Fluorescent probe targeting CA-IX

Abstract

The invention relates to the field of optical imaging. More specifically, it relates to fluorescent probes that target carbonic anhydrase 9 (CA-IX) and comprise a near infrared (NIR) cyanine dye. The invention also relates to methods of preparing these compounds, pharmaceutical compositions and kits comprising them and methods of using them as optical diagnostic agents in the imaging or treatment of diseases, such as solid tumors with hypoxic tissues comprising cells expressing CA-IX.

Description

Field of the Invention

[0001] The present invention relates to the field of optical imaging. More specifically, it relates to fluorescent probes capable of efficiently targeting the carbonic anhydrase IX enzyme (CA-IX) and comprising a heptamethine cyanine dye having near infrared (NIR) emission. The present invention also relates to methods of preparing these compounds, pharmaceutical compositions and kits comprising them, and methods of using them as optical diagnostic agents in the imaging or treatment of diseases, particularly solid tumors having hypoxic tissue comprising cells expressing CA-IX. Background Art

[0002] A dye is a chemical entity that absorbs photons of a specific wavelength after photoexcitation and re-emits a portion of this energy according to the quantum efficiency (usually at longer wavelengths). Specifically, a cyanine dye is a fluorescent organic molecule characterized by a delocalized electron system that spans a polymethyl bridge and is confined between two nitrogen atoms. Due to favorable optical properties, low toxicity, and good solubility in aqueous media, cyanine dyes can be used as contrast agents for biomedical imaging. Compared to dyes with fluorescent emission in the visible spectrum, cyanine dyes emitting in the near-infrared (NIR) region (700-900 nm) are particularly suitable for biomedical imaging applications due to the high penetration depth.

[0003] Among the NIR dyes currently used for biomedical imaging, indocyanine green (ICG) is a medical product approved for human use that can accumulate in pathological areas due to different homing mechanisms. Fluorophores such as fluorescein and ICG are distributed in tumor tissues by a combination of passive diffusion and enhanced permeability and retention (EPR) effects (Onda N. et al., Int J Cancer 2016, 139, 673-682). However, this first generation of fluorescent contrast agents requires a large mass dose (>1 mg / kg) to achieve proper visualization. In addition, false positive and false negative responses are common clinical findings associated with the use of these contrast agents (Tummers Q. et al., Plos One 2015).

[0004] Second generation contrast agents for optical imaging are currently under development, which utilize dyes conjugated to targeting agents to improve the sensitivity and specificity of detection. Molecular carriers such as small molecules, peptides, mAbs, and antibody fragments give probes tumor selectivity, while fluorescent dyes serve as detection moieties for spatial localization (Gioux S. et al., Mol Imaging 2010, 9 (5), 237-255). However, the in vivo behavior of dye-biomolecule conjugates may be strongly affected by the biological properties of the fluorescent moiety. For example, small structural modifications of cyanine Cy5 strongly regulate the accumulation of related bioconjugates in tumors and non-target tissues (Bunschoten A. et al., Bioconjugate Chem. 2016, 27, 1253-1258). Fluorescent contrast agents with low nonspecific accumulation and high selectivity for target tissues are preferred for use in living organisms.

[0005] Several examples of known cyanine dyes conjugated to low molecular weight targeting moieties are disclosed in the background art.For example, one of the targets of greatest interest for its potential in clinical settings is carbonic anhydrase IX (CA-IX).

[0006] CA-IX is a homodimeric glycoprotein located on the cell surface (Hilvo et al., J Biol Chem 2008; 283(41):27799-27809) and is a member of the large carbonic anhydrase (CA) family of zinc metalloenzymes. These enzymes participate in the maintenance of cellular acid-base homeostasis by catalyzing the reversible hydration of carbon dioxide (CO2 + H2O), which is converted into protons and bicarbonate (HCO 3- +H + )), resulting in a local decrease in pH.

[0007] To date, 16 CA isoforms have been characterized in mammals, which differ in their cellular localization, catalytic activity, sensitivity to different inhibitors, and tissue-specific distribution.

[0008] Among them, CA-IX has been shown to be an excellent biomarker for hypoxic response in tumors because its gene expression is promoted by a master regulator of hypoxia called hypoxia-inducible factor 1 (HIF-1), which is believed to be involved in maintaining the acidic environment of hypoxic cells (Wykoff et al., Cancer Res. 2000, 60, 7075-7083). Tumor hypoxia, mainly due to poor perfusion and anemia, is one of the key factors in inducing the development of cell clones with invasive and treatment-resistant phenotypes, which leads to rapid progression and poor prognosis of several cancer types. In fact, cancer cells survive in harsh environments by changing their gene expression (and in particular including genes involved in pH control among them).

[0009] Indeed, CA-IX plays an important role in the growth and metastasis of many tumors (including renal cancer, cervical cancer, colon cancer, prostate cancer, breast cancer, head and neck tumors, etc.) because its catalytic activity helps to lower the extracellular pH, thereby generating an acidic microenvironment that increases cancer cell proliferation and invasion.

[0010] Unlike other CAs, many studies have demonstrated that CA-IX is expressed only in a few normal tissues (i.e., intestinal and gastric mucosa, gallbladder, and testis), whereas it is overexpressed in many types of cancer cells, especially in solid tumors characterized by insufficient oxygen supply. Therefore, CA-IX represents a definitive biomarker of tumor hypoxia, which plays a key role in carcinogenesis, and which is considered a hallmark of the disease (Muz et al., Hypoxia (Auckl) 2015; 3: 83-92).

[0011] Targeting CA-IX using specific tools opens up new important areas for improving conventional therapy as well as early diagnosis and prognosis of malignant tumors.

[0012] Classes of CA-IX-targeting agents developed so far for imaging and / or therapeutic applications include monoclonal antibodies (e.g., G250, M75) or miniantibodies (e.g., A3 and CC7) and small chemical compounds such as inorganic ions, sulfonamide-based compounds, phenols, and coumarins. Some agents belonging to these classes of compounds are currently in clinical development.

[0013] Specifically, monoclonal antibodies (mabs) M75 and G250 represent the first solutions developed to target the CA-IX enzyme. Mab M75 binds to the PG-like domain of CA-IX on the N-terminus of the target, while mab G250 interacts with the catalytic domain of CA-IX. 124I-labeled chimeric version of G250 (referred to as cG250), which is used to detect clear cell renal cell carcinoma (ccRCC). For example, in WO2014 / 128258, the use of antibody cG250 in methods for diagnosing, predicting and / or classifying cancer diseases (including quantification of CA-IX expression) is described.

[0014] Although monoclonal antibodies are generally considered the ligand of choice for most tumor targeting applications, it is becoming increasingly clear that they have a number of disadvantages. Indeed, antibodies are characterized by slow and inefficient tumor penetration and long blood residence times, which necessitate the use of long-lived radioisotopes and imaging at late time points, exposing patients to high radiation burdens. 124 I-cG250 does not reach a tumor / blood ratio suitable for imaging until 2-7 days after injection into the patient. In addition, monoclonal antibodies can be immunogenic and therefore cannot be administered repeatedly in routine diagnostic procedures.

[0015] The use of small molecules can avoid these problems. Unlike large macromolecules, small molecules are rapidly cleared from the circulation and thus achieve a tumor / blood ratio suitable for imaging at an early time point. This fact in turn allows physicians to obtain diagnostic information much faster than using antibody-based imaging agents.

[0016] Among the small chemical compounds that target CA, the best studied and most effective class of inhibitors are the sulfonamides (ie, acetazolamide) because of their high affinity, availability, and ease of chemical manipulation.

[0017] Although they have been used in clinical applications for decades to treat conditions such as glaucoma, epileptic seizures, altitude sickness, and as diuretics, they have only recently been considered anti-tumor agents for their ability to inhibit CA-IX. The first compound to enter Phase I and Phase II clinical trials alone or in combination with other anti-tumor agents was indisulam, but it has been discontinued. The only CA inhibitor (CAI) currently in clinical development is SLC-0111, a urea-based benzenesulfonamide that has successfully completed Phase I studies to determine safety and tolerability in patients with advanced solid tumors (McDonald et al., Am J Clin Oncol 2020; 43(7): 484-490).

[0018] For imaging purposes, CAI has been used with various positron-emitting isotopes (e.g. 18 F. 64 Cu, 68 Ga, 111 In) or 99m Tc labeling to obtain PET / SPECT agents.

[0019] Acetazolamide is an FDA-approved drug for the treatment of glaucoma, epilepsy, altitude sickness, periodic paralysis, heart failure, etc. Acetazolamide is a first-generation carbonic anhydrase inhibitor, thus causing the accumulation of carbonic acid, and because its terminal sulfonamide group is deprotonated to -SONH at physiological pH - , and thus it is known to bind strongly to the Zn ions present in the catalytic pocket.

[0020] It is possible to hydrolyze the acetyl group of acetazolamide and replace it with a different linker, such as by acylation of the amino group, and still retain inhibitory activity and moderate / strong binding to the enzyme, depending of course on the type of substituent.

[0021] In 2015, Wichert et al. (Nat Chem 2015; 7(3): 241-249) described the selection of novel low nanomolar binders for CA-IX by innovative chemical library technology to achieve affinity maturation of ligands for specific targets. Among them, an acetazolamide derivative named "4a ligand" was identified by acyl chloride modification and click chemistry of acetazolamide after hydrolysis of the acetyl moiety in a strongly acidic aqueous medium. Ligand 4a showed high affinity for CA-IX (16.7 nM, measured by SPR), and its use as a delivery vehicle for tumor targeting has been reported.

[0022] Examples of NIR fluorescent dyes conjugated to "4a ligands" are disclosed in, for example, WO2020 / 229438 (e.g., compound 11 on page 13, having a linear heptamethine dye) and WO2021 / 259899 (e.g., compound 6 on page 19, having a cyclohexenyl heptamethine dye).

[0023] Acetazolamide and other sulfonamides have also been conjugated to various NIR fluorescent dyes with the goal of imaging hypoxia-induced CA-IX expression in tumor cells. In fact, among the fluorescent dyes targeting CA-IX functionalized with different sulfonamides and even cyclic secondary amides such as saccharin reported in the literature, the best results in terms of binding were always obtained by derivatization with acetazolamide. A preliminary in vivo experiment illustrates the potential of NIR fluorescent CAI and fluorescence molecular tomography (FMT) imaging for non-invasive quantification of CA-IX expression (Groves et al., Bioorg Med Chem Lett 2012; 22: 653-657).

[0024] Representative examples of NIR fluorescent probes targeting CA-IX enzymes are disclosed in WO2017 / 161195 (On Target Laboratories), which describes compounds comprising a ligand of CA-IX, a dye, and a linker formed by an amino acid, an amide, a urea group, or a polyethylene glycol derivative. Specifically, among the several compounds disclosed therein, the compound Az-8AOA-LMNIR2, also known as HypoxyFluor-1, is mentioned, which is currently being developed as a fluorescent probe for targeted imaging of tumors that highly express CA-IX, such as optical imaging and surgery involving CA-IX-positive tissues or tumors. For example, this compound is also mentioned in Mahalingam SM et al., Bioconjugate Chem 2018, 29, 3320-3331, where its application in fluorescent imaging of hypoxic tumors is described in detail.

[0025] In this compound, as in many other compounds disclosed in the literature, the acetazolamide targeting moiety is conjugated at the central (middle) position of the heptamethylene backbone of the dye. Typically, attachment of the ligand is performed on a central phenyl or -O-phenyl ring that is functionalized with a carboxyl, formamido or ester group, optionally by intervening a linker.

[0026] However, in several cases, this central position is affected by the higher instability of the central substituent, which can be removed in vivo by reaction with endogenous nucleophiles (i.e., it is susceptible to nucleophilic substitution in vivo), resulting in loss of the targeting unit and leading to potential in vivo degradation and inactivation of the probe. This drawback has been partially addressed by replacing the central CO bond with a CC bond, which is much more stable in vivo, but in this case the aqueous solubility of the dye tends to drop sharply due to the higher aggregation tendency.

[0027] In general, the use of tumor-targeted fluorescent dyes to aid surgeons in identifying tumor nodules that would otherwise go undetected, reduce the incidence of cancer-positive margins, and facilitate localization of malignant lymph nodes has proven to hold considerable promise for improving cancer debulking surgery. Unfortunately, the library of available fluorescent dyes targeting tumors expressing CA-IX is not yet extensive and diverse enough to allow for the determination of all types of hypoxic tumor cells, and thus there is a need to develop additional CA-IX-specific fluorescent dyes that, utilizing specific targeting at the biomolecular level, can ensure localization of all malignant lesions and precise tumor resection during cancer surgery.

[0028] Therefore, despite many efforts made so far, there is still an urgent and unmet need to develop efficient fluorescent probes that target CA-IX with high affinity and specificity for use in diagnosis and / or therapy.

[0029] Unlike the above-mentioned probes, the compounds of the present invention are characterized in that the Cy7 cyanine dye is conjugated to the acetazolamide moiety at a different position of the backbone, namely at position 1 of one of the indolenine rings, and thus they are more stable in vivo.

[0030] In addition, the compounds of the present invention have shown surprising improvements in affinity for the CA-IX target compared to other fluorescent probes with CA-IX targeting moieties at the same conjugation position. In addition, they have lower affinity for plasma proteins and therefore show improved bioavailability results in in vivo tests. SUMMARY OF THE INVENTION

[0032] In general, the present invention aims to provide a fluorescent probe targeting CA-IX that can be used as an optical imaging contrast agent and aims to solve the above-mentioned problems. Specifically, the present invention provides a fluorescent probe with optimal performance for different molecular imaging applications, which can interact with cells expressing CA-IX, accumulate in pathological cells and tissues, and specifically display fluorescent signals corresponding to pathological tissues, with a high signal-to-noise ratio and improved imaging efficacy at low mass doses.

[0033] The fluorescent probes of the present invention are characterized by a heptamethine cyanine dye conjugated to an acetazolamide moiety via different linkers, thereby providing strong binding results to the target and unexpectedly exhibiting optimal in vitro and in vivo behaviors, and are therefore particularly suitable for molecular imaging.

[0034] In particular, among the several advantages that can be achieved with the aid of the compounds according to the invention, one can highlight characteristics such as high selectivity for the target tissues and low accumulation due to nonspecific interactions with other tissues, high solubility in water, low binding to plasma proteins, negligible or no observed adverse reactions after systemic administration, good chemical and optical stability in plasma after administration.

[0035] Another aspect of the invention relates to such fluorescent probes for use as diagnostic agents, in particular for optical imaging of human or animal organs or tissues, for use in optical imaging methods, 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, short-wave infrared imaging, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, open field surgery, laser-guided surgery, or photoacoustic or sonofluorescent methods.

[0036] In addition, the present invention relates to a method for preparing the provided compounds and / or pharmaceutically acceptable salts thereof, and their use in preparing diagnostic agents.

[0037] According to another aspect, the present invention relates to a pharmaceutically acceptable composition comprising at least one compound of the present invention or a pharmaceutically acceptable salt thereof mixed with one or more physiologically acceptable carriers or excipients. The composition is particularly useful as an optical imaging agent to provide useful imaging of human or animal organs or tissues.

[0038] In another aspect, the present invention relates to a method for optical imaging of a body organ, tissue or region by using optical imaging techniques, comprising administering an effective amount of a compound of the present invention.

[0039] Description of the invention

[0040] Therefore, the first aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0041]

[0042] in

[0043] R 1 Selected from a straight or branched C1-C 10 Alkyl and a group of formula (II)

[0044]

[0045] X is a straight or branched C1-C 10 Alkylene;

[0046] L is a key or linker;

[0047] Each Y is independently a straight or branched C1-C 10 alkyl;

[0048] R 3 is selected from hydrogen and a group optionally substituted by -SO3H, phenyl or -O-phenyl;

[0049] n is an integer equal to 0 or 1.

[0050] Preferably, the linker L is of the formula -NH-(CH2) p -CO- or a di-residue of one or more moieties selected from the group consisting of amino acids such as, for example, glycine, alanine, β-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid, etc.; peptides comprising 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid; cysteic acid; polyethylene glycol, such as a compound of formula -NH-(O-CH2-CH2) p - or -NH-(O-CH2-CH2) p-CO- or a derivative thereof; amino-polyethylene glycol-carboxylic acid; diaminobutyric acid; and diaminopropionic acid; or it is a group -L1-L2-, wherein L1 is the di-residue of a diamine, such as, for example, the formula -NH-(O-CH2-CH2) p -NH-amino-polyethylene glycolamine or a diresidue of ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexamethylenediamine, etc.; and L2 is a diresidue of a dicarboxylic acid (such as, for example, succinic acid, glutaric acid, suberic acid, adipic acid, etc.); wherein p is an integer from 1 to 20.

[0051] More preferably, L is selected from the formula -NH-(CH2) p -CO- group; formula -NH-(O-CH2-CH2) p -CO-; and a diresidue comprising 1-5 amino acids, wherein p is an integer from 1 to 20.

[0052] Preferably, X is a linear or branched C1-C6 alkylene group.

[0053] The present invention also relates to processes for preparing compounds of formula (I) by means of synthetic transformation steps.

[0054] The invention further comprises compounds of formula (I) for use as fluorescent agents for detecting tumor margins in guided surgery.

[0055] Description of the drawings

[0056] Figure 1 Representative binding curves of compounds 1 and 2 of the present invention to HT-29 cells expressing CA-IX are shown, as well as representative binding curves obtained for reference compounds 1 (HF-1), 2 and 3 to HT-29 cells expressing CA-IX.

[0057] Figure 2 It shows that compound 1 competes with fully human recombinant MSC8 antibody for binding to HT-29 cells, selectively recognizing and blocking CA-IX isoforms. Panel A shows the binding of compound 1 (excitation 640nm, emission 780 / 60nm); Panel B shows the binding of MSC8, detected by secondary antibody (excitation 488nm, emission 533 / 30nm).

[0058] Figure 3 In vivo tumor to background ratios (TBRs) of representative compounds 1 and 2 and references 1 (HF-1), 2, and 3 from images acquired 24 hours after administration are shown.

[0059] Figure 4Shown are (a) ex vivo tumor to background ratios (TBR) and (b) organ to muscle ratios for representative compounds 1, 2 and reference 1 (HF-1), 2 and 3 from samples collected 24 hours after administration.

[0060] One-way analysis of variance and Dunnett's multiple comparison test were used to Figure 3 and 4 Statistical analysis of TBR in (a), where p<0.05 indicates values ​​significantly different from the reference (1 nmol / mouse).

[0061] definition

[0062] In this specification, and unless otherwise provided, the following terms and phrases used herein are intended to have the following meanings.

[0063] The term "diadole" refers to a chemical group in which hydrogen atoms at both terminal portions of a molecule are removed to form a bond.

[0064] The expression "straight or branched C1-C 10 "Alkyl" means an aliphatic hydrocarbon group having 1 to 10 carbon atoms in the chain, which may be straight or branched. For example, "C1-C8 alkyl" includes in its meaning a straight or branched chain containing 1 to 8 carbon atoms. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, pentyl and hexyl. Unless otherwise specified, a straight or branched alkyl group is a monovalent residual group. In some cases, it may be a "divalent" or "multivalent" residual group in which two or more hydrogen atoms are removed from the above hydrocarbon group and replaced, such as a methylene, ethylene, isopropylidene group, etc. In such a case, the expression "straight or branched C1-C8 alkyl" is used. 10 "Alkylene".

[0065] The term "hydroxyalkyl" denotes any of the corresponding alkyl chains in which one or more hydrogen atoms are replaced by a hydroxy group.

[0066] The term "protecting group" (Pg) represents a protective group suitable for retaining the function of the group combined with it. Specifically, the protecting group is used to retain amino, hydroxyl or carboxyl functions. Suitable protecting groups can 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 group, such as trifluoroacetyl, benzyl ester, allyl, or other substituents that are usually used to protect such functions, they are well-known to those skilled in the art (see, for example, general reference TW Green and PGM Wuts, Protective Groups in Organic Synthesis, Wiley, NY2007, the 4th edition, the 5th chapter).

[0067] In addition, the present invention also comprises the precursor or the intermediate compound of the compound of formula (I) or its salt that is applicable to prepare expectation.In such intermediate, any functional group, such as carboxylic acid or carboxamide, can be protected with suitable blocking group (Pg) as defined above, preferably with alkyl or ester group protection.If necessary, the hydroxyl of Y group can also be protected with suitable blocking group (Pg) in the process of preparing the compound of formula (I), forming for example acetoxyl group, alkoxyl group or ester group.

[0068] The expression "coupling agent" denotes, for example, an agent for forming 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 subsequent acylation of the amino group with an 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 agents, such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), ... tripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy-tripyrrolidinyl-phosphonium hexafluorophosphate (PyAOP), [ethylcyano(oxime)acetato-O2]tris-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), tripyrrolidinylphosphonium bromide hexafluorophosphate (PyBrOP), and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one (DEPBT); and ammonium / urea imonium reagents such as N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1-[1-(Cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU), 2-(2,5-dioxopyrrolidin-1-yl)-1,1,3,3-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 those skilled in the art.

[0069] The expression "activated carboxylic acid" refers to a derivative of a carboxyl group that is more susceptible to nucleophilic attack than the free carboxyl group; suitable derivatives may include, for example, anhydrides, thioesters, acyl halides, NHS esters and sulfo-NHS esters.

[0070] The term "moiety" or "residue" is intended herein to define the remaining portion of a given molecule once it has been suitably linked or conjugated to the rest of the molecule, either directly or via a suitable linker.

[0071] The term "imaging agent" refers to a detectable entity that, when used in conjunction with an appropriate diagnostic imaging technique, can be used for in vitro, ex vivo or in vivo visualization or detection of biological elements, including cells, biological fluids and biological tissues derived from a living mammalian patient, and preferably a human patient, and human organs, regions or tissues.

[0072] Preferably, the fluorescent probes of the present invention are capable of selectively targeting tumor cells or tissues expressing CA-IX. Specifically, they are capable of targeting tumors selected from brain cancer, breast cancer, head and neck cancer, ovarian cancer, prostate 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. In addition, the fluorescent probes of the present invention are capable of targeting the metastatic spread of the above cancers in tissues and organs different from the primary source. In addition, the fluorescent probes of the present invention are capable of targeting pre-neoplastic lesions and dysplasia in different tissues and organs.

[0073] Connector (L)

[0074] According to the present invention, L is an optionally present linker that separates the CA-IX targeting ligand (acetazolamide) from the dye.

[0075] The presence of a linker is particularly useful for certain embodiments where the acetazolamide moiety and the dye are at risk of adversely interacting with each other. In addition, the presence of a linker may be advantageous when the dye is relatively large and may interfere with the binding of the targeting moiety to the target site.

[0076] The linker can be flexible (e.g., comprising a linear alkyl chain) or rigid (e.g., comprising an amino acid with an aromatic group) to keep the dye away from the target. The linker can also alter the pharmacokinetics and metabolism of the conjugate of formula (I) used as an imaging agent in a living organism.

[0077] Hydrophilic linkers can reduce interactions with plasma proteins, reduce blood circulation time and promote excretion. For example, if the linker is a polyethylene glycol (PEG) moiety, the pharmacokinetics and blood clearance of the in vivo imaging agent may be altered. In such an embodiment, the linker can improve the clearance of the imaging agent from background tissues (e.g., muscle, blood), thereby producing better diagnostic images due to high target-to-background contrast. In addition, the introduction of specific hydrophilic linkers can shift the elimination of contrast agents from the liver to the kidneys, thereby reducing overall body retention.

[0078] Thus, in a preferred embodiment, the linker is of the formula -NH-(CH2) p -CO- or a di-residue of one or more moieties selected from the group consisting of amino acids such as, for example, glycine, alanine, β-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid, etc.; peptides comprising 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid; cysteic acid; polyethylene glycols such as the formula -NH-(O-CH2-CH2) p - or -NH-(O-CH2-CH2) p -CO- or a derivative thereof; amino-polyethylene glycol-carboxylic acid; diaminobutyric acid; and diaminopropionic acid; or it is a group -L1-L2-, wherein L1 is the di-residue of a diamine, such as, for example, the formula -NH-(O-CH2-CH2) p -NH-amino-polyethylene glycolamine or a diresidue of ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexamethylenediamine, etc.; and L2 is a diresidue of a dicarboxylic acid (such as, for example, succinic acid, glutaric acid, suberic acid, adipic acid, etc.); wherein p is an integer from 1 to 20.

[0079] More preferably, L is selected from the formula -NH-(CH2) p -CO- group; formula -NH-(O-CH2-CH2) p -CO-; and a diresidue comprising 1-5 amino acids, wherein p is an integer from 1 to 20.

[0080] The compound of above-mentioned formula (I) may have one or more asymmetric carbon atoms, also referred to as chiral carbon atoms, and may therefore produce diastereomers and optical isomers. Unless otherwise specified, the present invention further includes all such possible diastereomers and their racemic mixtures, their substantially pure enantiomers of resolution, all possible geometrical isomers and pharmaceutically acceptable salts thereof.

[0081] The present invention further relates to compounds of the above formula (I), wherein R 1 and / or R 3 The sulfonyl group may be in the form of a negatively charged ion or a pharmaceutically acceptable salt.

[0082] Detailed description of implementation plan

[0083] In a preferred embodiment, the invention relates to compounds of formula (I), wherein Y is independently selected from:

[0084]

[0085] More preferably, the present invention relates to compounds of formula (I), wherein Y is a group of formula (ii) as defined above. Preferably, said group (ii) has the following stereochemical configuration obtained by using D-reduced glucosamine in the preparation of the compound:

[0086]

[0087] More preferably, both Y are the above-mentioned group (ii).

[0088] In another preferred embodiment, R 1 It is a group -(CH2)4SO3H or -CH2CH2CH(CH3)SO3H.

[0089] In another preferred embodiment, R 1 is group (II) as defined above.

[0090] Preferably, X is a linear C1-C6 alkylene group.

[0091] In a preferred embodiment, X is a linear C5 alkylene group and L is a group -NH(CH2)7-CO- according to the following formula (Ia):

[0092]

[0093] Where R 1 , R 3 , Y and n are as defined above.

[0094] In a preferred embodiment, n is 0 and R 3 is hydrogen, represented by the following formula (Ib):

[0095]

[0096] Where R 1 , X, Y and L are as defined above.

[0097] In another preferred embodiment, n is 1 and R 3 is a group -O-phenyl optionally substituted by -SO3H, represented by the following formula (Ic):

[0098]

[0099] Where R 1 , X, Y and L are as defined above.

[0100] Particularly preferred are the compounds of formula (I) listed in Table I.

[0101] Table I also reports the structures of the following reference compounds: Reference compound 1 corresponding to a known fluorescent probe with affinity for CA-IX Hypoxyfluor-1 (HF-1); Reference compound 2 corresponding to compound 6 disclosed in WO2021 / 259899; Reference compound 3 corresponding to compound 11 disclosed in WO2020 / 229438. Both reference compounds 2 and 3 are disclosed as CA-IX targeting probes.

[0102] Table I - Preferred compounds of formula (I) and reference compounds 1-3

[0103]

[0104]

[0105] The present invention also relates to a process for the synthesis of compounds of formula (I) prepared as shown in the following part of the specification. The compounds of the present invention can be used as imaging agents in the detection of tumors in humans and animals. Therefore, the present invention provides compounds of formula (I) as defined above, which are used as fluorescent probes for detecting and delineating tumor tissue during diagnosis, interventional imaging and intraoperative procedures, in particular wherein the tumor is a tumor showing increased or variable CA-IX expression. Preferably, the object of imaging is a human.

[0106] The invention also provides compounds of formula (I) for use as fluorescent probes as defined above, wherein the detection and delineation of tumor tissue is performed under NIR radiation.

[0107] Preferably, the fluorescent probe of the present invention is capable of selectively targeting cells or tissues of tumors expressing CA-IX, wherein the tumor is selected from brain cancer, breast cancer, head and neck cancer, ovarian cancer, prostate 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 regional and distant metastases.

[0108] The probe of the present invention is capable of determining diseased tissue in vivo in a subject in need thereof. This can be achieved by administering a compound of formula (I) as defined above and irradiating the body of a subject in need thereof containing diseased tissue in vivo with light having at least one excitation wavelength in the NIR range of about 650 nm to about 850 nm. Direct observation of the fluorescence emitted from the administered compound in response to the at least one excitation wavelength determines the location and / or surface area of ​​the diseased tissue in the subject, wherein the administered compound specifically binds to the diseased tissue in the body part.

[0109] In particular, the present invention also provides a compound of formula (I) for use in a method for detecting the possible presence of a disease in a subject, the method comprising the steps of:

[0110] - 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 said compound to bind to cells expressing CA-IX;

[0111] - measuring a signal from the compound in a biological sample;

[0112] - comparing the signal to at least one control data set comprising a signal from a compound of formula (I) contacted with a biological sample not comprising the target cell type, to indicate the possible presence of a disease.

[0113] The present invention further provides a compound of formula (I) for use as an imaging agent in a method for imaging tissues and cells, the method comprising the steps of:

[0114] - contacting said tissue or cell with a compound of formula (I) as defined above;

[0115] - irradiating the tissue or cells at a wavelength absorbed by the imaging agent;

[0116] -Detection of near-infrared emission using a fluorescence camera.

[0117] Specifically, in a preferred embodiment, the present invention provides a compound of formula (I) for use in a method of performing imaging-guided surgery on a subject, the method comprising the steps of:

[0118] - administering a composition comprising a compound of formula (I) as defined above under conditions and for a time sufficient to allow said compound to accumulate at a given surgical site;

[0119] - using near infrared light to illuminate and visualize the compound;

[0120] - Surgical excision of the area that fluoresces after excitation with said near-infrared light.

[0121] Another aspect of the present invention relates to a pharmaceutical composition comprising a fluorescent probe of formula (I) or a salt thereof as defined above, and one or more pharmaceutically acceptable adjuvants, excipients or diluents.

[0122] Another aspect of the present invention relates to a diagnostic kit comprising a compound of formula (I) as defined above. In addition, the kit may contain additional adjuvants for realizing biomedical optical imaging applications. These adjuvants are, for example, suitable buffers, containers, detection reagents or instructions for use. The kit preferably contains all materials for intravenous administration of the compound of the present invention.

[0123] Depending on the disease to be treated and the location of the suspected disease to be diagnosed, an effective amount of the compound of the present invention may be administered by different routes prior to the imaging procedure.

[0124] For example, it can be administered to the organ or tissue to be imaged by a topical route (eg, transdermal), enteral route (eg, oral), or parenteral route (eg, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous).

[0125] In certain embodiments, the compounds of the invention may be administered by topical spraying or atomization of pharmaceutical compositions containing them and / or specially formulated for such use.

[0126] The composition is administered in a dosage effective to achieve the desired optical image of a tumor, tissue or organ, which dosage can vary widely depending on the compound used, the tissue undergoing the imaging procedure, the imaging device used, and the like.

[0127] The exact concentration of the imaging agent in the pharmaceutical composition depends on the experimental conditions and the desired results, but can generally be in the range of 1 pM to 0.1 mM. The optimal concentration is determined by system changes until satisfactory results with minimal background fluorescence are obtained. After administration, the imaging agent of the present invention is exposed to light or other forms of energy that can pass through tissue layers. 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 rest 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.

[0128] The preparation of the compound of formula (I) itself or its pharmaceutically acceptable salt form represents another aspect of the present invention. For example, the compound of the present invention can be prepared according to the method described in the following chapters and experimental section. General teachings on the preparation of cyanine skeletons can be found in Mujumdar RB et al., Bioconjugate Chem. 1993, 4 (2), 105-111, which relates to the synthesis and labeling of sulfonated indocyanine dyes.

[0129] However, in some cases, due to the presence of various functional moieties such as carboxylic acid or amide groups in the cyanines of the present invention, it is necessary to use protecting groups to direct the reaction at the desired functional groups.

[0130] In general, in order to avoid the removal of protecting groups, special care is required when handling cyanines under strong pH and temperature conditions, since in some cases the stability of the polymethylene backbone may be compromised, leading to severe degradation of the dye. Contrary to expectations, it has been found that the compounds of the present invention are very stable at alkaline pH (i.e. at about pH 11-12), and no or negligible degradation is observed during the removal of protecting groups.

[0131] Experimental Section

[0132] The present invention and specific embodiments thereof described in the following sections are merely exemplary and should not be viewed as limitations of the present invention: they show how the invention may be implemented and are intended to be illustrative rather than limiting of the scope of the invention.

[0133] Materials and Equipment

[0134] All commercially available reagents used in the synthesis were obtained from Sigma Aldrich and TCI and used directly without further purification. Reference compound 1 (HF-1) was prepared as described in Mahalingam SM et al., Bioconjugate Chem 2018, 29, 3320-3331, while reference compounds 2 and 3 were prepared as described in WO2021 / 259899 and WO2020 / 229438, respectively.

[0135] All reactions were monitored by HPLC (Agilent mod. 1100 / 1200) and HPLC-MS (Agilent mod. 1260, Quadrupole LC / MS Mod. 6120) equipped with absorption detectors set at different wavelengths (column: YMC-Triart Phenyl, 250 x 4.6 mm / S-5 μm / 12 nm), eluent: 0.1% ammonium acetate and acetonitrile.

[0136] Use a pre-packed silica gel C18 column ( SNAP or ), in an automated purification system ( Purification was performed by flash chromatography on HPLC (Rf+, Teledyne ISCO), typically eluting with a water / acetonitrile gradient.

[0137] The absorbance (Abs) of the compound of the present invention is measured using a double beam UV-VIS spectrophotometer (Lambda 40, Perkin Elmer). Emission / excitation (Em / Ex) spectra are carried out on a fluorescence spectrophotometer (FluoroLog-3 1IHR-320, Horiba Jobin Yvon) equipped with an F-3018 integrating sphere attachment. The excitation wavelength at the maximum absorbance of different dyes is used for measurement, and the sample is excited with a 450W xenon light source. Detected by a detector cooled by a photomultiplier tube (PMT-NIR) or by a TBX-04 detector. Carefully prepare the dye solution so that it has an absorbance lower than 0.1 (optical density) to minimize reabsorption.

[0138] Human colon adenocarcinoma HT-29 cells (ATCC) used for cell binding experiments were cultured in McCoy's 5A medium (Sigma-Aldrich) supplemented with 10% HyClone Fetal Clone III (Euroclone), 2 mM L-glutamine (Sigma-Aldrich), 100 IU / mL penicillin, 0.1 mg / mL streptomycin, 0.25 μg / mL amphotericin B (antibiotic-antimycotic solution, Life Technologies) and grown at 37° C. in a humidified atmosphere enriched with 5% CO 2. Cells were washed with DPBS (Sigma-Aldrich) without MgCl 2 and CaCl 2.

[0139] List of abbreviations

[0140] DCM Dichloromethane

[0141] DMF N,N-Dimethylformamide

[0142] DNSA 5-(dimethylamino)naphthalene-1-sulfonamide; dansamide

[0143] TEA Triethylamine

[0144] HPLC High Performance Liquid Chromatography

[0145] HSA Human serum albumin

[0146] NMR Nuclear Magnetic Resonance

[0147] t R Retention time (HPLC)

[0148] RT Room temperature

[0149] CV column volume

[0150] DMSO Dimethyl sulfoxide

[0151] PBS Phosphate buffered saline

[0152] DPBS Dulbecco's Phosphate Buffered Saline

[0153] FMOC 9-Fluorenylmethoxycarbonyl

[0154] C8-AZA 8-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide

[0155] NMM N-Methylmorpholine

[0156] HBTU O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0157] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0158] TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0159] TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0160] COMU (1-cyano-2-ethoxy-2-oxoethylenylaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate

[0161] Example 1: Synthesis of Compound 1

[0162] Preparation of intermediate compound C8-AZA

[0163]

[0164] In a round bottom flask, acetazolamide (1103.56 mg, 4.96 mmol) was suspended in 1M HCl (26 mL) and heated to reflux for 1.5 hours to give a clear solution. The mixture was concentrated under reduced pressure and passed through a pre-packed C18 silica gel column ( C18 (60 g) was purified by flash chromatography with a water-acetonitrile gradient (4 CV of 0% ACN, 2 CV of a linear gradient from 0 to 100% ACN). The fractions containing the pure intermediate compound 11 thus obtained (eluted with 100% water) were combined, concentrated under reduced pressure and freeze-dried to give a white solid (775.24 mg, 72% yield). HPLC purity: 97.8% at 270 nm, [M+H] + 181.1.

[0165] In a round-bottom flask cooled with an ice bath, Fmoc-8-aminocaprylic acid (500 mg, 1.311 mmol) was suspended in anhydrous DCM (30 mL) and a small amount of anhydrous DMF (20 μL, 0.262 mmol) was added. Then, thionyl chloride (2.85 mL, 39.33 mmol) was slowly added dropwise. The mixture was stirred at room temperature for about 30 minutes and complete dissolution was observed. The solvent and residual thionyl chloride were concentrated under reduced pressure. The yellow crude oil was immediately dissolved in a solution of compound 11 (283 mg, 1.311 mmol) and anhydrous pyridine (211 μL, 2.62 mmol) in anhydrous DMF (10 mL) and stirred at room temperature for 1.5 hours. The solution was then concentrated under reduced pressure and the intermediate compound 12 was precipitated with DCM. The white solid was filtered and dried overnight at 30 ° C under vacuum (294.25 mg, 41% yield). HPLC purity: 93% at 270 nm and 254 nm, MS: [M+H]+ 544.2.

[0166] Compound 12 (294.25 mg, 0.541 mmol) was then dissolved in anhydrous DMF (5.8 mL) in a round bottom flask and piperidine (1.16 mL) was added. After 30 minutes, cold ether (100 mL) was added; the solid was then filtered, dissolved in methanol, the solution was concentrated to a solid residue and further processed in ether (100 mL). Finally, the intermediate compound 13 (C8-AZA) was recovered as a white powder, which was dried under vacuum at room temperature overnight (163.56 mg, 94% yield). HPLC purity: 96.6% at 270 nm and 96.7% at 254 nm, MS: [M+H] + 322.2.

[0167] Preparation of intermediate compound (15)

[0168]

[0169] Compound 14 (70.1 mg, 0.0547 mmol, prepared as described in WO2021 / 259899) was dissolved in anhydrous DMF (8 mL). TSTU (32.9 mg, 0.109 mmol) and NMM (18 μL, 0.164 mmol) were added at room temperature, and the dark green solution was stirred at room temperature in the dark for 1 hour.

[0170] Then, the solution was quickly dropped into cold ethyl acetate (100 mL) under vigorous stirring in an ice bath, and the suspension was placed at -20°C for 30 minutes and then filtered. The dark green solid was washed with ethyl acetate (2 x 10 mL), dried under a stream of nitrogen and used in the next step without further purification (intermediate 15 conversion: 90% at 780 nm).

[0171] Synthesis of compound 1

[0172]

[0173] The solid of compound 15 was dissolved in anhydrous DMF (8 mL) and then a solution of compound 13 (21.1 mg, 0.0657 mmol) and DIPEA (48 μL, 0.273 mmol) in anhydrous DMF (1 mL) was added. The solution was stirred at room temperature in the dark for 1 hour. An additional 4 mg of compound 13 was added and the solution was stirred for another 30 minutes. The dark green solution was then dropped into cold ethyl acetate (150 mL), the precipitate thus obtained was dissolved in water and passed through a pre-packed C18 silica gel column (Biotage 30g) with a water-acetonitrile gradient (2 CV of 100% water, from 0 to 25% ACN in 2.5 CV, 5 CV at 25% ACN and 3 CV at 100% ACN). The fractions containing the pure product (eluted with 25% ACN) were combined, concentrated under reduced pressure and freeze-dried to give 53.3 mg of a dark green solid (HPLC purity: 97.5% at 780 nm and 98.4% at 254 nm, 61% yield).

[0174] Example 2: Synthesis of Compound 2

[0175] Preparation of intermediate compound (17)

[0176]

[0177] Compound 16 (50.3 mg, 0.0472 mmol, prepared as described in WO2020 / 229438) was dissolved in anhydrous DMF (5 mL). TSTU (21.3 mg, 0.0708 mmol) and NMM (12 μL, 0.108 mmol) were added at room temperature, and the dark green solution was stirred at room temperature in the dark for 1 hour.

[0178] Then, the solution was quickly dropped into cold ethyl acetate (25 mL) under vigorous stirring in an ice bath, and the suspension was placed at -10 ° C for 22 hours, and then filtered. The dark green solid was washed with ethyl acetate (2 x 10 mL), dried under a nitrogen stream, dissolved in a few mL of water and freeze-dried. The solid was then used in the next step without further purification (intermediate 17 conversion: 84% at 756 nm).

[0179] Synthesis of compound 2

[0180]

[0181] Compound 17 (58 mg, 0.041 mmol) was dissolved in anhydrous DMF (6 mL) and then a solution of compound 13 (10 mg, 0.0311 mmol) and DIPEA (32 μL, 0.196 mmol) in anhydrous DMF (2 mL) was added. The solution was stirred at room temperature in the dark for 2.5 hours. The dark green solution was then dropped into cold ether (30 mL), left at -20 ° C for several hours and then filtered. The precipitate thus obtained was dissolved in water and passed through a pre-packed C18 silica gel column (Biotage The product was purified three times by flash chromatography on 10% 4% ACN (30 g) with a water-acetonitrile gradient. The fractions containing the pure product (eluted with 35% ACN) were combined, concentrated under reduced pressure and freeze-dried to give 39 mg of a dark green solid (HPLC purity: 97% at 756 nm and 99% at 254 nm, 92% yield relative to C8-AZA).

[0182] Example 3: Optical Characterization

[0183] The compounds of the present invention were characterized in terms of their in vitro optical properties in aqueous media (i.e., water / PBS pH 7.4) and in clinical chemistry control serum (Seronorm, Sero SA), thereby simulating the chemical composition and optical properties of human serum. All solutions were freshly prepared. Specifically, the compounds of the present invention are characterized by an absorption maximum in the range of about 750 nm to 780 nm.

[0184] Table I shows the excitation and emission maxima of representative compounds of formula (I) in PBS and Seronorm and the molar extinction coefficient (ε) in water, and is compared with a known fluorescent probe targeting CA-IX named Hypoxyfluor-1 (HF-1, disclosed in, for example, BioconjugateChem 2018, 29, 3320-3331). Due to certain interactions between the probe and serum proteins, the absorption and emission of the compounds of the present invention in Seronorm show a red shift relative to PBS. In addition, the extinction coefficients of all compounds are higher than 200,000 mM -1 cm -1 .

[0185] Table I - Excitation / emission maxima and molar extinction coefficients (ε) of representative compounds of formula (I) and reference 1

[0186]

[0187] The compounds of the invention are characterized in that their emission maximum, when measured in Seronorm, is only slightly altered compared to that in PBS, indicating a moderate interaction with serum proteins.

[0188] Example 4: Receptor Binding Affinity

[0189] Affinity for the catalytic site of carbonic anhydrase

[0190] The binding affinity of the compounds of formula (I) to the catalytic site of carbonic anhydrase (CA) was determined to assess the targeting efficacy of the molecular vector even after labeling the targeting ligand with the fluorescent moiety of the probe.

[0191] Specifically, the receptor affinity of representative probes with acetazolamide moieties as targeting ligands to the CA catalytic site was determined by testing their binding to recombinant bovine CA-II enzyme (bCAII). Such enzymes are in fact highly available and have catalytic sites with high homology among different carbonic anhydrase isoforms (Aggarwal M. et al., J Enzyme Inhib Med Chem 2013; 28(2): 267-277).

[0192] The dissociation constant (K) of the compounds of formula (I) for bCAII was estimated by performing a fluorescence-based assay utilizing the decrease in fluorescence derived from displacement of carbonic anhydrase-bound dansylamide (DNSA) in the presence of competitor. D), and adapted to 96-well plates according to Wang SC et al., Biochem Mol Biol Educ. 2006, 34(5): 364-368. The assay is based on the fluorescence emission spectrum of DNSA, which shows different peaks depending on the molecular state: when DNSA is free in solution, the emission wavelength is about 570 nm, and when DNSA is bound to CA, the emission wavelength is about 460 nm. In both cases, DNSA is excited at about 320 nm. Therefore, excitation of the aromatic residue (Trp) of carbonic anhydrase at 285 nm produces emission at 340 nm, which is quenched in the presence of bound DNSA. Quenching occurs as FRET (fluorescence resonance energy transfer) to bound DNSA, which is in turn excited and emits at 460 nm. Unbound DNSA that is not near the excited Trp is not affected by this phenomenon and does not produce any background interfering fluorescence. Molecules that bind to CA in the same binding site of DNSA, such as sulfonamides or derivatives, will competitively displace DNSA from CA. The extent of displacement depends on the affinity of the competitor and can be followed by a decrease in fluorescence at 460 nm.

[0193] Specifically, 5-(dimethylamino)-1-naphthalenesulfonamide (Dansamide, DNSA, Sigma-Aldrich 218898) was reconstituted in 20 mM Tris pH 8, aliquoted, and stored at -20° C.; bCAII (recombinant, expressed in E. coli, Sigma-Aldrich) was aliquoted and stored at -20° C. Scalar concentrations of compounds of formula (I) (Compound 1: range 0-9.5 μM; Compound 2: range 0-5 μM), or reference compounds (Reference 2: range 0-21.1 μM; Reference 3: range 0-22.5 μM), or acetazolamide (as an internal control, range 0-22.5 μM) were incubated with 0.25 μM bovine CA-II and 5 μM DNSA in 20 mM Tris pH 8.

[0194] After excitation at 285 nm (Trp excitation wavelength), DNSA fluorescence emission at 462 nm was recorded in each well, followed by selective excitation of DNSA bound to bCAII by FRET. The saturation fraction of bCAII was experimentally determined by normalizing the fluorescence data between 1 (no competing compound) and 0 (maximum DNSA displacement due to the presence of bound competitor) according to the following equation:

[0195]

[0196] Where r is the saturation fraction, FL obs is the recorded fluorescence intensity, FL minis the fluorescence intensity recorded at the maximum compound concentration, and FL max is the fluorescence recorded in the absence of compound.

[0197] The K of the CA-binding compounds was then derived by mathematical fitting of the experimental data according to the following equation D (=KD comp ):

[0198]

[0199] where r is the saturation fraction (calculated from experimental data as described above), [DNSA] is the nanomolar concentration of DNSA, and KD DNSA is the dissociation constant of DNSA at equilibrium (previously experimentally determined = 169.6 nM), and [comp] is the nanomolar concentration of the CA targeting compound being analyzed. Mathematical fits were performed using Microsoft Excel Solver. Acetazolamide was included in each experiment as an internal control for the assay (acetazolamide K D =29.6±2.6 nM, previously estimated).

[0200] All compounds were tested in independent experiments at least in duplicate. Table II summarizes the experimental K D Results (mean values ​​with standard deviations from independent experiments) were compared with corresponding results obtained for reference compounds and unlabeled acetazolamide.

[0201] The tested probes showed very high affinity for the carbonic anhydrase receptor, showing a K D The values ​​were below 15 nM, approximately 2-5 fold lower than acetazolamide, and 4- and 5-fold lower than the corresponding reference compounds with the same cyanine dye but conjugated to different CA-IX targeting moieties (compound 1 was compared with reference 2, and compound 2 was compared with reference 3), respectively.

[0202] Table II - Binding affinity of representative compounds of formula (I) for CA-II receptors compared to unlabeled acetazolamide and compared to reference compounds 2 and 3.

[0203]

[0204] Cell binding assessment

[0205] To further confirm these results showing improved binding affinity, the binding of the probes to CA-IX was assessed using an in vitro model of the human colorectal adenocarcinoma cell line HT-29 (ATCC). Indeed, by Western blotting and flow cytometry, it was demonstrated that HT-29 cells constitutively express high levels of CA-IX under normoxic conditions. Indeed, by using the anti-human CA-IX antibody MAB2188 (R&D Systems) and the DAKO QUIFIKIT, it was estimated, according to the data sheet, that HT-29 cells express approximately 150,000 CA-IX molecules / cell. Therefore, HT-29 cells were used to measure the binding of all compounds to CA-IX, which is expressed on the cell surface in its physiological environment. In short, HT-29 cells were cultured with Cell dissociation reagent (Life Technologies) was used to dissociate the cells, and the cells were collected in DPBS and counted. 5 Place 100 μL of HT-29 cells in a 1.5 mL tube on ice and resuspend in 50-100 μL of cold FACS buffer (eBioscience TM Flow cytometry staining buffer, Invitrogen) containing scalar concentrations of compounds (HF-1, reference 2, and compound 1: range 0-20 μM; reference 3: range 0-24 μM; compound 2: range 0-10 μM). After incubation (1 hour 30 minutes on ice in the dark), cells were washed 3 times in 300 μL cold FACS buffer by centrifugation (5 min, 4 ° C, 350 RCF), and the supernatant was discarded. The cell pellet was then resuspended in 200 μL cold FACS buffer (or in an appropriate volume so that no more than 1000 recorded events per μL were obtained during FACS analysis) and analyzed by flow cytometer Accuri TM C6 (BD Biosciences) analysis. The average cell-associated fluorescence was plotted against the concentration, and the dissociation constant (K) at equilibrium was inferred by mathematical fitting using GraphPad Prism v.9 software according to the following equation: D ):

[0206]

[0207] Among them B max is the maximum specific binding, with the same unit as Y; K D is the equilibrium dissociation constant, in the same units as X; NS is the nonspecific binding divided by the slope, in units of Y units divided by X units; and background is the amount of nonspecific binding when no compound is added.

[0208] All compounds were analyzed in at least three independent experiments. Table III summarizes the calculated K values ​​of representative compounds 1 and 2 of the present invention. D and compared with the K obtained for reference compounds 2 and 3, respectively. D For comparison; representative binding curves are shown in Figure 1 shown.

[0209] Table III - Binding affinity of representative compounds 1 and 2 of the present invention to HT-29 cells expressing CA-IX compared to reference compounds 1, 2 and 3.

[0210]

[0211] This assay demonstrates that compounds of the invention bearing the C8-AZA moiety have high affinity for cells expressing CA-IX. Specifically, compound 1 of the invention exhibited significantly improved affinity for the target, with a K D 2-fold lower than the corresponding reference compound 2, while compound 2 of the present invention showed a K value more than 4-fold lower than the corresponding reference compound 3. D Compound 2 also showed a 2-fold lower K than reference 1 (HF-1). D , while compound 1 was comparable to reference 1.

[0212] Cell Specificity

[0213] For compound 1, additional in vitro binding tests were performed using a fully human recombinant MSC8 antibody to evaluate the specificity of targeting CA-IX, which has been shown to selectively recognize isoform IX of human CA and block CA-IX enzymatic activity (WO2011 / 139375). HT-29 cells were incubated on ice in suspension with compound 1 (at a given concentration of 0.125 μM) and different concentrations of MSC8 (0.1-fold, 1-fold, 10-fold and 20-fold relative to compound 1, i.e., 0.0125 / 0.125 / 1.25 / 2.5 μM); in addition, HT-29 cells were incubated with compound 1 alone, or also with individual MSC8 at various concentrations. The binding results of compound 1 and MSC8 were then recorded as cell-associated fluorescence intensity, where for MSC8, AlexaFluor was used. TM488 (ThermoFischer) labeled secondary antibodies and compared them with the fluorescence intensity generated by the two compounds alone (as 100%). In fact, MSC8 reduced the binding of compound 1 to HT-29 cells in a dose-dependent manner, indicating that it prevented compound 1 from entering the catalytic pocket of CA-IX. Vice versa, compound 1 also reduced the binding of MSC8 in a dose-dependent manner, with a particularly pronounced effect at a 10-fold concentration (i.e., 0.125 μM of compound 1 and 0.0125 μM of MSC8). The results are shown in Figure 2 middle.

[0214] Example 5: Cellular Uptake

[0215] Competition experiments were performed to analyze Receptor-mediated uptake , i.e., whether the uptake of the probe is mediated by binding to the target receptor CA-IX. The compounds were administered to cells (1 μM, 2 hours at 37°C) in the presence or absence of a 100-fold excess of acetazolamide, which competes with the co-administered compounds for CA-IX binding sites. Under the above conditions, the cell-associated fluorescence of each probe dropped to the values ​​reported in Table IV, indicating that most of the recorded cellular uptake was dependent on CA-IX targeting. Specifically, for representative compounds 1 and 2, the cell-associated fluorescence recorded in flow cytometry experiments was shown to be at least 90% dependent on probes bound to cell surface CA-IX, with the remaining approximately 10% attributed to nonspecific cellular uptake.

[0216] Table IV - Residual cellular uptake of representative compounds of formula (I)

[0217] % Uptake in the presence of 100-fold excess of acetazolamide Compound 1 6.5±2.2 Compound 2 11.3±1.7

[0218] Example 6: Binding to albumin

[0219] The affinity of the probes for human serum albumin (HSA) was determined to assess the impact of structural features on their albumin binding properties. Briefly, HSA (A9511, Sigma-Aldrich) was prepared at a 0.5 M stock solution in PBS and used to make a series of dilutions in PBS (0, 9.52, 10, 15, 20, 21, 22, 23) in the presence of 4.76 μM CA-IX-targeted probe (prepared from a 50 μM stock solution in PBS) in a total volume of 0.525 mL. -7 4.76·10 -6 ,9.52·10 -6 , 1.90·10 -5 ,3.81·10 -5 ,5.71·10 -5 ,9.52·10 -5 , 1.43·10-4 , 1.90·10 -4 ,2.86·10 -4 ,3.81·10 -4 M). The samples were centrifuged (10000 g for 30 min at 25°C) in a Microcon device (10 KDa MW cut-off, Amicon Ultra-0.5 centrifugal filter unit with Ultracel-10 membrane) and absorbance measurements of the filtrate were obtained using a spectrophotometer at the wavelength of maximum absorbance of the fluorophore.

[0220] Data analysis was performed by fitting the raw data to the following equation using Microsoft Excel Solver:

[0221]

[0222] Where ΔA / b = measured absorbance (b = 1 cm), K RL = K calculated by fitting A , Δε·R is calculated by fitting t , and [L] = HSA concentration. ΔA / b values ​​were experimentally obtained by subtracting the absorbance of each other sample from the absorbance of the "0 HSA sample". Table V reports the K values ​​of the obtained compounds for HSA A .

[0223] Table V: Affinity for human serum albumin (K A )

[0224] <![CDATA[HSA affinity (K A ,M -1 ) <!-- 23 -->]]> Reference 1 (HF-1) 156,300 Reference 2 14,300 Reference 3 41,700 Compound 1 12,500 Compound 2 9,500

[0225] Notably, representative compounds 1 and 2 showed lower binding to HSA compared to HF-1, which is similar to reference compounds 2 and 3, which share the same parent dye, respectively, and thus they also exhibited better expected bioavailability (K A It is 10 4 M -1 magnitude or lower).

[0226] Example 7: Tumor Imaging Efficacy

[0227] To evaluate the in vivo efficacy of the compounds of the invention, subcutaneously induced hypoxic HT-29 colorectal tumors were used as an animal model due to the well-known hypoxic tumor microenvironment. HT-29 cells were collected and washed once with serum-free medium. Five million HT-29 cells were suspended in 0.1 mL of serum-free medium and injected subcutaneously into the right flank of 5-week-old mice. Tumor development was followed by caliper measurements twice a week starting from 7 days after inoculation. Tumor volume was calculated according to the following formula: (L*W 2) / 2, where L and W are the maximum length and width of the tumor.

[0228] When the tumor is at least 200 mm 3 Volume, optical imaging experiments were performed by IVIS Spectrum system on animals bearing HT-29 tumors, which had previously been administered with References 1, 2 and 3 and Compounds 1 and 2, all at a dose of 1 nmol / mouse, with an administration volume of 0.1 mL (equivalent to about 5 mL / kg for 20 g-mice), at an injection rate of about 1 mL / min.

[0229] For each group, in vivo OI experiments were performed 0.5, 1, 2, 3, 4, 6 and 24 hours after intravenous administration of fluorescent compounds. During the imaging experiment, animals were maintained under gas anesthesia. For each fluorescent image at each time point, a region of interest (ROI) was drawn on the tumor and on the reference background healthy area (hind limb muscle) of the mouse to evaluate the signal intensity in the tissue (expressed as mean radiant efficiency). The ratio between the fluorescent signal in the tumor and the fluorescent signal in the background healthy tissue was then calculated (tumor to background ratio, TBR) to evaluate the contrast.

[0230] The results are shown in Figure 3 In and Table VI, the in vivo TBR values ​​for samples collected 24 hours after administration are reported. All compounds of the invention and references were tested at a dose of 1 nmol / mouse.

[0231] The compounds of the present invention show selective accumulation in tumors, and at the same dose of 1 nmol / mouse, the TBR value is higher than that of the reference compound. Specifically, they show TBR that is 2 to 8.5 times that of reference 1, and 1.5 to 2.5 times that of the corresponding references 2 and 3.

[0232] Table VI - In vivo TBR values ​​24 hours after administration (mean ± SD)

[0233] TBR (mean ± SD) Reference 1 (HF-1) 1.8±0.3 Reference 2 3.4±0.6 Reference 3 4.7±0.6 Compound 1 5.2±1.0 Compound 2 10.1±3.1

[0234] Example 8: Biodistribution and elimination

[0235] At the end of the in vivo OI course of 24 hours after intravenous administration, the in vitro biodistribution of representative compounds 1 and 2 and reference compounds (at 1 nmol / mouse) was evaluated in HT-29 excised tumors and tissues. At the end of the in vivo experiment, animals were euthanized by overdose of gas anesthesia and subsequent cervical dislocation. Tumor tissue, muscle and other organs of interest were taken out for in vitro fluorescence signal measurement. Fluorescence signal intensity was measured by IVIS Spectrum system. Regions of interest (ROIs) were drawn around each organ and tumor using Living Image Software (PerkinElmer), and the signal intensity in the tissue was evaluated (expressed as mean radiant efficiency). The ratio between the fluorescence signals in the organs and tumors was then calculated to evaluate the contrast with the hind limb muscles (representing the background).

[0236] The results are shown in Figure 4 (a) and Table VII, which demonstrate that for the compounds of the present invention injected at a dose of 1 nmol / mouse, the fluorescence signal observed in the tumor area is significantly higher than that of the background (hind limb muscle), and the ratio of tumor to background values ​​is in the range of about 13 to about 23. In contrast, it should be noted that reference 1 (HF-1) at the same dose of 1 nmol / mouse exhibited a much smaller difference between the fluorescence signals in the tumor and in the muscle (background). In addition, compounds 1 and 2 showed TBRs of about 1.5 times that of references 2 and 3, respectively.

[0237] Table VII - Ex vivo TBR values ​​(mean ± SD) for samples collected 24 hours after administration of Compounds 1, 2 and reference compounds.

[0238]

[0239] also, Figure 4 (b) and Table VIII show the accumulation and distribution of the fluorescent probe in anatomical organs, reporting the values ​​of the ex vivo muscle to organ ratio for samples collected 24 hours after administration of representative compounds 1, 2 and reference compounds (1 nmol / mouse). Compound fluorescence showed very low concentrations in most tissues except the kidney (probably due to the renal excretion pathway of the injected compound) and the liver. Some uptake can also be seen in the stomach (which is known to express CAIX in the gastric mucosa).

[0240] Table VIII - Organ to muscle ratio values ​​(mean ± SD) 24 hours after administration of compounds 1, 2 and reference compounds.

[0241]

[0242] References:

[0243] 1. Onda N. et al., Int J Cancer 2016, 139, 673-682

[0244] 2. Tummers Q. et al., PlosOne 2015 (doi.org / 10.1371 / journal.pone.0129766)

[0245] 3. Gioux S. et al., Mol Imaging 2010, 9(5), 237-255

[0246] 4. Bunschoten A. et al., Bioconjugate Chem. 2016, 27, 1253-1258

[0247] 5. Hilvo M. et al., J Biol Chem 2008; 283(41): 27799-27809

[0248] 6. Wykoff et al., Cancer Res. 2000, 60, 7075-7083

[0249] 7. Muz et al., Hypoxia (Auckl) 2015; 3: 83-92

[0250] 8. WO2014 / 128258

[0251] 9. McDonald et al., Am J Clin Oncol 2020; 43(7): 484-490

[0252] 10. Wichert et al., Nat Chem 2015; 7(3): 241-249

[0253] 11.Groves et al., Bioorg Med Chem Lett 2012;22:653-657

[0254] 12. WO2017 / 161195

[0255] 13. Mahalingam SM et al., Bioconjugate Chem 2018, 29, 3320-3331

[0256] 14. TW Green and PGM Wuts, Wiley, NY 2007, 4th edition, Chapter 5

[0257] 15.Aggarwal M. et al., J Enzyme Inhib Med Chem 2013, 28(2):267-277

[0258] 16. Wang SC et al., Biochem Mol Biol Educ. 2006, 34(5): 364-368

[0259] 17. WO2011 / 139375

[0260] 18. WO2021 / 259899

[0261] 19. WO2020 / 229438

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, in R 1 Selected from a straight or branched C1-C 10 Alkyl and a group of formula (II) X is a straight or branched C1-C 10 Alkylene; L is a key or linker; Each Y is independently a straight or branched C1-C 10 alkyl; R 3 is selected from hydrogen and a group optionally substituted by -SO3H, phenyl or -O-phenyl; n is an integer equal to 0 or 1.

2. The compound of formula (I) according to claim 1, wherein L is of the formula -NH-(CH2) p -CO- or a di-residue of one or more moieties selected from the group consisting of amino acids such as, for example, glycine, alanine, β-alanine, lysine, homolysine, ornithine, glutamic acid, aspartic acid, etc.; peptides comprising 2 to 10 amino acids in the L or D configuration; 4-aminomethylbenzoic acid; cysteic acid; polyethylene glycols such as the formula -NH-(O-CH2-CH2) p - or -NH-(O-CH2-CH2) p -CO- or a derivative thereof; amino-polyethylene glycol-carboxylic acid; diaminobutyric acid; and diaminopropionic acid; or it is a group -L1-L2-, wherein L1 is the di-residue of a diamine, such as, for example, the formula -NH-(O-CH2-CH2) p -NH-amino-polyethylene glycolamine or a diresidue of ethylenediamine, propylenediamine, putrescine, spermidine, spermine, hexamethylenediamine, etc.; and L2 is a diresidue of a dicarboxylic acid such as, for example, succinic acid, glutaric acid, suberic acid, adipic acid, etc.; wherein p is an integer from 1 to 20.

3. A compound of formula (I) according to claim 1 or 2, wherein L is of the formula -NH-(CH2) p -CO- or -NH-(O-CH2-CH2) p -CO-, wherein p is an integer from 1 to 20.

4. A compound of formula (I) according to claims 1 to 3, wherein Y is independently selected from:

5. A compound of formula (I) according to claim 4, wherein Y is a group of formula (ii) 6. A compound of formula (I) according to any one of the preceding claims, wherein R 1 It is a group -(CH2)4SO3H or -CH2CH2CH(CH3)SO3H.

7. A compound of formula (I) according to any one of the preceding claims, wherein X is a linear or branched C1-C6 alkylene group.

8. The compound of formula (I) according to claim 1, which is represented by formula (Ia): Where R 1 , R 3 , Y and n are as defined in claim 1.

9. The compound of formula (I) according to claim 1, which is represented by formula (Ib): Where R 1 , X, Y and L are as defined in claim 1.

10. The compound of formula (I) according to claim 1, which is represented by formula (Ic): Where R 1 , X, Y and L are as defined in claim 1.

11. A compound of formula (I) according to any one of the preceding claims for use as a fluorescent contrast agent for the detection and delineation of tumor tissue during diagnostics, interventional imaging and intraoperative procedures.

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

13. The compound of formula (I) for use according to claim 11, wherein the tumor is selected from brain cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, esophageal cancer, skin cancer, stomach cancer, pancreatic cancer, bladder cancer, oral cancer, lung cancer, kidney cancer, uterine cancer, thyroid cancer, liver cancer and colorectal cancer, including primary tumors and regional and distant metastases.

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

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

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