194] Yield: 36.5%

[0195] HPLC purity: 98.6% at 790 nm; 100% at 254 nm

[0196] MS: [M + H]+831.4

[0197] Preparation of intermediate compound (23}

[0198]

[0199] Intermediate 22 (223.8 mg, 0.269 mmol) was dissolved in DMSO (25 mL) and dropped into a suspension of sodium 4-hydroxybenzenesulfonate (529.27 mg, 2.69 mmol) and potassium carbonate (372.93 mg, 2.69 mmol) in DMSO (5 mL). The mixture was stirred in the dark at 40 °C for 3 hours, then precipitated in cold ethyl acetate (200 mL). The precipitate was filtered under suction, dissolved in water, quickly acidified at pH 8.5 with diluted HCI, and purified by flash chromatography on a pre-packed Cis silica column (Biotage® SFAR 60 g) with a water-acetonitrile gradient (the cyanine was eluted with 12% acetonitrile). Fractions containing the pure product were concentrated under reduced pressure and freeze-dried obtaining 210.6 mg of a green solid.

[0200] Yield: 80%

[0201] HPLC purity: 99.9% at 790 nm; 99.9% at 254 nm

[0202] MS: [M + H]+967.1

[0203] Preparation of intermediate compound (25}

[0204]

[0205] Intermediate 23 (208.4 mg, 0.215 mmol) was dissolved in DMF (20 mL), then HATU (93.3, 0.258 mmol) and DIPEA (78 pL, 0.451 mmol) were added. The mixture was cooled in an ice bath and a solution of D-glucamine (46.75 mg, 0.258 mmol) in DMF (6 mL) was slowly dropped. The mixture was stirred at 0 °C for 1.5 hours in the dark. Then the product was precipitated in cold diethyl ether (50 mL), stirring for 1 hour in an ice bath. The precipitate was filtered under suction, dissolved in water and purified by flash chromatography on a prepacked Cis silica column (Biotage® SFAR 60 g) with a water / acetonitrile gradient (the cyanine was eluted with 15% acetonitrile). Fractions containing the pure product were concentrated under reduced pressure obtaining 176 mg of a green solid corresponding to intermediate 24.

[0206] Yield: 72%

[0207] HPLC purity: 99.2% at 790 nm; 61.7% at 254 nm

[0208] MS: [M + H]+1130.8

[0209] Such intermediate 24 (119 mg, 0.106 mmol) was dissolved in water (30 mL) and hydrolysed at 40 °C for 5 hours maintaining pH 12 by continuous addition of 1 M NaOH (0.856 mL in total). The mixture was cooled down to room temperature, pH was adjusted to 8 with diluted HCI and the aqueous solution was purified by flash chromatography on a pre-packed Cis silica column (Biotage® SFAR 60 g) with a water-acetonitrile gradient (the cyanine was eluted with 8% acetonitrile). Fractions containing the pure product were combined, concentrated under reduced pressure and freeze-dried, obtaining 88.1 mg of a green solid corresponding to intermediate 25.

[0210] Yield: 75%

[0211] HPLC purity: 99.97% at 756 nm; 99.5% at 254 nm

[0212] MS: [M + H]+1116.3

[0213] Intermediate 25 (155.7 mg, 0.137 mmol) was dissolved in anhydrous DMF (50 mL), then NMM (60 pL, 0.548 mmol) and TSTU (82.49 mg, 0.274 mmol) were added. The dark green solution was stirred for 1.5 hour at RT. The product was precipitated in cold ethyl acetate (300 mL) stirring for 1 hour in an ice bath. The precipitate was filtered under suction, washed twice with ethyl acetate (10 mL) and dissolved in DMF (50 mL). A solution of 6- aminocaproic acid (18.87 mg, 0.144 mmol) and DIPEA (60 pL, 0.343 mmol) in 1 :2 DMF / DMSO (10 mL) was added. The solution was stirred in the dark for 45 hours. Then the product was precipitated with cold 1 : 1 ethyl acetate / diethyl ether mixture (500 mL) and, once recovered from the funnel, purified by flash chromatography on a pre-packed Cis silica column (Biotage® SFAR 60 g) with water-acetonitrile gradient (the cyanine was eluted with 10% acetonitrile). The pure product was freeze-dried obtaining 99.7 mg of a green solid corresponding to intermediate 26.

[0214] Yield: 60%

[0215] HPLC purity: 96.8% at 780 nm; 95.3% at 254 nm

[0216] MS: [M + H]+1230.6

[0217] Such intermediate 26 (113 mg, 0.0922 mmol) was dissolved in anhydrous DMF (15 mL), then TSTU (69.4 mg, 0.2305 mmol) and NMM (40.5 pL, 0.3688 mmol) were added. The dark green solution was stirred for 24 hour at RT. The product was precipitated in cold ethyl acetate (200 mL) stirring for 1 hour in an ice bath. The precipitate was filtered under suction, washed twice with ethyl acetate (10 mL) and dissolved in DMF (50 mL). A solution of EuK TFA salt (59.7 mg, 0.1383 mmol) and DIPEA (80.5 pL, 0.461 mmol) in DMF (10 mL) was added. The solution was stirred in the dark for 22 hours. Then the product was precipitated with cold diethyl ether (200 mL) stirring for 1 hour in an ice bath. The precipitate was filtered under suction, dissolved in water and purified by flash chromatography on a pre-packed Cis silica column (Biotage® SFAR 60 g) with a water / acetonitrile gradient (the product was eluted with 8% acetonitrile). The pure product was freeze-dried obtaining 64.3 mg of a green solid.

[0218] Yield: 46%

[0219] HPLC purity: 98.7% at 780 nm; 99.7% at 254 nm

[0220] MS: [M + H]+1530.4

[0221] Example 5: Optical characterization

[0222] The compounds of the invention were characterized in terms of their optical properties in vitro in water and in a clinical chemistry control serum (Seronorm, Sero SA), mimicking the chemical composition and optical properties of human serum. All solutions were freshly prepared. The compounds of the invention were characterized by absorption maxima comprised in the range from about 770 nm to 810 nm.

[0223] The excitation and emission maxima in water and Seronorm and the fluorescence quantum yield (QY%) of representative compounds of formula (I) are shown in Table I. The compounds of the invention displayed an increased quantum yield and a red shift in absorption and emission in Seronorm with respect to water, due to some interaction of the probes with serum proteins. Table I - Excitation / Emission maxima and quantum yield % of representative compounds of formula (I)

[0224] Example 6: Binding affinity to PSMA-expressing cell

[0225] 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 both western blot and flow 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). Conversely, PC-3 prostatic adenocarcinoma cells were used in all experiments as control, due to the lack of PSMA expression.

[0226] All probes were incubated in increasing concentrations (range 0 - 4 pM) either with PSMA-positive LNCaP cells and PSMA-negative PC-3 cells on ice, as well as their corresponding unconjugated dyes, which resulted negligible. Cell-associated fluorescence was collected by flow cytometry analysis and the binding constant at equilibrium was calculated by mathematical fitting. The observed tendency to a plateau was per se an indication of the probe specific binding to the cell surface, whereas no tendency to saturation was observed for the PSMA-negative PC-3 cells.

[0227] In more detail, LNCaP or PC-3 cells were detached with StemPro® Accutase® Cell Dissociation Reagent (Life Technologies), collected in DPBS and counted. At least 2- 105cells 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 (e.g. representative Compounds 1, 2, 3 or 4), two-fold serially diluted (4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.015, 0.008, 0.004, 0.002, 0.001 pM). 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 2 times in 500 pL of 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 analyzed 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

[0228] Y is the main cell-associated fluorescence;

[0229] X is the concentration of the Test Articles;

[0230] Bmax is the maximum specific binding in the same units as Y;

[0231] KD is the equilibrium dissociation constant, in the same units as X;

[0232] NS is the slope of nonspecific binding in Y units divided by X units; background is the amount of nonspecific binding with no added Test Article or Compound.

[0233] All compounds were analyzed in replicate independent experiments. The calculated KD are summarized in Table II, while binding curves are shown in Figure 1.

[0234] Table II - Binding constant of equilibrium (KD) to PSMA-positive LNCaP cells of representative compounds of formula (I). KD values are expressed in nM (mean ofKD resulting in replicate independent experiments, with SEM).

[0235] This assay confirmed that compounds of the invention are endowed of high affinity for PSMA-positive LNCaP cells, with KD lower than 20 nM. As expected, no specific binding was detected in PSMA-negative PC-3 cells, which displayed a linear non-saturating curve of cell- associated fluorescence, characterized by comparatively low cell-associated fluorescence values, at increasing probe concentrations (see Figure 1).

[0236] Example 7: Cellular uptake assessment

[0237] Cellular uptake experiments were performed using flow cytometry analysis to assess if probe internalization occurs after targeting to cell surface-expressed PSMA.

[0238] Different kind of in vitro tests were performed, aiming to appreciate specific features of the compounds of invention :

[0239] 1) time-dependent uptake (time course); 2) incubation at physiological temperature (37 °C) vs endocytosis-blocking temperature (on ice), to differentiate cell-associated fluorescence deriving from internalized compounds from the one due to residual cell surface-associated ones;

[0240] 3) uptake in presence of an excess of 2-PMPA, competing for PSMA binding site, to verify if the uptake of probes was mediated by binding to the target receptor.

[0241] The experiments were carried out according to the following general procedure.

[0242] Cells were plated in 24-well plates: 1.7-105cells / well for PSMA-positive LNCaP cells and 2-105cells / well for PSMA-negative PC3 cells were seeded two days before experiment.

[0243] Test Articles (e.g. representative Compounds 1, 2, 3 or 4) and Control Articles (the corresponding unconjugated dyes, i.e. the intermediate compounds 15 or 25) were diluted to the working concentration of 1 pM in serum-free medium with 25 mM HEPES pH 7.4. In competition experiments, a 100X excess of 2-PMPA (Sigma-Aldrich, dissolved in water at 2 mg / mL stock concentration and stored at 4 °C) was added in the same treatment.

[0244] Cells were rinsed twice in DPBS.

[0245] Treatment solutions (250 pL / well) were administered and cells were incubated for the indicated time at 37 °C in the dark, unless otherwise indicated. In a subset of experiments, after incubation with dyes / probes at either 37 °C (endocytic processes permissive temperature) or on ice (endocytosis blockage). Cell-associated fluorescence obtained after incubation at 37 °C is the sum of residual cell surface-bound and internalized compounds (total), whereas the fluorescence recorded after incubation on ice represents the sole cell surface-bound quote (residual).

[0246] At the end of treatment, cells were rinsed 2 times with cold DPBS and detached from the plate with 120 pL / well of StemPro® Accutase® Cell Dissociation Reagent (Thermo-Fischer). Few minutes in incubator at 37 °C were enough to obtain a complete cell suspension.

[0247] Plates were placed on ice and 500 pL of cold FACS buffer were added in each well to dilute and inactivate the dissociation reagent. The cell suspension of each well was collected and transferred to 1.5 mL tube, on ice.

[0248] Cells were centrifuged (5 minutes, 350 RCF, 4 °C). Then, the supernatant was discarded, and the cell pellet was resuspended in 100 pL of cold FACS buffer, or in a suitable volume in order to obtain no more than about 1000 recorded events per pL during FACS analysis.

[0249] At the end of analysis, each sample was plotted in graphs, where X axis = fluorescence intensity (FL4-A) in log scale and Y axis = counts. Usually, a gaussian distribution of the cell population was observed. The data retrieved were the mean fluorescence of each population and the CV%. In those cases where the CV% was higher than 50%, a gate on the fluorescence intensity axis was established to exclude the tails of the gaussian distribution of the cell population; no more than 5% all events in each sample were excluded, in most cases less than 1%. The resulting mean fluorescence of the cell population was considered the fluorescence intensity value of the sample.

[0250] Every treatment was performed in independent two replicates and, among replicates, mean fluorescence intensity with corresponding standard deviation was calculated.

[0251] Normalized cell-associated fluorescence was calculated with the following formula:

[0252] (FL treated — FL untreated') Normalized FL = - - -

[0253] FL untreated

[0254] Time-dependent uptake

[0255] Experiments of time-dependent uptake were assessed in both PSMA-positive LNCaP and PSMA-negative PC-3 cells by flow cytometry, from 1 to 4 hours of cell treatment with probe concentration of 1 pM. They were performed both on ice and at 37 °C. At the physiological temperature (37 °C), the conjugated compounds of the invention were internalized by LNCaP cells, resulting in total cell-associated fluorescence increasing over time, from 1 hour to 4 hours of observation. The increase in total-cell associated fluorescence occurring after treatment with the corresponding unconjugated dyes was visible, indicating that no target- mediated internalization mechanisms occurred, despite negligible respect to those driven by PSMA-targeting. The residual cell-associated fluorescence of LNCaP cells treated with the probes on ice remained roughly constant over time, as expected in the case of blocked endocytic processes, while the binding of the unconjugated dyes was mainly undetectable (see Figure 2).

[0256] To deeply assess the cell behavior of the compounds of the invention, some parameters, such as the slope of internalization, the percentage of internalization and the targeting advantage, were estimated from the time-dependent uptake outcomes.

[0257] The slope of internalization was calculated by applying a linear fitting to cell-associated fluorescence values over time obtained after cell incubation at 37 °C (simple linear regression by GraphPad Prism v.9 software; fluorescence vs hours of uptake).

[0258] The percentage of internalization was calculated by considering the total cell-associated fluorescence recorded after 2 hours of cell treatment at 37 °C (representing the sum of the residual cell surface-bound and internalized compounds) and cell-associated fluorescence recorded after cell treatment on ice (representing the residual cell surface-bound quote).

[0259] The targeting advantage expresses the ratio between the total cell-associated fluorescence after 4 hours of cell treatment at 37 °C with the targeting probes and the corresponding unconjugated dyes. Of note, since the cell-associated fluorescence recorded for the unconjugated dyes was well-distinguishable from the untreated cells at 4 hours of treatment, this time point was selected for the calculation of the targeting advantage parameter.

[0260] The results highlighted that all probes tended to accumulate into LNCaP cells over time, with similar rate of internalization. Considering the percentage of internalization, all probes displayed high values (> 50%) of percentage of internalized probes respect to the residual ones bound on the cell surface. The targeting advantage (fold-over-dye) parameter, expressing the cellular uptake advantage of PSMA-targeting probes over the corresponding unconjugated dyes, showed that all probes were preferentially internalized by a target- mediated mechanism in LNCaP cells (see Table III).

[0261] Table III - Internalization potential of representative compounds of formula (I) (mean ± std.dev.)

[0262] In PSMA-negative PC-3 cells, the uptake of both PSMA-targeting probes and unconjugated dyes was at least 30 times lower. In particular, after 4 hours treatment, the normalized cell-associated fluorescence of all probes was comparable to the uptake of the unconjugated dyes and much lower (< 4 fluorescence AU) with respect to the internalization in LNCaP cells (> 50 fluorescence AU), indicating that, in PSMA-negative cells, the PSMA- targeting probes were mainly internalized by target-independent mechanisms. The residual cell-associated fluorescence of cells treated with probes or unconjugated dyes on ice was mainly undetectable. These results are reported in Table IV.

[0263] Table IV - Cellular uptake, expressed as normalized cell-associated fluorescence at 37°C after 4 hours: comparison of cell treatment in PSMA-positive LNCaP and in PSMA-negative PC-3 cells (Mean ± std dev).

[0264] Uptake competition experiments

[0265] The dependence of the internalization of the probes on PSMA targeting in LNCaP cells was assessed by cell treatment in presence of an excess of 2-PMPA, a well-known PSMA inhibitor. The cellular uptake of both targeting probes and corresponding unconjugated dyes was tested after 2 hours cell treatment at 37 °C, alone or in combination with 100X excess of 2-PMPA inhibitor ( / .e. 1 pM PSMA-targeting probe and 100 pM 2-PMPA), competing for the access to PSMA binding site. As illustrated in Figure 3, in this condition the cell-associated fluorescence of each probe decreased to values (mean), expressed as the percentage of the cell-associated fluorescence obtained in absence of 2-PMPA.

[0266] Therefore, the excess of 2-PMPA competitively reduced the uptake of all targetingprobes, indicating that their cellular internalization was mostly mediated by the interaction with PSMA protein. The remaining quote of cell-associated fluorescence in presence of the competitor might be ascribed to no-target-mediated uptake mechanisms (the same that are in place for the unconjugated dyes). Indeed, the uptake of unconjugated dyes was unaffected by the presence of the competitor, further indicating that, in the case of PSMA targeting compounds, the excess 2-PMPA blocked the cell-surface binding and the receptor-mediated internalization.

[0267] Example 8: Binding Affinity (Ka) to Human Serum Albumin (HSA)

[0268] 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. It was assessed by UV / VIS spectrophotometry after an ultrafiltration or peak-shift method.

[0269] Briefly, for the ultrafiltration method, optimal for compound with low affinity for HSA, 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, 3.81-10’4M), in presence of 50 pL from a 50 pM stock solution of the Test Articles (i.e. representative compound 2, 3 or 4), in a total volume of 0.525 mL. The samples were centrifuged (10,000 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 spectrophotometer at the maximum absorbance wavelength of each Test Articles.

[0270] The peak shift method, optimal for compounds which strongly interact with HAS, was applied when the Test Article maximum peak of absorbance in PBS significantly differed (at least 10 nm) from a solution containing HSA: to this purpose, the absorbance spectra of 50 pM solutions of Test Article (i.e. representative compound 1), in either PBS or PBS / 40 pM HSA were compared. In this condition, a series of HSA dilutions in PBS were prepared from a 0.5 M stock solution (0, IO’6, 5-10’6, IO’5, 2-10’5, 4-10’5, 6-10’5, 8-10’5, 10’4, 1.5-10’4, 2-10’4, 3-10-4, 4-10’4M), in presence of 50 pL from a 50 pM stock solution of the Test Article, in a total volume of 0.5 mL. The absorbance of each solution was calculated at the maximum absorbance peak obtained for the "0 HSA sample" (dye without HSA).

[0271] For both methods, the affinity constant (KA, M-1) was calculated by fitting the raw data with the following formula:

[0272] AA Ac ■ KRL[L] ■ Rtb KRL[L] + 1 where: AA / b = Absorbance measured (b = 1 cm)

[0273] KRL = KA calculated by fitting

[0274] Ae ■ Rt was calculated by fitting

[0275] [L] = Albumin concentration

[0276] In the peak-shift method, AA / b was obtained subtracting the absorbance of the control "0 HSA sample" to the absorbance of each other samples (AAX = Asampiei - Asampiex) .

[0277] Both methods have demonstrated to provide comparable results, but the measurement of the affinity constant is more precise when the suitable method is used as a function of the affinity level of the compound.

[0278] The results of the estimated association constants at equilibrium (KA) of the targeting probes are summarized in Table V.

[0279] Table V - Estimated association constant of equilibrium (KA) of representative compounds 1-4

[0280] Example 9: Tumor imaging efficacy

[0281] Optical Imaging (01) experiments on LNCaP tumor bearing animals were performed after administration of Compounds 1, 2, 3, and 4, all at a dose of 10 nmol / mouse in an administration volume of 0.1 mL (corresponding to about 5 mL / kg, considering a 20 g-mouse) at an injection rate of about 1 mL / min, by an Explorer Air® I (SurgVision) imaging system when the tumor reached at least 50 mm3volume.

[0282] For each group, in vivo OI experiments were performed at 2 hours after intravenous administration of the fluorescent compounds. During OI experiments animals were maintained under gas anesthesia. At the end of the in vivo experiments, 2 hours after treatment, the animals were sacrificed, tumor and organs were excised and ex vivo signals were measured. Regions of interest (ROIs) were drawn on the tumor and on a reference background healthy region (hind limb muscle) of the mouse for each fluorescence image at every time point to evaluate signal intensity in the tissues (expressed as counts). The ratio between the fluorescence signal in the tumor and background healthy tissue (TBR) was then calculated to assess the contrast. Tumor morphology and PSMA expression were confirmed through histological analysis on excised tumors. The results are displayed in Figure 4 and in Table VI, reporting the values of ex vivo TBR from samples collected at 2 hours after administration. All the compounds of the invention, tested at a dose of 10 nmol / mouse, showed selective accumulation in the tumor. Table VI - Values of ex vivo TBR (mean ± SD) at 2 hours after administration.

[0283] References:

[0284] 1. Davis MI, et al. Proc Natl Acad Sci USA 2005; 102(17): 5981-6

[0285] 2. Jones W, et al., Cancers 2020; 12(6): 1367

[0286] 3. Kaewput et al., J. Clin. Med. 2022; 11 : 2738

[0287] 4. EP3636635 Al

[0288] 5. Benesova et al., J.Nucl.Med. 2015; 56: 914-920

[0289] 6. Galerna HA et al., Eur J Surg Oncol. 2022; 48(4): 810-821

[0290] 7. Gioux S. et al., Mol Imaging 2010; 9(5): 237-255

[0291] 8. Bunschoten A. et al., Bioconjugate Chem. 2016; 27: 1253-1258

[0292] 9. WO2017 / 044584

[0293] 10. WO2017 / 184383

[0294] 11. W02009 / 026177

[0295] 12. W02010 / 108125

[0296] 13. T.W. Green and P.G.M. Wuts, Protective Groups in Organic Synthesis, Wiley, N.Y.

[0297] 2007, 4thEd., Ch. 5

[0298] 14. Mujumdar R.B. et al., Bioconjugate Chem. 1993; 4(2): 105-111

[0299] 15. Maresca K.P. et al., J. Med. Chem. 2009; 52(2): 347-357

Claims

CLAIMS1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,wherein each R1is independently a straight or branched C1-C10 alkyl substituted by a group - SO3H;R2is selected from -SO3H, a group -CONH-Y wherein Y is a straight or branched C1-C10 alkyl substituted with at least two hydroxyl groups, and a group of formula (II)R3is selected from hydrogen and a group phenyl or -O-phenyl, optionally substituted with -SO3H;L is a bond or a linker;T is a PSMA-targeting moiety of formula (III):wherein X is an amino acid or a derivative thereof; n is an integer equal to 0 or 1.

2. The compound of formula (I) according to claim 1, wherein X is selected from lysine, glutamic acid, 3-(2-furyl)-alanine and 2-(2'-propynyl)-alanine.

3. The compound of formula (I) according to claim 1, wherein L is a group of formula -NH- (CH2)P-CO- or a diradical of one or more moieties selected from the group consisting of an amino acid or a derivative thereof; a peptide comprising from 2 to 10 amino acids inL or D configuration; 4-aminomethylbenzoic acid, cysteic acid; a polyethylene glycol or a derivative thereof; amino-polyethylene glycol-carboxylic acid; trans-4- (aminomethyl)cyclohexanecarboxylic acid; diaminobutyric acid; diaminopropionic acid, and combinations thereof; or it is a group -L1-L2- wherein -Li- is a diradical of a diamine and -L2- is a diradical of a dicarboxylic acid; wherein p is an integer comprised between 1 and 20.

4. The compound of formula (I) according to claim 3, wherein L is selected from a group of formula -NH-(CH2)p-CO-; a polyethylene glycol of formula -NH-(O-CH2-CH2)p-CO-; and a diradical comprising from one to five amino acids, wherein p is an integer comprised between 1 and 20, or it is th5. The compound of formula (I) according to any of the preceding claims, wherein R2is - SO3H.

6. The compound of formula (I) according to any of the preceding claims, wherein R2is a group -CONH-Y, wherein Y is selected from the group consisting of7. The compound of formula (I) according to claim 6 wherein Y is a group (ii), preferably the stereoisomer thereof8. The compound of formula (I) according to any preceding claim wherein both R1are agroup -(CH2)4-SO3H.

9. The compound of formula (I) according to claim 1, which is represented by the formulawherein R1, R2, L and T are as defined in claim 1.

10. The compound of formula (I) according to claim 1, which is represented by the formula (lb)wherein R1, R2, L and T are as defined in claim 1.

11. The compound of formula (I) according to claim 1 for use as fluorescence contrast agent for the detection and demarcation of tumor tissue during diagnostic, interventional imaging and intraoperative procedures.

12. The compound of formula (I) for use according to claim 11 wherein the detection and demarcation of the tumor tissue is carried out under NIR radiation .

13. The compound of formula (I) for use according to claim 11, wherein said tumor is 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, uterinecancer, thyroid cancer, liver cancer, and colorectal cancer, including both primary tumors and regional and distant metastases.

14. A pharmaceutical diagnostic composition comprising a compound of formula (I) as defined in claim 1, or a salt thereof, and at least one pharmaceutically acceptable adjuvant, excipient or carrier or diluent.

15. A diagnostic kit comprising at least one compound of formula (I) as defined in claim1, or a pharmaceutical composition as defined in claim 14, together with additional adjuvants thereof for implementing a biomedical optical imaging application.

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