Psma-targeted fluorescent probes

Abstract

This invention relates to the field of optical imaging. More specifically, it relates to fluorescent probes capable of effectively targeting prostate-specific membrane antigen (PSMA) and comprising heptamethrin dye having near-infrared (NIR) emission. The invention also relates to methods for preparing these compounds, pharmaceutical compositions and kits for introducing these compounds, and methods for using them as optical diagnostic agents in the imaging or treatment of diseases, particularly prostate cancer.

Description

[0001] Invention Field

[0002] This invention relates to the field of optical imaging. More specifically, it relates to fluorescent probes capable of effectively targeting prostate-specific membrane antigen (PSMA) and comprising a heptamethine anthocyanin dye with near-infrared (NIR) emission. The invention also relates to methods for preparing these compounds, pharmaceutical compositions and kits for introducing these compounds, and methods for using them as optical diagnostic agents in the imaging or treatment of diseases, particularly prostate cancer. Background Technology

[0003] Prostate-specific membrane antigen (PSMA)

[0004] PSMA is a type II transmembrane glycoprotein of 750 residues (approximately 84 kDa) with a short N-terminal cytoplasmic tail, a single transmembrane helix, and an extracellular portion. It exists as a symmetrical homodimer with enzymatic activity and is known to possess N-acetylation, α-linked acid dipeptidase (NAALAD) and folic acid hydrolase (FOLH) activities, respectively hydrolyzing the γ-peptide bond between N-acetylaspartate and glutamate in the abundant neuropeptide N-acetylaspartate glutamate (NAAG) and the γ-glutamyl bond in pteroyl polyglutamate. The substrate-binding cavity is located deep within the PSMA structure and is formed by the action of three identified domains: a protease domain (coordinated to two zinc atoms); a apical domain; and a C-terminal dimerization domain (Davis MI et al., Proc. Natl. Acad. Sci. USA 2005; 102(17):5981-6). PSMA undergoes constitutive endocytosis from the plasma membrane via clathrin-coated pits, while ligand-induced internalization has been characterized after antibody or antibody fragment binding. In benign prostatic cells, PSMA is localized in the cytoplasm and apical side of prostatic epithelial cells (Jones W. et al., Cancers 2020; 12(6):1367). With malignant transformation, PSMA translocates from the cytoplasm to the luminal surface of the prostatic ducts, where it presents a large extracellular domain to its ligands. Since its ligands are internalized via endocytosis, it may possess transport functions.

[0005] PSMA is expressed in almost all prostate cancers, with increased expression in poorly differentiated, metastatic, and hormone-resistant cancers. Its expression level is approximately 1000 times higher than the physiological levels found in other tissues such as the kidneys, small intestine, or brain. PSMA is abundantly expressed in all stages of prostate cancer, is present on the cell surface, and is not released into circulation.

[0006] PSMA is also expressed in other cancers, due to overexpression in cancer-associated angiogenesis structures such as bladder cancer, pancreatic cancer, lung cancer, and renal cell carcinoma. Physiological expression of PSMA has been confirmed at lower levels in other tissues, including the healthy prostate, duodenum, kidney, salivary and lacrimal glands, neuroendocrine system, proximal renal tubules, liver, and brain. These characteristics and expression patterns make it a valuable target for molecular imaging in diagnosis, staging, follow-up, surgery, and treatment (Kaewput et al., J. Clin. Med. 2022; 11:2738). Therefore, targeting PSMA with specific tools may open up important new avenues for improving conventional therapies for malignancies, as well as early diagnosis and prognosis.

[0007] Currently developed classes of PSMA-targeted agents for imaging (e.g., PET imaging) and / or therapeutic applications in patients with prostate cancer include monoclonal antibodies and small compounds. Some agents belonging to these classes of compounds have been commercialized or are currently in clinical development. In particular, 111 In-Carolomab-Penditin ( 111 In-capromab-pendetide)(ProstaScint ® AYTU Bioscience Inc. was the first commercially available anti-PSMA antibody to target PSMA to be approved by the U.S. FDA in 1996.

[0008] However, although monoclonal antibodies are the ligands of choice for most tumor-targeting applications, their use has several drawbacks: in fact, they are characterized by slow and ineffective tumor penetration and a long delay between injection and imaging due to their long half-life, which leads to high accumulation in inflamed tissues and significant radiation exposure. Furthermore, they may be immunogenic, hindering repeated administration for routine diagnostic procedures.

[0009] These problems can be circumvented by using small molecules, which show the same precise localization of prostate cancer lesions, but with faster tumor uptake and excretion, thus reducing radiation exposure and allowing physicians to obtain diagnostic information much more quickly (Jones W. et al.). ， Cancers 2020; 12(6):1367).

[0010] Among small compounds, currently characterized pharmacological inhibitors of PSMA include: phosphonate-based derivatives, such as (phosphonomethyl)glutarate (2-PMPA); urea-based derivatives, like N-acetylaspartoylglutamate (NAAG) analogs, in which two amino acids (glutamate (E) and / or lysine (K)) are linked by a urea bond via their -NH2 groups; thiol-based derivatives; and hydroxamic acid derivatives. In recent decades, several urea-based derivatives (e.g., EuK, EuE, and other “EuX” groups) have been developed as highly effective inhibitors of PSMA (EP3636635 A1).

[0011] In many cases, EuK and EuE binding motifs have been functionalized with several spacer groups to enhance their bioactivity. In particular, the introduction of nonpolar chains has been shown to improve the interaction between the targeting motif and the enzyme's hydrophobic pocket: one example being in Bene... The use of the peptide mimic glutamic acid-urea-lysine-3-(2-naphthyl)-alanine-tranexamic acid is described in ová et al., J. Nucl. Med. 2015;56:914-920.

[0012] The initial inhibitors were mainly used as 68 Ga-labeled radiotracers were developed and are considered a breakthrough due to their superior characteristics, such as high tumor contrast. [Approved by the US FDA] 68 Ga]Ga-PSMA-11 and [ 18 F]F-DCFPyL was used for positron emission tomography (PET) imaging to identify suspected metastases or recurrences in patients with prostate cancer. 68 Ga]Ga-PSMA-11 (also known as HBED-CC, HBED, PSMA-HBED, or Prostamedix™) is now the most widely used radiopharmaceutical for prostate PET-CT imaging, capable of detecting even very small metastases (Kaewput et al., J. Clin. Med. 2022; 11:2738).

[0013] In many cases, prostate cancer surgery can serve as a compromise between complete tumor removal and preservation of essential structures, improving the diagnostic accuracy of prostate cancer detection. However, prostate surgery relies entirely on white light endoscopy, which does not allow for sensitive and specific local and regional visualization of the extent and presence of the tumor. In fact, this limited ability to differentiate between cancerous and non-cancerous tissue during surgery explains the high recurrence rate (30-40%) in patients with locally advanced cancer. Accurate real-time identification of prostate cancer during surgery, such as using near-infrared (NIR) fluorescence, could enhance the detection of tumor tissue during surgery and enable complete tumor removal while avoiding damage to essential structures.

[0014] Fluorescent labeling

[0015] A fluorophore (or dye) is a chemical entity that absorbs photons of a specific wavelength upon photoexcitation and, depending on quantum efficiency, typically re-emits some of that energy at longer wavelengths. In particular, cyanine dyes are fluorescent organic molecules characterized by a delocalized electron system spanning a polyacetylenic bridge and confined between two nitrogen atoms. Due to their favorable optical properties, low toxicity, and good solubility in aqueous media, cyanine dyes, especially those emitting in the near-infrared (NIR) region (700–900 nm), can be used as contrast agents for biomedical imaging due to their high penetration depth.

[0016] Currently available clinical fluorescent probes, such as indocyanine green (ICG), have limited utility for imaging tumor tissues due to their distribution in tissues via a combination of passive diffusion and enhanced permeability and retention (EPR) effects. Typically, this first-generation fluorescent contrast agent requires large doses (>1 mg / kg) for appropriate tumor visualization. Although ICG may be suitable for certain indications, visualization is hampered by nonspecific binding and lack of tumor specificity (Galema HA et al., Eur J Surg Oncol. 2022; 48(4):810-821). To date, various fluorescent conjugates consisting of targeting carriers (antibodies, peptides, particles, and other small molecules) coupled with fluorescent NIR dyes have been developed or are being explored and validated for locating and visualizing malignant lesions that interact with target cell surface proteins or the cancer cell microenvironment (Gioux S. et al., Mol Imaging 2010; 9(5):237-255). Their in vivo behavior may be strongly influenced by the biological properties of the fluorescent moiety. For example, small structural modifications of anthocyanin Cy5 strongly modulate the accumulation of the corresponding bioconjugates in both tumor and off-target tissues (Bunschoten A. et al., Bioconjugate Chem. 2016; 27:1253-1258). Fluorescent contrast agents with low nonspecific accumulation and high target tissue selectivity would be preferred for use in living organisms.

[0017] An example of anthocyanin dyes conjugated to a low molecular weight portion of PSMA is described in WO2017 / 044584 (On Target Laboratories), which in particular discloses the compound OTL-78, which is currently in clinical development and contains the NIR dye S0456, which is conjugated to the PSMA-targeting portion EuE at the meso position via a linker formed by 8-aminooctanoic acid and Phe-Tyr dipeptide.

[0018] However, centrally located couplings may suffer from higher instability due to the central substituent, which can be removed in vivo by reacting with endogenous nucleophiles (i.e., it is prone to in vivo nucleophilic substitution), leading to the loss of the targeting unit and potentially causing in vivo degradation and inactivation of the probe. 。 This drawback has been partially addressed by replacing the central CO bond with a C-C bond, which has higher in vivo stability. However, in this case, the water solubility of the dye is often significantly reduced due to the higher tendency to aggregate.

[0019] Other examples of fluorescent probes characterized by conjugation at the 1 or 3 position of indole are disclosed in patent applications WO2017 / 184383 (Intuitive Surgical Operations) and WO2009 / 026177 (Purdue Research Foundation), which disclose different PSMA-binding conjugates, in both cases containing a PSMA-binding ligand linked to a known NIR sulfocyanine dye.

[0020] Another major class of PSMA-targeting compounds also includes a few examples of conjugates with fluorescent dyes, disclosed in WO2010 / 108125 (J. Hopkins).

[0021] Unfortunately, the available dye libraries for targeting PSMA-expressing tumors are not extensive or diverse enough. Therefore, despite efforts to date, there remains an urgent and unmet need to develop efficient fluorescent probes with high affinity and specificity for targeting PSMA for diagnostic and / or therapeutic purposes.

[0022] For the aforementioned probes, the compounds of the present invention are characterized by having different functionalization modes and / or Cy7 anthocyanins conjugated at alternative sites on the anthocyanin backbone to the PSMA-targeting portion. In addition to greater stability during their preparation, such probes have surprisingly demonstrated high affinity for the target PSMA and optimal intracellularization and tumor imaging efficacy. Invention Overview

[0024] Overall, the object of this invention is to provide fluorescent probes that can be used as optical imaging contrast agents and are designed to address the aforementioned problems by targeting PSMA. Specifically, this invention provides fluorescent probes that possess optimal properties for various molecular imaging applications, are capable of interacting with PSMA-expressing cells to accumulate in pathological cells and tissues, and specifically display fluorescent signals corresponding to pathological tissues with high signal-to-noise ratios and improved imaging efficacy at low mass doses.

[0025] Preferred conjugation sites for cyanine dyes disclosed in the art are indicated by the 1- or 3-position of a pseudoindole group of a linear or cyclohexenyl-centered Cy7 cyanine, for example, by conjugation at an alkyl chain functionalized with a carboxylic acid or ester. In some cases, Cy7 cyanine with a cyclohexenyl-centered Cy7 cyanine can also be conjugated at the central (meso) position of the heptamethrin skeleton, typically by attachment to a phenyl or phenoxy ring, which may be functionalized at the para-position with a carboxyl, carboxamide, or ester group, optionally by the insertion of a linker. These cyanine compounds generally have a symmetrical structure, with -SO3H, alkyl, or alkyl-SO3H groups at other positions.

[0026] In contrast, the fluorescent probe of the present invention comprises an asymmetric dye and is characterized in that the heptamethrin anthocyanin dye is conjugated to the PSMA targeting portion at different positions on the anthocyanin backbone, namely at the 5 position of at least one pseudoindole ring, providing strong binding to the target and unexpectedly exhibiting optimal in vitro and in vivo behavior, thus being particularly suitable for molecular imaging.

[0027] Specifically, among the several advantages that can be achieved by means of the compounds of the present invention, the following features can be emphasized, such as: high selectivity for uptake by target tissues and cells, low accumulation due to nonspecific interactions with other tissues, high solubility in water, and low binding to albumin.

[0028] Another aspect of the invention relates to fluorescent probes used as diagnostic reagents, particularly in methods of optical imaging of human or animal organs or tissues, wherein the imaging is tomographic imaging of organs, monitoring of organ function, including angiography, urinary tract imaging, bile duct imaging, neuroimaging, intraoperative cancer identification, fluorescence-guided surgery, fluorescence lifetime imaging, shortwave infrared imaging, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, openfield surgery, laser-guided surgery, or photoacoustic or sonofluorescence methods.

[0029] Furthermore, the present invention relates to methods for preparing the provided compounds and / or their pharmaceutically acceptable salts, and their use in the preparation of diagnostic agents.

[0030] According to another aspect, the present invention relates to pharmaceutically acceptable compositions comprising at least one compound of the invention or a pharmaceutically acceptable salt thereof, mixed with one or more physiologically acceptable carriers or excipients. The compositions are particularly suitable for use as optical imaging agents to provide useful imaging of human or animal organs or tissues.

[0031] In another aspect, the present invention relates to a method for optical imaging of body organs, tissues or regions using optical imaging techniques, the method comprising using an effective dose of the compound of the present invention.

[0032] Invention Description

[0033] Therefore, the first aspect of the present invention relates to compounds of formula (I) or pharmaceutically acceptable salts thereof.

[0034] (I)

[0035] in

[0036] Each R 1 Independently, straight-chain or branched C1-C chains substituted with the group -SO3H10 alkyl;

[0037] R 2 Selected from -SO3H, where Y is a straight-chain or branched C1-C chain substituted with at least two hydroxyl groups. 10 Alkyl groups -CONH-Y and (II) groups

[0038] (II);

[0039] R 3 Selected from hydrogen and phenyl or -O-phenyl groups optionally substituted with -SO3H;

[0040] L represents a bond or linker;

[0041] T represents the PSMA targeting portion of equation (III):

[0042] (III)

[0043] Where X is an amino acid or a derivative thereof; and

[0044] n is an integer equal to 0 or 1.

[0045] Preferably, X is an amino acid group selected from lysine, glutamic acid, etc., or a derivative thereof selected from 3-(2-furanyl)-alanine, 2-(2'-propynyl)-alanine, etc.

[0046] More preferably, X is lysine or glutamic acid. Even more preferably, it is lysine.

[0047] Preferably, L is of the formula -NH-(CH2). p -CO- group or a bimolecular group selected from one or more of the following: amino acids or derivatives thereof; peptides containing 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid; sulfoalanine; polyethylene glycol 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 -L1- is a bimolecular group of a diamine and -L2- is a bimolecular group of a dicarboxylic acid; wherein p is an integer between 1 and 20.

[0048] More preferably, the linker L is a group -NH-(CH2). p-CO- or a dimer selected from one or more of the following: amino acids such as, for example, glycine, alanine, β-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid, etc., or amino acid derivatives such as, for example, 3-(2-naphthyl)-alanine; peptides containing 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid; sulfoalanine; polyethylene glycol of the formula -NH-(O-CH2-CH2). p -or -NH-(O-CH2-CH2) p -CO- or its derivatives; 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-, where L1 is a diamine (e.g., selected from the formula -NH-(O-CH2-CH2)). p -NH-amino-polyethylene glycolamine, ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexamethylenediamine, etc.) bimolecular group, and L2 is a dicarboxylic acid (e.g., selected from succinic acid, glutaric acid, octanoic acid, adipic acid, etc.) bimolecular group; where p is an integer between 1 and 20.

[0049] More preferably, L is selected from the formula -NH-(CH2). p -CO- group; formula -NH-(O-CH2-CH2) p -CO- polyethylene glycol; and a bimolecular group containing one to five amino acids, wherein p is an integer between 1 and 20.

[0050] In another preferred embodiment, L is a combination of tranexamic acid and an amino acid derivative, such as a group of formula (L3), for example:

[0051] (L3),

[0052] Corresponding to, for example, in Bene The tranexamic acid-(3-(2-naphthyl)-alanine) linker described in Ová et al., J Nucl Med 2015;56: 914-920. The present invention also relates to a method for preparing compounds of formula (I) by means of a synthetic transformation step.

[0053] The present invention also includes compounds of formula (I), which are used as fluorescent agents for detecting tumor margins in fluorescence-guided surgery.

[0054] Attached Figure Description

[0055] Figure 1 The binding curves of representative compounds 1-4 of the present invention with PSMA-positive LNCaP cells and PSMA-negative PC-3 cells are shown.

[0056] Figure 2 The cellular uptake of compounds 1–4 (black bars) in LNCaP over time is shown on ice (where endocytosis is blocked) and at 37°C (the temperature at which internalization is permitted). For each compound, the corresponding uptake of the unconjugated dye is reported (striped bars).

[0057] Figure 3 Cellular uptake of representative compounds 1–4 in the presence or absence of competitive excess 2-PMPA is shown.

[0058] Figure 4 The tumor imaging efficacy of representative compounds 1-4 of the present invention, expressed as tumor-to-background ratio (TBR), was demonstrated in in vivo experiments on animals bearing LNCaP tumors.

[0059] definition

[0060] In this specification, and unless otherwise specified, the following terms and phrases as used herein are intended to have the following meanings.

[0061] The term "dibasic" refers to a chemical group in which hydrogen atoms at both ends of a molecule are removed to form a bond.

[0062] The expression "straight chain or branched chain C1-C" 10 "Alkyl" refers to an aliphatic hydrocarbon group, which can be straight-chain or branched, having 1 to 10 carbon atoms. For example, "C1-C8 alkyl" includes, within its meaning, a straight-chain or branched group containing 1 to 8 carbon atoms. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, pentyl, hexyl, etc. Unless otherwise specified, the straight-chain or branched alkyl group is a monovalent group. In some cases, it can be a "divalent" or "polyvalent" group, wherein two or more hydrogen atoms are removed from and substituted from the aforementioned hydrocarbon group, such as methylene, ethylene, isopropylene, etc. In such cases, the expression "straight-chain or branched C1-C8 alkyl" can be used. 10 Alkylene".

[0063] The term "hydroxyalkyl" refers to any of the corresponding alkyl chains in which one or more hydrogen atoms are replaced by hydroxyl groups.

[0064] The term "protecting group" (Pg) refers to a protective group used to maintain the function of the group to which it is attached. Specifically, a protecting group is used to protect amino, hydroxyl, or carboxyl functional groups. Suitable protecting groups may include, for example, benzyl, carbonyl such as formyl, 9-fluoromethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), isopropoxycarbonyl or allyloxycarbonyl (Alloc), alkyl such as tert-butyl or triphenylmethyl, sulfonyl, acetyl groups such as trifluoroacetyl, benzyl ester, allyl, or other substituents commonly used to protect such functional groups, which are well known to those skilled in the art (see, for example, the general reference TW Green and PGM Wuts, Protective Groups in Organic Synthesis, Wiley, NY, 2007, 4th edition, Chapter 5).

[0065] Furthermore, the present invention also relates to precursor or intermediate compounds suitable for preparing desired compounds of formula (I) or their salts. In such intermediates, any functional group, such as a carboxylic acid or formamide, can be protected with a suitable protecting group (Pg) as defined above, preferably with an alkyl or ester group. If necessary, during the preparation of compounds of formula (I), the hydroxyl group of the Y group can also be protected with a suitable protecting group (Pg) to form, for example, an acetoxy, alkoxy, or ester group.

[0066] The term "coupling agent" refers to a reagent used, for example, to form an amide bond between a carboxyl moiety and an amino moiety. The reaction can consist of two consecutive steps: activation of the carboxyl moiety, followed by acylation of the amino group with the activated carboxylic acid. Non-limiting examples of such coupling agents are selected from: carbodiimides, such as N,N′-diisopropylcarbodiimide (DIC), N,N′-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC); phosphonium reagents, such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BO6). P), (benzotriazol-1-yloxy)tripyrrolidinyl phosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy-tripyrrolidinyl phosphonium hexafluorophosphate (PyAOP), [cyano(hydroxyimino)ethyl acetate-O2]tri-1-pyrrolidinyl phosphonium hexafluorophosphate (PyOxim), bromotripyrrolidinyl phosphonium hexafluorophosphate (PyBrOP), and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one (DEPBT); and ammonium / ureonium-ammonium reagent Agents, such as N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)ureonium tetrafluoroborate (TBTU), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)ureonium hexafluorophosphate (HBTU), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)ureonium hexafluorophosphate (HATU), O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethylureonium hexafluorophosphate (HCTU), 1-[1- [(cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-ureon hexafluorophosphate (COMU), 2-(2,5-dioxopyrrolidone-1-yl)-1,1,3,3-tetramethylisoureon tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(N-succinimide)ureon hexafluorophosphate (HSTU), and fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TFFH) or other compounds well known to those skilled in the art.

[0067] The term "activated carboxylic acid" refers to a derivative of the carboxyl group that is more susceptible to nucleophilic attack than a free carboxyl group; suitable derivatives may include, for example, acid anhydrides, thioesters, acyl halides, NHS esters, and sulfonated NHS esters.

[0068] The terms “part” or “residue” used here are intended to define the remaining part of a given molecule once it has been properly linked or conjugated to the rest of the molecule, either directly or by means of a suitable linker.

[0069] The term "imaging agent" refers to a detectable entity that, when used in combination with suitable diagnostic imaging techniques, can be used for the in vitro imaging of biological elements (including cells, bodily fluids, and biological tissues derived from living mammalian patients, preferably human patients) and human organs, regions, or tissues. 、 Visualization or detection in vitro or in vivo.

[0070] Preferably, the fluorescent probes of the present invention can selectively bind to tumor cells or tissues expressing PSMA. Specifically, they can bind to 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, kidney cancer, uterine cancer, thyroid cancer, liver cancer, and colorectal cancer, or diseases related to cancer-associated angiogenesis. Furthermore, the fluorescent probes of the present invention can target the metastasis of the aforementioned cancers in tissues and organs different from their primary origin. Moreover, the fluorescent probes of the present invention can target precancerous lesions and dysplasia in different tissues and organs.

[0071] Target portion (T)

[0072] According to the present invention, the targeting group (T) is a molecule that binds to a PSMA biological target with specific selectivity and promotes the accumulation of contrast agents in specific tissues or body sites expressing PSMA proteins, thereby allowing for the detection and imaging of cancer, particularly prostate cancer. Generally, it is represented by natural or synthetic molecules used in biological systems.

[0073] Such specific binding can be achieved by ligands, such as small molecules, proteins, peptides, peptide mimics, enzyme substrates, antibodies, or fragments or aptamers thereof, interacting with specific biological targets expressed on the surface of target tissues or cells.

[0074] Preferably, such a targeting moiety is represented by a small molecule. Among the known pharmacological inhibitors of PSMA that have been characterized so far, the following categories can be mentioned: phosphonate-based derivatives, such as (phosphonomethyl)glutarate (2-PMPA); urea-based derivatives, like N-acetylaspartic glutamate (NAAG) analogs, in which two amino acids (glutamate (E) and / or lysine (K)) are linked by a urea bond through their -NH2 groups; thiol-based derivatives; and isohydroxamic acid derivatives.

[0075] In a preferred embodiment, the targeting portion is represented by the carrier glutamate-urea-lysine (EuK) or other PSMA-binding carriers of the formula “EuX” as described in EP3636635A1, where glutamate is linked to another amino acid or analog via a bridging urea. For example, it can be represented by EuFA (glutamate-urea-3-(2-furanyl)-alanine), EuPG (glutamate-urea-2-(2'-propynyl)-alanine), EuE (glutamate-urea-glutamate), or other urea-based peptide mimics.

[0076] Connecting base (L)

[0077] According to the present invention, L is an optional linker that separates the PSMA targeting ligand from the dye.

[0078] The presence of a linker is particularly important for some implementations where there is a risk of adverse interactions between the ligand (e.g., a urea-based peptide mimic inhibitor) and the dye. Furthermore, the presence of a linker can be advantageous when the dye is relatively large and may interfere with the binding of the targeting moiety to the target site.

[0079] Linker groups can be flexible (e.g., containing straight-chain alkyl chains) or rigid (e.g., containing amino acids with aryl groups), causing the dye to be oriented away from the target. Linker groups can also alter the pharmacokinetics and metabolism of formula (I) conjugates used as imaging agents in living organisms.

[0080] Hydrophilic linkers can reduce interactions with plasma proteins, shorten blood circulation time, and promote excretion. For example, if the linker is a polyethylene glycol (PEG) moiety, the pharmacokinetics and blood clearance of the imaging agent in vivo may be altered. In such embodiments, the linker can improve the clearance of the imaging agent from background tissues (i.e., muscle, blood), resulting in better diagnostic images due to the high contrast between the target and the background. Furthermore, the introduction of specific hydrophilic linkers can shift the elimination of contrast agents from the liver to the kidneys, thereby reducing systemic retention.

[0081] Therefore, in a preferred embodiment, the linker (when present) is the group -NH-(CH2). p -CO- or a dimer selected from one or more of the following: amino acids such as, for example, glycine, alanine, β-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid, etc., or amino acid derivatives such as, for example, 3-(2-naphthyl)-alanine; peptides containing 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid; sulfoalanine; polyethylene glycol of the formula -NH-(O-CH2-CH2). p -or -NH-(O-CH2-CH2) p-CO- or its derivatives; amino-polyethylene glycol-carboxylic acid; trans -4-(aminomethyl) Cyclohexanecarboxylic acid (tranexamic acid); diaminobutyric acid; and diaminopropionic acid and combinations thereof, or it is a group -L1-L2-, where L1 is a diamine (such as, for example, -NH-(O-CH2-CH2)). p -NH-amino-polyethylene glycolamine) dimethyl or ethylenediamine 、 The dimer of propylenediamine, putrescine, spermidine, spermine or hexamethylenediamine, etc., and L2 is a dimer of dicarboxylic acid (such as succinic acid, glutaric acid, octanoic acid, adipic acid, etc.); where p is an integer between 1 and 20.

[0082] More preferably, L is selected from the group -NH-(CH2). p -CO-; formula -NH-(O-CH2-CH2) p -CO- polyethylene glycol; and a bimolecular group of one to five amino acids, where p is an integer between 1 and 20.

[0083] In another preferred embodiment, L is a combination of tranexamic acid and an amino acid derivative, such as, for example, in Bene The tranexamic acid-(3-(2-naphthyl)-alanine) linker described in ová et al., J Nucl Med 2015;56: 914-920.

[0084] The compounds of formula (I) above may have one or more asymmetric carbon atoms (also referred to as chiral carbon atoms), and may thus produce diastereomers and optical isomers. Unless otherwise specified, the invention also includes all such possible diastereomers and their racemic mixtures, their substantially pure enantiomers, all possible geometric isomers and their pharmaceutically acceptable salts.

[0085] The present invention also relates to compounds of formula (I) above, wherein R 1 R 2 and / or R 3 The sulfonyl group can be in the form of a negatively charged ion or a pharmaceutically acceptable salt.

[0086] Detailed description of the implementation plan

[0087] In a preferred embodiment, the present invention relates to R 2 Compounds of formula (I) with the group -SO3H.

[0088] In another preferred embodiment, the invention relates to R 2 It is a compound of formula (I) with the group -CONH-Y, wherein Y is selected from

[0089] (i) (ii) (iii)

[0090] (iv) (v) and (vi).

[0091] More preferably, the present invention relates to R 2 The compound is of formula (I) with the group -CONH-Y and Y being a group of formula (ii) as defined above. Preferably, group (ii) has the following stereochemical configuration, which is obtained by using D-glucosamine in the preparation of the compound:

[0092] (ii-a)

[0093] In another preferred embodiment, the invention relates to R 2 Compounds of formula (I) with group (III) as defined above. More preferably, R 2 It is a group of the following formula (IIIa), where L is as defined above:

[0094] (IIIa).

[0095] Preferably, L is a bond or group -NH-(CH2)5-CO-.

[0096] More preferably, L is represented by equation (L3).

[0097] (L3).

[0098] In another preferred embodiment, the two R 1 All of them are the group –(CH2)4-SO3H.

[0099] In a preferred embodiment, n is 1, and R 3 The -O-phenyl group substituted with -SO3H is represented by the following formula (Ia).

[0100] (Ia),

[0101] Where R 1 R 2 L and T are as defined above.

[0102] In another preferred embodiment, n is 0 and R 3 It is hydrogen, as represented by the following formula (Ib).

[0103] (Ib)

[0104] Where R 1 R 2 L and T are as defined above.

[0105] A more preferred embodiment of the present invention relates to a compound represented by the following formula (Ic).

[0106] (Ic).

[0107] The particularly preferred and representative compounds of the present invention are those of formula (I) listed in Table I.

[0108] Table I – Representative compounds of formula (I)

[0109]

[0110] The present invention also relates to a method for synthesizing compounds of formula (I) prepared as described below.

[0111] The compounds of the present invention can be used as imaging agents for detecting tumors in both humans and animals. Therefore, the present invention provides compounds of formula (I) as defined above, which are used as fluorescent probes for the detection and demarcation of tumor tissue during diagnostic, interventional imaging, and intraoperative procedures, particularly wherein the tumor is a tumor exhibiting elevated or altered PSMA expression. Preferably, the subject being imaged is a human.

[0112] The present invention also provides compounds of formula (I) which are used as fluorescent probes as defined above, wherein the detection and demarcation of tumor tissue are performed under NIR radiation.

[0113] Preferably, the fluorescent probe of the present invention can selectively connect to cells or tissues of tumors expressing PSMA, such as prostate cancer or tumors 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, kidney cancer, uterine cancer, thyroid cancer, liver cancer, and colorectal cancer, including primary tumors and both regional and distant metastases.

[0114] The probe of the present invention is capable of identifying lesion tissue in a subject in vivo. This can be achieved by administering a compound of formula (I) as defined above and irradiating the body of the subject containing lesion tissue in vivo with light having at least one excitation wavelength in the NIR range of about 650 nm to about 850 nm. The location and / or surface area of ​​the lesion tissue in the subject are determined by directly observing the fluorescence emitted by the administered compound in response to at least one excitation wavelength, which specifically binds to the lesion tissue at the body site.

[0115] Specifically, the present invention also provides a compound of formula (I) as an imaging agent in a method for detecting the possible presence of a disease in a subject, the method comprising the following steps:

[0116] - A certain amount of the compound of formula (I) as defined above shall be administered to a subject requiring diagnosis, within the time and conditions that allow the compound to bind to cells expressing PSMA;

[0117] - Measure the signal from the compound in the biological sample;

[0118] - The signal is compared with at least one control dataset containing signals from compounds of formula (I) that have been in contact with biological samples that do not contain the target cell type, to indicate the possible presence of disease.

[0119] The present invention also provides a compound of formula (I) as an imaging agent in a method for imaging tissues and cells, the method comprising the following steps:

[0120] - To contact the tissue or cells with a compound of formula (I) as defined above;

[0121] - Irradiate the tissue or cells at wavelengths absorbed by the imaging agent;

[0122] - Near-infrared emission was detected using a fluorescence camera.

[0123] Specifically, in a preferred embodiment, the present invention provides a method for performing image-guided surgery on a subject, the method comprising the following steps:

[0124] - Apply a composition comprising a compound as defined above (I) under conditions and time sufficient to allow the compound to accumulate at a given surgical site.

[0125] - Irradiate and visualize the compound using near-infrared light;

[0126] - Surgical excision and / or back-table fluorescence-guided imaging of areas that fluoresce under near-infrared light excitation.

[0127] Furthermore, the present invention relates to compounds of formula (I) as imaging agents used in the methods described above.

[0128] Another aspect of the invention relates to pharmaceutical compositions 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.

[0129] Another aspect of the invention relates to a diagnostic kit comprising at least one compound of formula (I) as defined above, or a pharmaceutical composition thereof. Furthermore, the kit may contain additional adjuvants for performing biomedical optical imaging applications. These adjuvants may be, for example, suitable buffer solutions, containers, detection reagents, or instructions for use. The kit preferably comprises all materials for intravenous administration of the compounds of the invention.

[0130] Based on the location of the disease to be treated and the suspected disease to be diagnosed, an effective amount of the compound of the present invention can be administered via various routes prior to the imaging procedure. For example, it can be administered to the organ or tissue to be imaged via local routes such as percutaneous, enteral routes such as oral, or parenteral routes such as intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous. In some embodiments, the compound of the present invention can be administered by local spraying or atomization of a pharmaceutical composition comprising the compound of the present invention and / or specifically formulated for this purpose.

[0131] The composition is administered at a dose that effectively obtains the desired optical image of a tumor, tissue, or organ. This dose can vary widely depending on, for example, the compound used, the tissue undergoing the imaging procedure, and the imaging device used. The exact concentration of the imaging agent in the pharmaceutical composition depends on the experimental conditions and desired results, but typically ranges from 1 pM to 0.1 mM. The optimal concentration is determined through systematic variation until satisfactory results and minimal background fluorescence are obtained. Once administered, the imaging agent of the invention is exposed to a light source or other form of energy that can penetrate the tissue layer. Preferably, the radiation wavelength or band matches the excitation wavelength or band of the photosensitizer and has low absorption by non-target cells and the remainder of the subject, including blood proteins. Typically, the optical signal can be detected by observation or instrumentation, and its response is related to fluorescence or light intensity, distribution, and lifetime.

[0132] The preparation of compounds of formula (I) as is or in the form of pharmaceutically acceptable salts represents another aspect of the invention. The compounds of the invention can be prepared, for example, according to the methods described in the experimental section. General teachings on the preparation of anthocyanin skeletons can be found in Mujumdar RB et al., Bioconjugate Chem. 1993; 4(2):105-111, which relates to the synthesis and labeling of sulfonylindocyanine dyes.

[0133] In some cases, due to the presence of different functional groups, such as carboxylic acid or amide groups, in the anthocyanins of this invention, it may be necessary to use protecting groups to guide the reaction at the desired functional group. Generally, special care is required when operating anthocyanins under the strong pH and temperature conditions necessary for removing the protecting groups, as the stability of the polyacetylenic backbone may be affected in some cases, while the dye undergoes severe degradation.

[0134] Contrary to expectations, the compounds of the present invention were found to be very stable at alkaline pH (i.e., at about pH 11-12), and no degradation or negligible degradation was observed during the removal of the protecting group.

[0135] Experimental Section

[0136] The invention and specific embodiments thereof described in the following sections are merely exemplary and should not be considered as limiting the invention: they show how the invention can be practiced and are intended to be illustrative rather than limiting the scope of the invention.

[0137] Materials and equipment

[0138] All commercially available reagents used in the synthesis were obtained from Sigma Aldrich and TCI and were used without further purification. Other known starting materials were prepared according to procedures described in the literature: for example, the Glu-urea-Lys (EuK) moiety was synthesized as described in Maresca KP et al., J. Med. Chem. 2009; 52(2): 347–357; the glutamic acid-urea-lysine-3-(2-naphthyl)-alanine-tranexamic acid moiety followed the Bene The procedure described by Ová et al., Journal of Nuclear Medicine; 56(6):914-920, is carried out by solid-phase peptide chemistry.

[0139] All reactions were monitored by HPLC-UV (Agilent Model 1100) and HPLC-UV-MS (Agilent Model 1260, MS detector single quadrupole Model 6120), equipped with absorption detectors or DAD detectors set at different wavelengths (column: YMC-Triart phenyl, 250 x 4.6 mm / S - 5 µm / 12 nm, eluent: 0.1% ammonium acetate or 10 mM ammonium formate and acetonitrile).

[0140] Use pre-filled KP-Sil columns or silicone C18 columns (Biotage) ® SNAP or SFÄR, Teledyne RediSepGold ® C18Aq), in an automated purification system (CombiFlash) ® Rapid chromatographic purification is performed on Rf+ (Teledyne ISCO), typically using methanol / ethyl acetate or water / acetonitrile gradients, respectively.

[0141] Absorption spectra were recorded using a UV-VIS spectrophotometer (Lambda 365, Perkin Elmer) with an acquisition range of 500–1000 nm. Emission spectra and absolute quantum yield were obtained using a FluoroLog-3 1IHR-320 spectrophotometer equipped with a 450 W xenon source and an F-3018 integrating sphere attachment (Horiba Jobin Yvon). Detection was performed using a photomultiplier tube (PMT-NIR, R5509) cooled detector.

[0142] Human prostate cancer LNCaP cells (CRL-1740, ATCC) and human prostate adenocarcinoma PC-3 cells (CRL-1435, ATCC) used in cell experiments were cultured in RPMI medium (Euroclone) supplemented with 10% HyClone Fetal Clone III (Euroclone), 2 mM glutamine (Sigma-Aldrich), 100 IU / mL penicillin, 0.1 mg / mL streptomycin, and 0.25 µg / mL amphotericin B (antibiotic-antifungal solution, Life Technologies) and Ham's F-12K nutrient mixture (Euroclone), respectively. Cells were grown at 37°C in a humidified atmosphere rich in 5% CO2. DPBS (Sigma-Aldrich) without MgCl2 and CaCl2 was used for cell washing.

[0143] Samples for cell experiments were analyzed using the Accuri™ C6 flow cytometer (BD Biosciences) according to the following general parameters: Event detection threshold: 2,000,000 for FSC-H; Gating of live cells based on physical parameters (reasonable FSC-A, low SSC-A, exclusion of doublets); At least 10,000 valid events per sample (i.e., within the established gate); Flow rate: "medium"; Event count / µL ≤ 1000; Excitation: 640 nm laser; Each sample was collected in the FL4 channel (780 / 60 nm filter).

[0144] To evaluate the in vivo efficacy of the compounds of this invention, in situ induced LNCaP prostate cancer was used as an animal model, due to the well-known overexpression of PSMA on prostate cancer cells. LNCaP cells were collected and washed twice with PBS. One million cells were resuspended in 20 μL of serum-free culture medium and injected into the left ventral lobe of the prostate of each 7-week-old male mouse under general anesthesia. Starting 14 days post-cell implantation, anatomical images were acquired at least twice weekly by magnetic resonance imaging (MRI) to monitor tumor development. The MRI acquisition protocol was as follows:

[0145] a) Locating sequences to verify the animal's correct location;

[0146] b) Optimize and adjust the scheme (baseline frequency, first-order and second-order shims, radio frequency pulses);

[0147] c) T2-weighted spin echo sequences (along axial, coronal, and sagittal geometry) are used to obtain a complete set of reference images for defining the geometry of subsequent sequences;

[0148] d) High-resolution T2-weighted spin echo image (TR = 2500 ms, TE = 30.15 ms, rare factor = 8, FOV = 35 mm x 35 mm, matrix size = 256 x 256, number of layers = 25, layer thickness 0.8 mm, average order = 4).

[0149] List of abbreviations

[0150]

[0151] Example 1: Synthesis of Compound 1

[0152] Preparation of intermediate compound (11)

[0153]

[0154] 3-Methylbut-2-one (1.5 mL, 0.014 mol) and sodium acetate (1.64 g, 0.020 mol) were added to a suspension of 4-hydrazinobenzenesulfonic acid (2.09 g, 0.011 mol) in glacial acetic acid (10 mL). After stirring at 110 °C for 4.5 hours, the orange solution was cooled to RT and precipitated in cold diisopropyl ether (100 mL). The solid was filtered under suction and dissolved in methanol along with SiO2. The methanol was removed, and the slurry in silica gel was loaded onto a silica gel column and eluted with 30% methanol in ethyl acetate to give intermediate 10 (2.46 g), a light brown solid.

[0155] Yield: 94%

[0156] HPLC purity: 90% 270 nm

[0157] MS: [M+H] + 240.1

[0158] The mixture of intermediate 10 (1.32 g, 5.51 mmol) and 1,4-butanesulfonyl lactone (0.84 mL, 7.58 mmol) in sulfolane (2 mL) was heated at 120 °C for 24 hours. The mixture was then cooled to RT, and cold ethyl acetate (50 mL) was added, followed by stirring in an ice bath for 1 hour. The precipitate was then filtered under suction and washed twice with cold ethyl acetate (2 × 50 mL). The crude product was dissolved in methanol, dried under vacuum, and passed through a pre-packed KP-Sil column (Biotage). ® The intermediate 11 (0.573 g) was purified by rapid column chromatography on a SNAP plate (60 g) and eluted with 50% methanol / ethyl acetate. The pink solid intermediate 11 was recovered.

[0159] Yield: 28%

[0160] HPLC purity: 98.3% at 270 nm

[0161] MS: [M+H] + 376.0

[0162] Preparation of intermediate compound (13)

[0163]

[0164] 3-Methylbut-2-one (146 mL, 0.130 mol) and sodium acetate (11 g, 0.130 mol) were added to a suspension of 4-hydrazinobenzoic acid (10 g, 0.053 mol) in glacial acetic acid (150 mL). After stirring at 135 °C for 3 hours, the brown solution was cooled to RT, concentrated under reduced pressure, and suspended in 30 mL of a 9:1 water / methanol mixture. The solid (intermediate 12) was filtered under suction and washed with another 40 mL of 9:1 water / methanol, then dissolved in 200 mL of saturated NaHCO3 solution and extracted with dichloromethane (3 × 125 mL). The organic layer was concentrated under reduced pressure, and the orange solid was dried in an oven at 45 °C for 18 hours to obtain 9.4 g of a pale pink solid.

[0165] Yield: 87%

[0166] HPLC purity: 98% at 270 nm

[0167] MS: [M+H] +204.8

[0168] The mixture of intermediate 12 (4.01 g, 19.7 mmol) and 1,4-butanesulfonyl lactone (2.4 mL, 23.5 mmol) in butyronitrile (4 mL) was heated at 120 °C for 54 h. The mixture was cooled to RT and cold acetone (100 mL) was added, and the mixture was stirred in an ice bath for 1 h. The precipitate was then filtered under suction and washed twice with cold ethyl acetate (2 × 50 mL). The crude product was dissolved in 0.1% ammonium acetate aqueous solution and subjected to a pre-packed C18 silica column (Biotage). ® Purification was performed by rapid column chromatography (SNAP, 60 g), eluting the desired product with water and removing byproducts with acetonitrile. Desalting was carried out 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), followed by washing with water (2 CV). The fraction containing the desired product was concentrated under reduced pressure and lyophilized to give 4.8 g of pink solid, corresponding to intermediate 13.

[0169] Yield: 72%

[0170] HPLC purity: 98% at 270 nm

[0171] MS: [M+H] + 342.1

[0172] Preparation of intermediate compound (14)

[0173]

[0174] 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 an 8:2 acetic anhydride / glacial acetic acid mixture (18 mL) at 55 °C. Then, 2-chlorocyclohexyl-1-ene-1,3-dicarboxaldehyde (287.22 mg, 1.664 mmol) was added, and the mixture was heated at 100 °C for 2.5 h in the dark. The mixture was cooled to RT, and the solvent was removed under reduced pressure. The viscous residue was partially dissolved in methanol (2 mL), stirred in an ice bath for 1 hour, and then precipitated in cold diethyl ether (150 mL). The precipitate was filtered under suction, dissolved in water, and precipitated on a pre-packed C18 silica column (Biotage). ® The intermediate compound 14 was purified by rapid column chromatography using a water-acetonitrile gradient (eluting anthocyanins with 15% acetonitrile) on a SFÄR 60 g plate. The intermediate compound 14 was freeze-dried to give 328 mg of green solid.

[0175] Yield: 29%

[0176] HPLC purity: 98.2% at 790 nm; 96% at 254 nm.

[0177] MS: [M+H] + 851.2

[0178] Preparation of intermediate compound (15)

[0179]

[0180] Intermediate 14 (328 mg, 0.385 mmol) was dissolved in DMSO (10 mL) and added dropwise to 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 overnight in the dark at RT. The mixture was then precipitated in cold ethyl acetate (225 mL) with stirring in an ice bath for 1 hour. The precipitate was filtered under suction, dissolved in water, rapidly acidified to pH 7.5 with dilute HCl, and then passed through a pre-packed C18 silica column (Biotage). ® The product was purified by rapid column chromatography (SFÄR 60 g) using a water / acetonitrile gradient (eluting anthocyanins with 10% acetonitrile). The fraction containing the pure product was concentrated under reduced pressure and lyophilized to give 242.75 mg of a green solid corresponding to intermediate compound 15.

[0181] Yield: 63.7%

[0182] HPLC purity: 99.1% at 790 nm; 97.4% at 254 nm.

[0183] MS: [M+H] + 989.1

[0184] Synthesis of Compound 1

[0185]

[0186] Intermediate 15 (40 mg, 0.0371 mmol) was dissolved in anhydrous DMF (7 mL), followed by the addition of HATU (17.67 mg, 0.0464 mmol), DIPEA (12.9 µL, 0.0742 mmol), and Intermediate 16, namely EuK(tBu)3 (21.76 mg, 0.0445 mmol). The dark green solution was stirred in the dark at RT for 1 hour, followed by the addition of cold ether (50 mL), and the mixture was stirred in an ice bath for 30 minutes. The precipitate (Intermediate 17) was filtered under suction and dissolved directly in TFA (1 mL). The brown solution was stirred in the dark at RT for 30 minutes, followed by the addition of cold ether (50 mL), and the mixture was stirred in an ice bath for 30 minutes. The precipitate was filtered under suction, dissolved in water, and the pH was immediately adjusted to 8.5 with 1 N NaOH. The solution was loaded onto a pre-packed C18 silica column (Biotage). ® The product was purified by rapid column chromatography (SFÄR 30 g) using a water-acetonitrile gradient (eluting anthocyanins with 10% acetonitrile). The fraction containing the pure product was concentrated under reduced pressure and lyophilized to give 32.86 mg of green solid.

[0187] Yield: 68%

[0188] HPLC purity: 99.6% at 780 nm; 98.7% at 254 nm.

[0189] MS: [M+2H] 2+ 645.7, [M-2H] 2- 643.8

[0190] Example 2: Synthesis of Compound 2

[0191] Preparation of peptide mimics glutamic acid-urea-lysine-3-(2-naphthyl)-alanine-tranexamic acid

[0192]

[0193] Intermediate 18a was obtained via a well-known procedure in solid-phase peptide chemistry, following Benedict's method. The procedure described by Ová et al. in 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 purification was performed on a Waters HPLC system equipped with an MS detector and a photodiode array detector. An Atlantis dC was used. 18OBD preparation column, 100 Å, 5 μm, 19 mm x 150 mm column, was used for analysis. Final compounds were analyzed using an Acquity QDa MS detector and a TUV detector, as well as a UPLC Kinetex. ® The analysis was performed using a UPLC-H-Class system with a 1.7 mm F5 100 Å 100 × 2.1 mm column. Solvent A consisted of an aqueous solution of 0.01% TFA, and solvent B consisted of an acetonitrile solution of 0.01% TFA. Fractions containing the desired pure product were combined, evaporated under reduced pressure, and lyophilized to recover 55 mg of white solid.

[0194] MS: [M+H] + 656.4

[0195] Synthesis of Compound 2

[0196]

[0197] In a 50 mL centrifuge tube, intermediate 15 (18 mg, 0.0182 mmol) was dissolved in anhydrous DMF (2 mL), followed by the addition of NMM (4.4 µL, 0.040 mmol) and TSTU (8.22 mg, 0.0273 mmol). The dark green solution was stirred at RT for 1 h. Cold ethyl acetate (25 mL) was added, and the suspension was centrifuged at 4010 rpm and 5 °C for 10 min. The organic layer was then decanted, and the solid was resuspended twice in ethyl acetate (10 mL) and centrifuged again. The crude solid was finally dried under a nitrogen atmosphere, dissolved in 10 mL of a DMF / DMSO mixture, and added to a solution of glutamic acid-urea-lysine-3-(2-naphthyl)-alanine-tranexamic acid (intermediate 18: 14 mg, 0.0182 mmol) and DIPEA (7.92 µL, 0.0455 mmol) in anhydrous DMF (1 mL). The solution was stirred overnight in the dark at RT. Then, cold ethyl acetate (25 mL) was added, and the precipitate was filtered off by suction. The crude material was dissolved in water and acetonitrile (pH adjusted to 7.5) and passed through a pre-packed C18 silica column (Biotage). ® The product was purified twice by rapid column chromatography using a 0.1% ammonium acetate / acetonitrile gradient (eluting anthocyanins with 20% acetonitrile) on a SFÄR 30 g column. The purified product was dissolved in water and purified by chromatography on a pre-packed C18 silica column (Biotage). ® The sample (30 g) was desalted with 0.1% formic acid (5 CV), washed with water (5 CV), and eluted with an acetonitrile-water gradient (eluting anthocyanins with 20% acetonitrile). The purified product was freeze-dried to give 7.04 mg of green solid.

[0198] Yield: 24%

[0199] HPLC purity: 98.7% at 780 nm; 98.0% at 254 nm.

[0200] MS: [M+2H] 2+ 813.7

[0201] Example 3: Synthesis of Compound 3

[0202] Preparation of ligand EuK-C6 (intermediate 20)

[0203]

[0204] EuK( t -Bu)3 (intermediate 16: 75 mg, 0.154 mmol) was dissolved in anhydrous DMF (5 mL), then HATU (88 mg, 0.23 mmol), NMM (34 µL, 0.308 mmol) and N- (tert-Butyloxycarbonyl)-6-aminohexanoic acid (39 mg, 0.169 mmol). The yellow solution was stirred at RT for 1 hour, 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 reduced pressure and dissolved directly in TFA (3 mL). The solution was stirred at RT for 3 hours, 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 then passed through a pre-packed C18 silica column (Biotage). ® The product was purified by rapid column chromatography (SFÄR 12 g) using a water / acetonitrile gradient (eluting the product with 10% acetonitrile). The fraction containing pure intermediate compound 20 was concentrated under reduced pressure and lyophilized to give 44 mg of white solid.

[0205] Yield: 52%

[0206] HPLC purity: 90% at 220 nm

[0207] MS: [M+ H] + 433.5.

[0208] Synthesis of Compound 3

[0209]

[0210] In a 50 mL centrifuge tube, intermediate 15 (23.6 mg, 0.0327 mmol) was dissolved in anhydrous DMF (3 mL), followed by the addition of NMM (7.9 µL, 0.0719 mmol) and TSTU (14.77 mg, 0.049 mmol). The dark green solution was stirred at RT for 1 h. Cold ethyl acetate (25 mL) was added, and the suspension was centrifuged at 4010 rpm and 5 °C for 10 min. The organic layer was then decanted, and the solid was resuspended twice in ethyl acetate (10 mL) and centrifuged again. The crude solid was finally dried under a 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 µL, 0.2289 mmol) in DMSO (2 mL). The solution was stirred at RT in the dark overnight. Then, 25 mL of cold ethyl acetate was added and the precipitate was filtered off under suction. The crude substance was dissolved in water (pH adjusted to 7.5) and purified twice by rapid column chromatography on a pre-packed C18 silica column (Biotage® SFÄR 30 g) using a water / acetonitrile gradient (eluting anthocyanins with 20% acetonitrile). The purified product was lyophilized to give 22.87 mg of a green solid.

[0211] Yield: 49.8%

[0212] HPLC purity: 99.1% at 780 nm; 97.6% at 254 nm.

[0213] MS: [M+2H] 2+ 702.3

[0214] Example 4: Synthesis of Compound 4

[0215] Preparation of intermediate compound (22)

[0216]

[0217] Concentrated H₂SO₄ (0.820 mL) was added to a solution of intermediate 13 (500 mg, 1.47 mmol) in methanol (8 mL), and the solution was refluxed for 2 hours. The mixture was cooled to RT and concentrated under reduced pressure, diluted with water (adjusted to pH 2 with 1 M NaOH), and purified by rapid column chromatography on a pre-packed C18 silica column (Biotage® SFÄR 60 g) using a water / acetonitrile gradient (eluting the desired compound with 15% acetonitrile). After lyophilization, intermediate 21 (468 mg) was obtained as a light brown solid.

[0218] Yield: 88%

[0219] HPLC purity: 99.3% at 270 nm

[0220] MS: [M+H] + 354.1

[0221] 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 mixture of acetic anhydride and glacial acetic acid (30 mL) at 50 °C. Then, 2-chlorocyclohexyl-1-ene-1,3-dicarboxaldehyde (251.67 mg, 1.441 mmol) was added, and the mixture was heated at 100 °C for 1 hour in the dark. The mixture was cooled to RT, and the solvent was removed under reduced pressure. The viscous residue was partially dissolved in methanol (2 mL), stirred in an ice bath for 1 hour, and then precipitated in cold diethyl ether (200 mL). The precipitate was filtered under suction, dissolved in water, and then precipitated in a pre-packed C14 container. 18 Purification was performed by rapid column chromatography on a silica column (Biotage® SFÄR 60 g) using a water / acetonitrile gradient (eluting anthocyanins with 20% acetonitrile). Intermediate compound 22 was freeze-dried to give 362.9 mg of a green solid.

[0222] Yield: 36.5%

[0223] HPLC purity: 98.6% at 790 nm; 100% at 254 nm.

[0224] MS: [M+H] + 831.4

[0225] Preparation of intermediate compound (23)

[0226]

[0227] Intermediate 22 (223.8 mg, 0.269 mmol) was dissolved in DMSO (25 mL) and added dropwise to 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, and then precipitated in cold ethyl acetate (200 mL). The precipitate was filtered under suction, dissolved in water, rapidly acidified to pH 8.5 with dilute HCl, and purified by rapid column chromatography on a pre-packed C18 silica column (Biotage® SFÄR 60 g) using a water-acetonitrile gradient (eluting anthocyanins with 12% acetonitrile). The fraction containing the pure product was concentrated under reduced pressure and lyophilized to give 210.6 mg of green solid.

[0228] Yield: 80%

[0229] HPLC purity: 99.9% at 790 nm; 99.9% at 254 nm.

[0230] MS: [M+H] + 967.1

[0231] Preparation of intermediate compound (25)

[0232]

[0233] Intermediate 23 (208.4 mg, 0.215 mmol) was dissolved in DMF (20 mL), followed by the addition of HATU (93.3 mg, 0.258 mmol) and DIPEA (78 µL, 0.451 mmol). The mixture was cooled in an ice bath, and a solution of D-glucosamine (46.75 mg, 0.258 mmol) in DMF (6 mL) was slowly added dropwise. The mixture was stirred in the dark at 0 °C for 1.5 h. The product was then precipitated in cold diethyl ether (50 mL) and stirred in an ice bath for 1 h. The precipitate was filtered under suction, dissolved in water, and then packaged in a pre-packed C14 container. 18 Purification was performed by rapid column chromatography on a silica column (Biotage® SFÄR 60 g) using a water / acetonitrile gradient (eluting anthocyanins with 15% acetonitrile). The fraction containing the purified product was concentrated under reduced pressure to give 176 mg of a green solid corresponding to intermediate 24.

[0234] Yield: 72%

[0235] HPLC purity: 99.2% at 790 nm; 61.7% at 254 nm.

[0236] MS: [M+H]+ 1130.8

[0237] Intermediate 24 (119 mg, 0.106 mmol) was dissolved in water (30 mL) and hydrolyzed at 40 °C for 5 hours, maintaining pH 12 by continuously adding 1 M NaOH (total 0.856 mL). The mixture was cooled to room temperature, the pH was adjusted to 8 with dilute HCl, and the aqueous solution was placed in a pre-filled C... 18 Purification was performed by rapid column chromatography on a silica column (Biotage® SFÄR 60 g) using a water-acetonitrile gradient (eluting anthocyanins with 8% acetonitrile). Fractions containing the pure product were combined, concentrated under reduced pressure, and lyophilized to give 88.1 mg of a green solid corresponding to intermediate 25.

[0238] Yield: 75%

[0239] HPLC purity: 99.97% at 756 nm; 99.5% at 254 nm.

[0240] MS: [M+H] + 1116.3

[0241] Synthesis of Compound 4

[0242]

[0243] Intermediate 25 (155.7 mg, 0.137 mmol) was dissolved in anhydrous DMF (50 mL), followed by the addition of NMM (60 µL, 0.548 mmol) and TSTU (82.49 mg, 0.274 mmol). The dark green solution was stirred at RT for 1.5 h. The product was precipitated in cold ethyl acetate (300 mL) and stirred in an ice bath for 1 h. The precipitate was filtered under suction, washed twice with ethyl acetate (10 mL), and dissolved in DMF (50 mL). A solution of 6-aminohexanoic acid (18.87 mg, 0.144 mmol) and DIPEA (60 µL, 0.343 mmol) in a 1:2 DMF / DMSO (10 mL) was added. The solution was stirred in the dark for 45 h. The product was then precipitated with a cold 1:1 ethyl acetate / ether mixture (500 mL), and after recovery from the funnel, it was precipitated in a pre-packed C14 container. 18 Purification was performed by rapid column chromatography on a silica column (Biotage® SFÄR 60 g) using a water-acetonitrile gradient (eluting anthocyanins with 10% acetonitrile). The purified product was lyophilized to give 99.7 mg of a green solid corresponding to intermediate 26.

[0244] Yield: 60%

[0245] HPLC purity: 96.8% at 780 nm; 95.3% at 254 nm.

[0246] Mass spectrometry: [M+H] + 1230.6

[0247] Intermediate 26 (113 mg, 0.0922 mmol) was dissolved in anhydrous DMF (15 mL), followed by the addition of TSTU (69.4 mg, 0.2305 mmol) and NMM (40.5 µL, 0.3688 mmol). The dark green solution was stirred at RT for 24 h. The product was precipitated in cold ethyl acetate (200 mL) with stirring in an ice bath for 1 h. 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 µL, 0.461 mmol) in DMF (10 mL) was added. The solution was stirred in the dark for 22 h. The product was then precipitated with cold diethyl ether (200 mL) with stirring in an ice bath for 1 h. The precipitate was filtered under suction, dissolved in water, and then precipitated in a pre-packed C14 container. 18 Purification was performed by rapid column chromatography on a silica column (Biotage® SFÄR 60 g) using a water / acetonitrile gradient (eluting the product with 8% acetonitrile). The purified product was lyophilized to give 64.3 mg of green solid.

[0248] Yield: 46%

[0249] HPLC purity: 98.7% at 780 nm; 99.7% at 254 nm.

[0250] MS: [M+H] + 1530.4

[0251] Example 5: Optical Characterization

[0252] The optical properties of the compounds of the present invention were characterized in vitro in water and in a clinical chemistry control serum (Seronorm, Sero SA) with similar chemical composition and optical properties to simulated human serum. All solutions were freshly prepared. The compounds of the present invention are characterized by a maximum absorbance in the range of about 770 nm to 810 nm.

[0253] Table I shows the maximum excitation and emission values ​​and fluorescence quantum yield (QY%) of representative compounds of formula (I) in water and Seronorm. Due to some interactions between the probe and serum proteins, the compounds of the present invention exhibit increased quantum yield and redshift of absorption and emission in Seronorm compared to water.

[0254] Table I - Maximum excitation / emission and quantum yield % for representative compounds of formula (I)

[0255]

[0256] Example 6: Binding affinity with cells expressing PSMA

[0257] After internal validation of PSMA expression at the cell surface in its physiological environment, LNCaP prostate cells were selected as an in vitro model to evaluate the specific binding of the compounds of this invention to PSMA. Indeed, both Western blotting and flow cytometry confirmed the constitutive expression of high levels of PSMA in LNCaP cells, estimating approximately 130,000 PSMA molecules per cell using DAKO QUIFIKIT (Agilent). Conversely, PC-3 prostate adenocarcinoma cells were used as a control in all experiments due to the lack of PSMA expression.

[0258] All probes were incubated on ice at incremental concentrations (range 0–4 µM) with PSMA-positive LNCaP cells and PSMA-negative PC-3 cells, and their corresponding unconjugated dyes were treated in the same way (the results were negligible). Cell-related fluorescence was collected by flow cytometry, and the binding constant at equilibrium was calculated by mathematical fitting. The observed plateau trend itself indicates that the probes specifically bind to the cell surface, while no saturation trend was observed for PSMA-negative PC-3 cells.

[0259] More specifically, using StemPro ® Accutase ® LNCaP or PC-3 cells were isolated using a cell dissociation reagent (Life Technologies), collected in DPBS, and counted. At least 2.10 5Each cell was placed in a 1.5 mL tube on ice and resuspended in 100 µL of cold FACS buffer (eBioscience™ flow cytometry staining buffer, Invitrogen) containing two-fold serial dilutions (4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.015, 0.008, 0.004, 0.002, 0.001 µM) of the test sample (e.g., representative compound 1, 2, 3, or 4). Unstained samples incubated with FACS buffer were used to record basal autofluorescence of the cells. After incubation (2 hours on ice in the dark), the cells were washed twice in 500 µL of cold FACS buffer by centrifugation (5 min, 4°C, 350 RCF), and the supernatant was discarded. The cell pellet was then resuspended in 100 µL of cold FACS buffer (or resuspended in an appropriate volume to obtain no more than 1000 recorded events per µL during FACS analysis) and analyzed using an Accuri™ C6 flow cytometer (BD Biosciences). Mean cell-related fluorescence was plotted as a percentage of concentration, and the dissociation constant (Kc) at equilibrium was inferred by mathematical fitting using GraphPad Prism v.9 software (single site, total). D Specifically, the equations used are as follows:

[0260]

[0261] in

[0262] Y is the main cell-associated fluorescence;

[0263] X represents the concentration of the test sample;

[0264] B max It exhibits the highest specificity binding, with the same unit as Y;

[0265] K D It is the equilibrium dissociation constant, with the same unit as X;

[0266] NS is the nonspecific binding slope, expressed as Y units divided by X units;

[0267] background This is the amount of nonspecific binding without the addition of the test sample or compound.

[0268] All compounds were analyzed in repeated independent experiments. Calculated K D The results are summarized in Table II, and also shown in the curves. Figure 1 middle.

[0269] Table II - Equilibrium binding constants (K0) of representative compounds of formula (I) to PSMA-positive LNCaP cells D )。KD Value nM represents the average value of KD obtained from repeated independent experiments, with SEM. .

[0270]

[0271] This assay confirmed that the compound of the present invention has a high affinity for PSMA-positive LNCaP cells, K D Below 20 nM. As expected, no specific binding was detected in PSMA-negative PC-3 cells, which exhibited a linear, unsaturated curve of cell-related fluorescence, characterized by relatively low cell-related fluorescence values ​​at increasing probe concentrations (see [link to study]). Figure 1 ).

[0272] Example 7: Cellular Uptake Assessment

[0273] Cell uptake assays were performed using flow cytometry to assess whether probe internalization occurred after PSMA was expressed on the target cell surface.

[0274] Various in vitro tests were conducted to understand the specific characteristics of the compounds of this invention:

[0275] 1) Time-dependent intake (time process);

[0276] 2) Incubate at physiological temperature (37°C) and endocytosis blocking temperature (on ice) to distinguish cell-associated fluorescence derived from internalized compounds from cell-associated fluorescence due to residual cell surface compounds;

[0277] 3) Take-up was performed in the presence of 2-PMPA with an excess of competing PSMA binding sites to verify whether probe take-up was mediated by binding to the target receptor.

[0278] The experiment was conducted according to the following general procedures.

[0279] Cells were seeded in 24-well plates: for PSMA-positive LNCaP cells, 1.7 × 10⁻⁶ cells were seeded. 5 1 cell / well, and for PSMA-negative PC3 cells 2.10 5 10 cells / well, inoculated two days before the experiment.

[0280] The test sample (e.g., representative compound 1, 2, 3, or 4) and the control (the corresponding unconjugated dye, i.e., intermediate compound 15 or 25) were diluted to a working concentration of 1 µM in serum-free medium containing 25 mM HEPES (pH 7.4). In the competition assay, 100X excess of 2-PMPA (Sigma-Aldrich, dissolved in water at a stock concentration of 2 mg / mL and stored at 4°C) was added to the same treatment.

[0281] Wash the cells twice in DPBS.

[0282] Unless otherwise specified, apply the treatment solution (250 µL / well) and incubate cells at 37°C in the dark for the indicated time. In subgroups of experiments, this is after incubation with the dye / probe at 37°C (the temperature permissible for endocytosis) or on ice (endocytosis is blocked). Cell-related fluorescence obtained after incubation at 37°C represents the sum (total) of residual cell surface-bound and internalized compounds, while fluorescence recorded after incubation on ice represents the share of cell surface-bound compounds only (residual).

[0283] At the end of the treatment, the cells were washed twice with cold DPBS and treated with 120 µL / well StemPro. ® Accutase ® Thermo-Fischer cell dissociation reagent separates the cells from the plate. A few minutes in an incubator at 37°C is sufficient to obtain a complete cell suspension.

[0284] Place the plate on ice and add 500 µL of cold FACS buffer to each well to dilute and inactivate the dissociation reagent. Collect the cell suspension from each well and transfer it to a 1.5 mL tube on ice.

[0285] Centrifuge the cells (5 min, 350 RCF, 4 °C). Then, discard the supernatant and resuspend the cell pellet in 100 µL of cold FACS buffer, or in an appropriate volume to obtain no more than 1000 recorded events per µL during FACS analysis.

[0286] At the end of the analysis, each sample was plotted as a graph, with the X-axis representing fluorescence intensity on a logarithmic scale (FL4-A) and the Y-axis representing counts. Typically, a Gaussian distribution of the cell population was observed. The extracted data were the mean fluorescence and CV% for each population. For those cases where the CV% was higher than 50%, a gating system was set on the fluorescence intensity axis to exclude the tails of the Gaussian distribution of the cell population; no more than 5% of all events in each sample were excluded, and in most cases less than 1%. The mean fluorescence of the resulting cell population was considered the fluorescence intensity value of the sample.

[0287] Each treatment was performed in two independent replicates, and the mean fluorescence intensity and the corresponding standard deviation were calculated in each replicate.

[0288] Calculate normalized cell-related fluorescence using the following formula:

[0289]

[0290] Time-dependent intake

[0291] Time-dependent uptake assays were evaluated in both PSMA-positive LNCaP and PSMA-negative PC-3 cells by flow cytometry, with cells treated at a probe concentration of 1 µM for 1 to 4 hours. The experiments were performed on ice and at 37°C. At physiological temperature (37°C), observations from 1 to 4 hours showed that the conjugated compound of the present invention was internalized in LNCaP cells, resulting in an increase in total cell-related fluorescence over time. An increase in total cell-related fluorescence was observed after treatment with the corresponding unconjugated dye, but this increase was negligible compared to the increase driven by PSMA targeting, indicating that no target-mediated internalization mechanism occurred. Residual cell-related fluorescence in probe-treated LNCaP cells on ice remained substantially constant over time, as expected under conditions where endocytosis was blocked, while binding of the unconjugated dye was largely undetectable (see [link to relevant documentation]). Figure 2 ).

[0292] To further evaluate the cellular behavior of the compounds of this invention, several parameters, such as internalization slope, internalization percentage, and targeting advantage, were estimated from time-dependent uptake results.

[0293] The internalization slope was calculated by applying a linear fit to cell-related fluorescence values ​​over time obtained after cell incubation at 37°C (simple linear regression was performed using GraphPad Prism v.9 software; fluorescence relative to uptake time).

[0294] The percentage of internalization was calculated by taking into account the total cell-related fluorescence (representing the sum of residual cell surface-bound compounds and internalized compounds) recorded after treating cells at 37°C for 2 hours and the cell-related fluorescence (representing the residual cell surface-bound fraction) recorded after treating cells on ice.

[0295] Targeting advantage represents the ratio between total cell-related fluorescence (CRF) four hours after treatment with the targeting probe and the corresponding unconjugated dye at 37°C. Notably, this time point was chosen for calculating the targeting advantage parameter because the CRF recorded by the unconjugated dye was well distinguishable from that of untreated cells at 4 hours of treatment.

[0296] The results highlight that all probes tend to accumulate in LNCaP cells over time, exhibiting similar internalization rates. Considering the percentage of internalization, all probes showed a higher percentage (>50%) of internalized probes compared to residual probes bound to the cell surface. The targeting advantage (fold relative to dye) parameter, representing the cellular uptake advantage of PSMA-targeting probes relative to their corresponding unconjugated dyes, shows that all probes are preferentially internalized in LNCaP cells via target-mediated mechanisms (see Table III).

[0297] Table III – Internalization potential (average) of representative compounds of formula (I) ±standard deviation)

[0298]

[0299] In PSMA-negative PC-3 cells, uptake of both the PSMA-targeting probe and the unconjugated dye was at least 30-fold lower. Specifically, after 4 hours of treatment, normalized cell-associated fluorescence (CAL) of all probes was comparable to that of the unconjugated dye and significantly lower (< 4 AU) than that of internalization (> 50 AU) in LNCaP cells, indicating that in PSMA-negative cells, the PSMA-targeting probe is internalized primarily through a target-independent mechanism. Residual CAL was largely undetectable in cells treated on ice with either the probe or the unconjugated dye. These results are reported in Table IV.

[0300] Table IV - Cellular uptake, expressed as normalized cell-associated fluorescence after 4 hours at 37°C: PSMA positive Comparison of cell treatments in LNCaP and PSMA-negative PC-3 cells (mean ± standard deviation) .

[0301]

[0302] Intake competition experiment

[0303] The dependence of probe internalization on PSMA targeting was assessed in LNCaP cells by treating cells with an excess of 2-PMPA (a well-known PSMA inhibitor). Cellular uptake of both the targeting probe and the corresponding unconjugated dye was tested after treating cells for 2 hours at 37°C, alone or in combination with a 100X excess of a 2-PMPA inhibitor competing for PSMA binding site accessibility (i.e., 1 µM PSMA-targeting probe and 100 µM 2-PMPA). Figure 3 As shown, under these conditions, the cell-associated fluorescence of each probe decreased to the value (average), which is expressed as a percentage of cell-associated fluorescence obtained in the absence of 2-PMPA.

[0304] Therefore, excess 2-PMPA competitively reduced the uptake of all targeting probes, indicating that their internalization is primarily mediated by interaction with PSMA proteins. In the presence of a competitor, the remaining share of cell-associated fluorescence could be attributed to non-target-mediated uptake mechanisms (also present in unconjugated dyes). Indeed, the uptake of unconjugated dyes was unaffected by the presence of a competitor, further suggesting that, with respect to PSMA-targeting compounds, excess 2-PMPA blocks cell surface binding and receptor-mediated internalization.

[0305] Example 8: Binding affinity (K) with human serum albumin (HSA) a )

[0306] The binding affinity of the probe of this invention to human serum albumin (HSA) was determined to assess the influence of structural features on its albumin-binding properties. Evaluation was performed by UV / VIS spectrophotometry following ultrafiltration or peak shifting methods.

[0307] In short, for the optimal ultrafiltration method for compounds with low affinity for HSA, HSA (A9511, Sigma-Aldrich) is prepared as a 0.5 M stock solution in PBS to obtain a series of dilutions in PBS (0, 9.5 M, 2.1 M). -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 The total volume was 0.525 mL, with 50 µL of 50 µM test sample (i.e., representative compound 2, 3, or 4) in stock solution. The samples were centrifuged (10,000 g, 25 °C, for 30 min) in a Microcon apparatus (10 kDaMW cutoff, Amicon Ultra-0.5 centrifuge filter unit with Ultracel-10 membrane), and the absorbance of the filtrate was measured using a spectrophotometer at the maximum absorption wavelength for each test sample.

[0308] When the peak absorbance of the test sample in PBS differs significantly (by at least 10 nm) from that in the HSA-containing solution, the optimal peak shift method for compounds strongly interacting with HSA is employed. For this purpose, the absorbance spectra of a 50 µM test sample (i.e., representative compound 1) in PBS or a PBS / 40 µM HSA solution were compared. Under these conditions, a series of dilutions of HSA in PBS (0, 10, ...) were prepared from a 0.5 M stock solution. -6 5.10 -6 10 -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 4.10-4 M), which is a total volume of 0.5 mL in the presence of 50 µL of 50 µM test sample stock solution. The absorbance of each solution was calculated at the maximum absorbance peak obtained for the "0 HSA sample" (dye without HSA).

[0309] For both methods, the affinity constant (K) is calculated by fitting the original data using the following formula. A M -1 ):

[0310]

[0311] in:

[0312] ΔA / b = Measured absorbance (b = 1 cm)

[0313] K RL =K calculated through fitting A

[0314] Δε · R t It is calculated through fitting.

[0315] [L] = albumin concentration

[0316] In the peak shift method, ΔA / b is obtained by subtracting the absorbance of each other sample from the absorbance of the control "0 HSA sample" (ΔAX = A). 样品1 – A 样品X ).

[0317] Both methods have shown to provide comparable results, but the measurement of the affinity constant is more accurate when the appropriate method is used according to the affinity level of the compound.

[0318] Table V summarizes the estimated equilibrium association constant (K) of the target probe. A The result of ).

[0319] Table V - Estimated equilibrium binding constants (K) of representative compounds 1-4 A )

[0320]

[0321] Example 9: Tumor Imaging Efficiency

[0322] Following administration of compounds 1, 2, 3, and 4 at a total dose of 10 nmol / mouse, in an administration volume of 0.1 mL (equivalent to approximately 5 mL / kg for a 20 g mouse), and at an injection rate of approximately 1 mL / min, when tumors reached at least 50 mm... 3When measuring volume, optical imaging (OI) experiments were performed on animals bearing LNCaP tumors using the Explorer Air® I (SurgVision) imaging system.

[0323] For each group, in vivo oxygenation (OI) experiments were performed 2 hours after intravenous administration of the fluorescent compound. Animals were kept under gas anesthesia during the OI experiments. At the end of the in vivo experiments, 2 hours after treatment, animals were sacrificed, tumors and organs were removed, and the ex vivo signal was measured.

[0324] At each time point, for each fluorescence image, a target region (ROI) was plotted on the mouse tumor and a reference healthy background area (hind limb muscle) to evaluate signal intensity (expressed as counts) in the tissue. The tumor-to-background fluorescence signal ratio (TBR) was then calculated to assess contrast. Histological analysis of the resected tumor confirmed tumor morphology and PSMA expression.

[0325] The result is Figure 4 As shown in Table VI, which reports the in vitro TBR values ​​from samples collected 2 hours after administration. All compounds of this invention showed selective accumulation in tumors when tested at a dose of 10 nmol / mouse.

[0326] Table VI – In vitro TBR values ​​at 2 hours after application (mean ± SD).

[0327]

[0328] References:

[0329] 1. Davis MI, et al . Proc Natl Acad Sci USA 2005; 102(17): 5981-6

[0330] 2. Jones W, et al ., Cancers 2020; 12(6): 1367

[0331] 3. Kaewput et al ., J. Clin. Med. 2022; 11: 2738

[0332] 4. EP3636635 A1

[0333] 5. Bene ová et al ., J.Nucl.Med. 2015; 56: 914-920

[0334] 6. Galema HA et al ., Eur J Surg Oncol. 2022; 48(4): 810-821

[0335] 7. Gioux S et al ., Mol Imaging 2010; 9(5): 237-255

[0336] 8. Bunschoten A. et al ., Bioconjugate Chem. 2016; 27: 1253-1258

[0337] 9. WO2017 / 044584

[0338] 10. WO2017 / 184383

[0339] 11. WO2009 / 026177

[0340] 12. WO2010 / 108125

[0341] 13. T.W. Green and P.G.M. Wuts, Protective Groups in OrganicSynthesis, Wiley, N.Y. 2007, 4 th Ed., Ch. 5

[0342] 14. Mujumdar R.B. et al ., Bioconjugate Chem. 1993; 4(2): 105-111

[0343] Maresca K.P et al ., J. Med. Chem. 2009; 52(2): 347–357

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, in Each R 1 Independently, straight-chain or branched C1-C chains substituted with the group -SO3H 10 alkyl; R 2 Selected from -SO3H, where Y is a straight-chain or branched C1-C chain substituted with at least two hydroxyl groups. 10 Alkyl groups -CONH-Y, and groups of formula (II) ； R 3 Selected from hydrogen and phenyl or -O-phenyl groups optionally substituted with -SO3H; L represents a bond or linker; T represents the PSMA targeting portion of equation (III): ， Where X is an amino acid or its derivative; 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-furanyl)-alanine and 2-(2'-propynyl)-alanine.

3. The compound of formula (I) according to claim 1, wherein L is of formula -NH-(CH2). p -CO- or a bimolecular group selected from one or more of the following: amino acids or derivatives thereof; peptides containing 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid, sulfoalanine; polyethylene glycol or derivatives 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 -L1- is a dimethyl group of a diamine and -L2- is a dimethyl group of a dicarboxylic acid; wherein p is an integer between 1 and 20.

4. The compound of formula (I) according to claim 3, wherein L is selected from groups of formula -NH-(CH2)p-CO-; polyethylene glycol of formula -NH-(O-CH2-CH2)p-CO-; and a bimolecular group comprising one to five amino acids, wherein p is an integer between 1 and 20, or it is a group of formula (L3). 。 5. A compound of formula (I) according to any one of the preceding claims, wherein R 2 It is -SO3H.

6. A compound of formula (I) according to any one of the preceding claims, wherein R 2 The radical is -CONH-Y, where Y is selected from... (i)、 (ii)、 (iii)、 (iv), (v) and (vi).

7. The compound of formula (I) according to claim 6, wherein Y is a group (ii), preferably its stereoisomer (ii-a). (ii-a)。 8. A compound of formula (I) according to any of the preceding claims, wherein the two Rs 1 All of them are radicals -(CH2)4-SO3H.

9. The compound of formula (I) according to claim 1, which is represented by formula (Ia). (him) Where R 1 R 2 L and T are as defined in claim 1.

10. The compound of formula (I) according to claim 1, which is represented by formula (Ib). (One) Where R 1 R 2 L and T are as defined in claim 1.

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

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

13. The compound of formula (I) for the use according to claim 11, wherein the 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, kidney cancer, uterine cancer, thyroid cancer, liver cancer, and colorectal cancer, including primary tumors and both 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, carrier, or diluent.

15. A diagnostic kit comprising at least one compound of formula (I) as defined in claim 1, or a pharmaceutical composition as defined in claim 14, and additional adjuvants for performing biomedical optical imaging applications.

Images