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. The compounds have formula (I). # imgabs0 #

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] Dyes are chemical entities that absorb photons of a specific wavelength after light excitation and re-emit a portion of this energy according to the quantum efficiency (usually at longer wavelengths). Specifically, cyanine dyes are fluorescent organic molecules characterized by a delocalized electron system that spans multiple methyl bridges 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 with dyes with fluorescent emission in the visible spectrum, cyanine dyes that emit in the near-infrared (NIR) region (700-900nm) are particularly suitable for biomedical imaging applications due to their 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. 124 I-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. 124I-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 carbonic anhydrase, 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] 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).

[0023] Preferred conjugation sites with cyanine dyes disclosed in the art are represented by the 1 or 3 positions of the indolenine group in linear Cy5-Cy7 cyanines and Cy7 cyanines with a cyclohexenyl central ring, usually by conjugation at an alkyl chain functionalized with a carboxylic acid or ester. Cy7 cyanines with a cyclohexenyl central ring can also be conjugated to the acetazolamide targeting moiety located at the center (middle) position of the heptamethine backbone, usually by attachment to a benzene ring, optionally by attachment to the heptamethine chain through an inserted oxygen atom, and functionalized in the para position with a carboxyl, formamido or ester group, optionally by inserting a linker. These compounds usually have a symmetrical structure with -SO3H or alkyl-SO3H groups at other positions.

[0024] WO2017 / 161195 (On Target Laboratories) discloses an example of such a derivative, which describes the compound Az-8AOA-LMNIR2, also known as HypoxyFluor-1, which is currently being developed as a fluorescent probe for targeted imaging of tumors with high expression of carbonic anhydrase (CA) IX enzymes, such as optical imaging and surgery involving CA-IX positive tissues and tumors. This compound is also mentioned in, for example, Mahalingam SM et al., Bioconjugate Chem 2018, 29, 3320-3331, where its application in fluorescence imaging of hypoxic tumors is described in detail.

[0025] However, in several cases, conjugation at the central position suffers from 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.

[0026] In general, the use of tumor-targeted fluorescent dyes to help surgeons identify otherwise undetectable tumor nodules, 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 identification of all types of hypoxic tumor cells, thus creating a need for the development of additional CA-IX-specific fluorescent dyes that, utilizing specific targeting at the biomolecular level, can ensure visualization and precise tumor resection of all malignant lesions during cancer surgery.

[0027] 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.

[0028] Unlike the above-mentioned probes, the compounds of the present invention are characterized by Cy7 cyanine, which is conjugated to the acetazolamide moiety at an alternative position of the cyanine backbone, i.e., at the 5-position of at least one indolenine ring. Probes conjugated at said position have surprisingly shown higher affinity for the target CA-IX enzyme and higher stability during their preparation. SUMMARY OF THE INVENTION

[0030] 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.

[0031] The fluorescent probe of the present invention is characterized by a heptamethine cyanine dye, which is conjugated to an acetazolamide moiety at a specific position of the cyanine backbone (i.e., at the 5-position of at least one indolenine ring), thereby providing strong binding results to the target and unexpectedly exhibiting optimal in vitro and in vivo behaviors, and is therefore particularly suitable for molecular imaging.

[0032] 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 albumin, negligible or no adverse reactions observed after systemic administration, good chemical and optical stability in plasma after administration.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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, which comprises administering an effective amount of a compound of the present invention.

[0037] Description of the invention

[0038] Therefore, the first aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0039]

[0040] in

[0041] Each R 1 is independently a straight or branched C1-C10 alkyl;

[0042] R 2 Selected from -SO3H, -CONH-Y (wherein Y is a straight or branched C1-C1- 10 Alkyl) and a group of formula (II)

[0043]

[0044] R 3 is selected from hydrogen and a group optionally substituted by -SO3H, phenyl or -O-phenyl;

[0045] L is a key or linker;

[0046] n is an integer equal to 0 or 1.

[0047] Preferably, the linker L is a group -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 of 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.

[0048] 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.

[0049] The present invention also relates to processes for preparing compounds of formula (I) by means of synthetic transformation steps.

[0050] The invention further comprises compounds of formula (I) for use as fluorescent agents for detecting tumor margins in guided surgery.

[0051] Description of the drawings

[0052] Figure 1 Binding curves of representative compounds 1, 2 and 3 of the present invention and reference compound HF-1 to HT-29 cells expressing CA-IX are shown.

[0053] Figure 2 Competition of the binding of representative compounds 1, 2 and 3 to HT-29 cells by the anti-CA-IX antibody clone MSC8 is shown. In these experiments, compounds 1, 2 and 3 were used at a concentration of 0.1 μM, either alone or in combination with 0.01, 0.1 or 1 μM MSC8.

[0054] Figure 3 Shown are the time-dependent uptake of representative compounds 1, 2 and 3 and the corresponding unconjugated dyes into HT-29 cells at 37°C and after incubation on ice.

[0055] Figure 4 Linear graphs showing the cellular uptake of representative compounds 1, 2, and 3 at 37°C are shown.

[0056] Figure 5 Shown are in vivo tumor to background ratios (TBR) for representative compounds 1, 2, and 3 and reference compound HF-1 in images acquired 24 hours after administration.

[0057] Figure 6 Shown are (a) ex vivo tumor to background ratios (TBR) and (b) organ to muscle ratios for representative compounds 1, 2 and 3 and reference compound HF-1 in samples collected 24 hours after administration.

[0058] One-way analysis of variance (Dunnett's multiple comparison test) was used to Figure 5 and 6 Statistical analysis of TBR in (a), where p<0.05 indicates significant values ​​different from the reference HF-1 (1 nmol / mouse).

[0059] definition

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

[0061] 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.

[0062] 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, hexyl, and the like. 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, and the like. In such a case, the expression "straight or branched C1-C8 alkyl" is used. 10 "Alkylene".

[0063] The term "hydroxyalkyl" denotes any of the corresponding alkyl chains in which one or more hydrogen atoms are replaced by a hydroxy group.

[0064] The term "blocking group" (Pg) represents a protective group suitable for retaining the function of the group combined with it. Specifically, the blocking group is used to retain amino, hydroxyl or carboxyl functions. Suitable blocking groups can include, for example, benzyl, carbonyl, such as formyl, 9-fluoromethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), isopropoxycarbonyl or allyloxycarbonyl (Alloc), alkyl, for example tert-butyl or triphenylmethyl, sulfonyl, acetyl group, such as trifluoroacetyl, benzyl ester, allyl, or are usually used in protecting other substituents of such function, which 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).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Preferably, the fluorescent probes of the present invention are capable of selectively attaching to tumor cells or tissues expressing CA-IX. Specifically, they are capable of attaching to 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 associating the metastatic spread of the above cancers in tissues and organs different from the primary source. Furthermore, the fluorescent probes of the present invention are capable of attaching to pre-neoplastic lesions and dysplasia in different tissues and organs.

[0071] Connector (L)

[0072] According to the present invention, L is an optionally present linker that separates the CA-IX targeting ligand (acetazolamide) from the dye.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] Thus, in a preferred embodiment, when present, the linker is a group -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.

[0077] More preferably, L is selected from the group -NH-(CH2) p -CO-; formula -NH-(O-CH2-CH2) p -CO-; and 1-5 amino acid diresidues, wherein p is an integer from 1 to 20.

[0078] 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.

[0079] The present invention further relates to compounds of the above formula (I), wherein R 1 , R 2 and / or R 3 The sulfonyl group may be in the form of a negatively charged ion or a pharmaceutically acceptable salt.

[0080] Detailed description of implementation plan

[0081] In a preferred embodiment, the present invention relates to compounds of formula (I), wherein R 2 It is the group -SO3H.

[0082] In another preferred embodiment, the present invention relates to compounds of formula (I), wherein R 2is a group -CONH-Y, wherein Y is selected from:

[0083]

[0084] More preferably, the present invention relates to compounds of formula (I), wherein R 2 is a group -CONH-Y and Y is a group of formula (ii) as defined above. Preferably, the group (ii) has the following stereochemical configuration obtained by using D-glucosamine in the preparation of the compound:

[0085]

[0086] In another preferred embodiment, the present invention relates to compounds of formula (I), wherein R 2 is a group (II) as defined above. More preferably, R 2 is a group of the following formula (IIa):

[0087]

[0088] In another preferred embodiment, the two R 1 They are all groups -(CH2)4-SO3H.

[0089] In another preferred embodiment, L is a group -NH-(CH2)7-CO-, represented by the following formula (Ia):

[0090]

[0091] Where R 1 , R 2 , R 3 and n are as defined above.

[0092] In a preferred embodiment, n is 0 and R 3 is hydrogen, represented by the following formula (Ib):

[0093]

[0094] Where R 1 , R 2 and L are as defined above.

[0095] 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):

[0096]

[0097] Where R 1 , R 2and L are as defined above.

[0098] Particularly preferred are the compounds of formula (I) listed in Table I.

[0099] Table I also reports the structure of the reference compound Hypoxyfluor-1 (HF-1), which is a known fluorescent probe with affinity for CA-IX.

[0100] Table I - Preferred compounds of formula (I) and reference compound HF-1

[0101]

[0102]

[0103] The present invention also relates to a synthetic process for preparing a compound of formula (I) as shown in the following description. 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 a compound of formula (I) as defined above, which is used as a fluorescent probe 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.

[0104] 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.

[0105] Preferably, the fluorescent probe of the present invention is capable of selectively connecting to 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.

[0106] 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.

[0107] 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:

[0108] - 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;

[0109] - measuring a signal from the compound in a biological sample;

[0110] - 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.

[0111] 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:

[0112] - contacting said tissue or cell with a compound of formula (I) as defined above;

[0113] - irradiating the tissue or cells at a wavelength absorbed by the imaging agent;

[0114] -Detection of near-infrared emission using a fluorescence camera.

[0115] 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:

[0116] - 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;

[0117] - using near infrared light to illuminate and visualize the compound;

[0118] - Surgical excision of the area that fluoresces after excitation with said near-infrared light.

[0119] 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.

[0120] 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.

[0121] 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 invention may be administered by different routes prior to the imaging procedure.

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] However, in some cases, due to the presence of various functional moieties such as carboxylic acid or amide groups in the cyanines of the invention, it may be necessary to use protecting groups to direct the reaction at the desired functional groups.

[0128] In general, special care is required when manipulating cyanines under the strong pH and temperature conditions required for the removal of protecting groups, 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 the protecting groups.

[0129] Experimental Section

[0130] 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.

[0131] Materials and Equipment

[0132] All commercially available reagents used in the synthesis were obtained from Sigma Aldrich and TCI and used directly without further purification. Reference compound (HF-1) was prepared as described in Mahalingam SM et al., Bioconjugate Chem 2018, 29, 3320-3331.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] List of abbreviations

[0138] DCM Dichloromethane

[0139] DMF N,N-Dimethylformamide

[0140] TEA Triethylamine

[0141] HPLC High Performance Liquid Chromatography

[0142] NMR Nuclear Magnetic Resonance

[0143] t R Retention time (HPLC)

[0144] RT Room temperature

[0145] CV column volume

[0146] DMSO Dimethyl sulfoxide

[0147] PBS Phosphate buffered saline

[0148] C8-AZA 8-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide

[0149] NMM N-Methylmorpholine

[0150] HBTU O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0151] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0152] TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0153] TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0154] COMU (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbonium hexafluorophosphate

[0155] Example 1: Synthesis of Compound 1

[0156] Preparation of intermediate compound (13)

[0157]

[0158] To a solution of compound 11 (502 mg, 1.478 mmol) prepared as described in Example 2 in methanol (7.8 mL) was added concentrated H2SO4 (0.810 mL) and the solution was refluxed for 2 hours. The mixture was cooled to room temperature and concentrated under reduced pressure, diluted with water (pH 2) and passed through a pre-packed C18 silica gel column ( 60g) was purified by flash chromatography with a water-acetonitrile gradient (eluting the desired compound with 28% acetonitrile). After freeze drying, compound 12 was obtained as a light brown solid (447 mg, 86% yield, HPLC purity: 98.7% at 270nm, MS: [M+H] + 354.1).

[0159] Compound 12 (475 mg, 1.345 mmol) and pentadienaldehyde diphenylamine hydrochloride (383 mg, 1.345 mmol) were dissolved in a 4:1 acetic anhydride: glacial acetic acid mixture (10 mL) and heated at 120°C in the dark for 1 hour. Then, compound 11 (456 mg, 1.345 mmol) and potassium acetate (660 mg, 6.725 mmol) were added and diluted with another 10 mL of a 4:1 acetic anhydride: glacial acetic acid mixture. After being kept at 120°C for 30 minutes, the reaction was stopped. The mixture was cooled to room temperature and the solvent was removed by decompression. Cold ether (150 mL) was added and the mixture was stirred in an ice bath for 1 hour. The precipitate was filtered under suction, dissolved in water / acetonitrile and passed through a pre-packed C18 silica gel column ( The product was purified twice by flash chromatography on 400 μl (60 g) with a water-acetonitrile gradient (eluting the cyanine with 30% acetonitrile). The fractions containing the pure product were combined, concentrated under reduced pressure and freeze-dried to give 242 mg of a blue solid (24% yield, HPLC purity: 99.6% at 756 nm and 94.7% at 254 nm, MS: [M+H] + 755.1).

[0160] Preparation of intermediate compound (15)

[0161]

[0162] Compound 13 (242 mg, 0.321 mmol) was coupled with D-glucosamine (70 mg, 0.385 mmol) in the presence of HBTU (146 mg, 0.385 mmol) and DIPEA (112 μL, 0.642 mmol) in DMF (13 mL) and stirred at room temperature for 1 hour in the dark. Then, most of the solvent was removed under reduced pressure and the product was precipitated in cold ethyl acetate (60 mL) and stirred in an ice bath for 1 hour. The precipitate was filtered under suction and used in the next step without further purification (313 mg, HPLC purity: 93.9% at 756 nm, MS: [M+H] + 918.2).

[0163] Compound 14 (313 mg, 0.321 mmol theoretical value) was dissolved in water (50 mL) and hydrolyzed at 40°C for 7.5 hours, maintaining pH = 12 by continuous addition of 1 M NaOH (2.76 mL in total). The mixture was cooled to room temperature, the pH was adjusted to 7.5 with dilute HCl and the aqueous solution was passed through a pre-packed C18 silica gel column ( 30g) was purified by flash chromatography with a water / acetonitrile gradient (eluting the cyanine with 15% acetonitrile). The fractions containing the pure product were combined, concentrated under reduced pressure and freeze-dried to give 135 mg of a blue solid (35% yield over 2 steps, HPLC purity: 99.5% at 756nm and 98% at 254nm, MS: [M+H] + 904.2).

[0164] Preparation of 8-amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)octanamide (C8-AZA)

[0165]

[0166] 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 16 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.

[0167] 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). 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 hydrolyzed acetazolamide (compound 16) (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 product 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.

[0168] In a round bottom flask, the solid of compound 17 thus isolated (294.25 mg, 0.541 mmol) was dissolved in anhydrous DMF (5.8 mL) and piperidine (1.16 mL) was added. Cold ether (100 mL) was added after 30 min; the solid was then filtered, dissolved in methanol, the solution was concentrated to a solid residue and treated with ether (100 mL). Finally, the pure product 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.

[0169] Synthesis of compound 1

[0170]

[0171] In a 50 mL centrifuge tube, compound 15 (15 mg, 0.016 mmol) was dissolved in dry DMF (1.5 mL) and NMM (4 μL, 0.036 mmol) and TSTU (7.5 mg, 0.024 mmol) were added. The dark blue / green solution was stirred at room temperature in the dark for 1 hour, reaching a conversion of 88.1% (at 756 nm). Cold ethyl acetate (25 mL) was added and the suspension was centrifuged at 4010 rpm and 5 ° C for 10 min. Then, the organic layer was decanted, and the solid was suspended in ethyl acetate (10 mL) twice and centrifuged again. Finally, the crude solid was dried under nitrogen atmosphere and then dissolved in a solution of 8-amino-N-(5-sulfamoyl-1,3,4-thiadiazole-2-yl)octanamide (C8-AZA) (6.7 mg, 0.021 mmol) and DIPEA (12 μL, 0.066 mmol) in anhydrous DMF (2 mL). The solution was stirred at room temperature for 1 hour in the dark. Since the NHS ester was not completely converted to the desired product, an excess of C8-AZA (2.5 mg, 0.008 mmol) was added. The solution was stirred for another 30 minutes and then it was added dropwise into cold ether (25 mL). The precipitate was filtered under suction, the crude solid was dissolved in water / acetonitrile and passed through a pre-packed C18 silica gel column ( The fractions containing the pure product were combined, concentrated under reduced pressure and freeze-dried to give 12.5 mg of a blue solid (62% yield, HPLC purity: 98.2% at 756 nm and 97.6% at 254 nm, MS: [M+H] + 1207.2).

[0172] Example 2: Synthesis of Compound 2

[0173] Preparation of intermediate compound 19

[0174]

[0175] To a suspension of 4-hydrazinobenzenesulfonic acid (2.09 g, 0.011 mol) in glacial acetic acid (10 mL) was added 3-methylbutane-2-one (1.5 mL, 0.014 mol) and sodium acetate (1.64 g, 0.020 mol). After stirring at 110 °C for 4.5 hours, the orange solution was cooled to room temperature and precipitated in cold diisopropyl ether (100 mL). The solid was filtered under suction and dissolved with methanol together with SiO2. The methanol was removed and the silica gel was loaded onto a silica gel column and eluted with 30% methanol in ethyl acetate to produce compound 18 as a light brown solid (2.46 g, 94% yield, HPLC purity: 90% 270 nm, MS: [M+H] + 240.1).

[0176] A mixture of compound 18 (1.32 g, 5.51 mmol) and 1,4-butane sultone (0.84 mL, 7.58 mmol) in sulfolane (2 mL) was heated at 120 ° C for 24 hours. Then, the mixture was cooled to room temperature and cold ethyl acetate (50 mL) was added and stirred in an ice bath for 1 hour. Then, the precipitate was filtered under suction and washed twice with cold ethyl acetate (2 x 50 mL). The crude product was dissolved in methanol, dried under vacuum and passed through a pre-packed KP-Sil column ( SNAP, 60 g) and eluted with 50% methanol-ethyl acetate. Compound 19 was recovered as a pink solid (0.573 g, 28% yield, HPLC purity: 98.3% 270 nm, MS: [M+H] + 376.0).

[0177] Intermediate Compounds Preparation of 11

[0178]

[0179] 3-Methylbutane-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 room temperature, concentrated under reduced pressure and suspended in 30 mL of a 9:1 water-methanol mixture. The solid (compound 20) 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 x 125 mL). The organic layer was concentrated under reduced pressure and the orange solid was dried in an oven at 45 ° C for 18 h to give 9.4 g (87% yield, HPLC purity: 98% 270 nm, MS: [M+H] + 204.8).

[0180] A mixture of compound 20 (4.01 g, 19.7 mmol) and 1,4-butane sultone (2.4 mL, 23.5 mmol) in butyronitrile (4 mL) was heated at 120 ° C for 54 hours. The mixture was cooled to room temperature and cold acetone (100 mL) was added and stirred in an ice bath for 1 hour. Then, the precipitate was filtered under suction and washed twice with cold ethyl acetate (2 x 50 mL). The crude product was dissolved in 0.1% ammonium acetate aqueous solution and passed through a pre-packed C18 silica gel column ( SNAP, 60g) was purified by flash chromatography, the desired product was eluted with water, and the by-products were removed with acetonitrile. Desalting was performed on the same silica gel C18 column, the product dissolved in sodium acetate solution was loaded, and washed with 0.05% aqueous formic acid (2 CV), and then washed with water (2 CV). The fractions containing the desired product were concentrated under reduced pressure and freeze-dried to give 4.8g of a pink solid (72% yield, HPLC purity: 98% at 270nm, MS: [M+H] + 340.1).

[0181] Preparation of intermediate compound 21

[0182]

[0183] Compound 19 (100 mg, 0.266 mmol) and pentadienaldehyde diphenylamine hydrochloride (75 mg, 0.266 mmol) were stirred in a 2:1 acetic anhydride-glacial acetic acid mixture (15 mL) at 50 ° C for 1 hour in the presence of sodium acetate (20 mg, 0.252 mmol). Then, compound 11 (144 mg, 0.423 mmol) was added, followed by pyridine (0.36 mL, 4.520 mmol), and the dark mixture was refluxed in the dark for 4 hours. The green solution was cooled to room temperature and the solvent was evaporated under reduced pressure to obtain a viscous residue. Methanol (2 mL) was added to dissolve a portion of the solid, which was then precipitated in cold ether (50 mL) and stirred in an ice bath for 1 hour. The precipitate was filtered under suction, dissolved in water and charged on a pre-packed C18 silica gel column ( 30g) was purified three times with a water-acetonitrile gradient (compound 19 was eluted with 12% acetonitrile). Finally, compound 21 was freeze-dried to give 84 mg of a green solid (40% yield, HPLC purity: 99% at 756nm and 98% at 254nm, MS: [M+H] + 777.2).

[0184] Synthesis of compound 2 (by activation with standard coupling reagents)

[0185]

[0186] Compound 21 (9.8 mg, 0.0126 mmol) was dissolved in anhydrous DMF (4 mL), and then COMU (8.1 mg, 0.0189 mmol), C8-AZA (6.1 mg, 0.0189 mmol) prepared as described in Example 1, and DIPEA (4.5 μL, 0.0252 mmol) were added. The dark green solution was stirred at room temperature in the dark for 30 minutes, then cold ether (50 mL) was added and the mixture was stirred in an ice bath for 30 minutes. The precipitate was filtered under suction, dissolved in water and passed through a pre-packed C18 silica gel column ( 12g) was purified by flash chromatography with a water-acetonitrile gradient (eluting cyanine with 25% acetonitrile). The fractions containing the pure product were concentrated under reduced pressure and freeze-dried to give 7.1 mg of a green solid (52% yield, HPLC purity: 97.3% at 756nm and 98.5% at 254nm, MS: [M+H] + 1080.2).

[0187] Synthesis of compound 2 (via activation to NHS ester)

[0188] Compound 21 (10.1 mg, 0.0129 mmol) was dissolved in anhydrous DMF (4 mL), and TSTU (4.9 mg, 0.0162 mmol) and NMM (3.5 μL, 0.032 mmol) were added. The dark green solution was stirred in the dark at room temperature for 1 hour. Cold ethyl acetate (40 mL) was then added and the mixture was stirred in an ice bath for 30 minutes. The precipitate was filtered under suction and washed with ethyl acetate (2 x 10 mL). The solid (about 90% of NHS active ester) was dissolved in anhydrous DMF (4 mL), and C8-AZA (6.2 mg, 0.019 mmol) and DIPEA (11 μL, 0.0645 mmol) were added and the solution was stirred in the dark for 1 hour. The product was precipitated by adding cold ether (50 mL). The solid was filtered under suction, dissolved in water and passed through a pre-packed C18 silica gel column ( 12g) was purified by flash chromatography with a water-acetonitrile gradient (eluting cyanine with 25% acetonitrile). The fractions containing the pure product were concentrated under reduced pressure and freeze-dried to give 11.5 mg of a green solid (82% yield, HPLC purity: 98.5% at 756nm and 98.5% at 254nm, MS: [M+H] + 1080.2).

[0189] Example 3: Synthesis of Compound 3

[0190] Preparation of intermediate compound 22

[0191]

[0192] Compound 19 (100 mg, 0.266 mmol), compound 11 (90 mg, 0.266 mmol) and potassium acetate (72 mg, 0.744 mmol) were dissolved in a 2:1 acetic anhydride: glacial acetic acid mixture (10 mL) at 50 ° C. Then, 2-chlorocyclohex-1-ene-1,3-dicarbaldehyde (55 mg, 0.319 mmol) was added and the mixture was heated at 100 ° C for 1.5 hours in the dark. The mixture was cooled to room temperature and the solvent was removed under reduced pressure. The viscous residue was partially dissolved in methanol (2 mL) and stirred in an ice bath for 1 hour before precipitating in cold ether (70 mL). The precipitate was filtered under suction, dissolved in water (pH 3), and passed through a pre-packed C18 silica gel column ( 30g) was purified by flash chromatography with a water-acetonitrile gradient (eluting cyanine with 25% acetonitrile). A second purification was performed on the same C18 silica gel column with a water-acetonitrile gradient (eluting cyanine with 18% acetonitrile) at pH 8. Finally, compound 22 was freeze-dried to give 63 mg of a green solid (28% yield, HPLC purity: 99% at 780nm and 95% at 254nm, MS: [M+H] + 851.2).

[0193] Synthesis of compound 3

[0194]

[0195] Compound 22 (57 mg, 0.067 mmol) was dissolved in DMSO (5 mL) and added dropwise to a suspension of 4-hydroxybenzenesulfonic acid (131 mg, 0.670 mmol) and potassium carbonate (92 mg, 0.670 mmol) in DMSO (5 mL). The mixture was stirred overnight at room temperature in a dark place. Then, the mixture was precipitated in cold ethyl acetate (200 mL) and stirred in an ice bath for 1 hour. The precipitate was filtered under suction, dissolved in water, rapidly acidified with dilute HCl at pH 4, and filtered by a pre-packed C18 silica gel column ( 12g) was purified by flash chromatography on a water-acetonitrile gradient (eluting the cyanine with 20% acetonitrile). The fractions containing the pure product were concentrated under reduced pressure and freeze-dried to give 58 mg of a green solid (88% yield, HPLC purity: 99% at 780nm and 94% at 254nm, MS: [M+H] + 989.1).

[0196] In a 25 mL centrifuge tube, compound 23 (14.3 mg, 0.015 mmol) was dissolved in anhydrous DMF (2 mL), and then NMM (4 μL, 0.033 mmol) and TSTU (7 mg, 0.023 mmol) were added. The dark green solution was stirred at room temperature for 1 hour to obtain a conversion of 92.5% at 780 nm. Cold ethyl acetate (20 mL) was added and the suspension was centrifuged at 4010 rpm and 5 ° C for 10 min. Then, the organic layer was decanted, and the solid was suspended in ethyl acetate (10 mL) twice and centrifuged again. Finally, the crude solid was dried under a nitrogen atmosphere and then dissolved in a solution of C8-AZA (6.1 mg, 0.019 mmol) and DIPEA (13 μL, 0.075 mmol) in anhydrous DMF (2 mL). The solution was stirred at room temperature in the dark for 1 hour, but since some NHS ester was still present, an excess of C8-AZA (2.4 mg, 0.0075 mmol) was added. The reaction was stirred at room temperature in the dark overnight. Cold ethyl acetate (25 mL) was then added and the precipitate was filtered under suction. The crude material was dissolved in water and acetonitrile (pH adjusted to 7.5) and filtered through a pre-packed C18 silica gel column ( 12g) was purified twice by flash chromatography with a water-acetonitrile gradient (eluting cyanine with 28% acetonitrile). The pure product was freeze-dried to give 7.9 mg of a green solid (42% yield, HPLC purity: 98.9% at 780nm and 97.9% at 254nm, MS: [M+H] + 1292.1).

[0197] Example 4: Optical Characterization

[0198] 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.

[0199] 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 .

[0200] Table I - Excitation / emission maxima and molar extinction coefficients (ε) of representative compounds of formula (I)

[0201]

[0202] Example 5: Receptor Binding Affinity

[0203] Affinity for the catalytic site of carbonic anhydrase

[0204] 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.

[0205] 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 CAII 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).

[0206] 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, while when DNSA is bound to CA, the emission wavelength shifts to 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 by FRET (fluorescence resonance energy transfer) to bound DNSA, which is in turn excited and emits at 460 nm.

[0207] Unbound DNSA that is not in the vicinity of 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.

[0208] 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 the compound of formula (I) (range 0-9.5 μM) and acetazolamide (range 0-22.5 μM) as an internal control were incubated with 0.25 μM bCAII and 5 μM DNSA in 20 mM Tris pH 8.

[0209] 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 to 1 (no compound) and 0 (maximum DNSA displacement due to the presence of binding competitor) according to the following equation:

[0210]

[0211] Where r is the saturation fraction, FL obs is the recorded fluorescence intensity, FL min is the fluorescence intensity recorded at the maximum compound concentration, and FL maxis the fluorescence recorded in the absence of compound.

[0212] 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 ):

[0213]

[0214] 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).

[0215] All compounds were tested in triplicate. Table II summarizes the results of the assays performed and, for comparative purposes, reports the corresponding results obtained for unlabeled acetazolamide and for the known carbonic anhydrase targeting agent Hypoxyfluor-1 (HF-1).

[0216] The tested probes exhibited very high affinity for the carbonic anhydrase receptor, showing a K D The values ​​were below 7 nM, approximately 5-7 times lower than acetazolamide, and 1.5-2.5 times lower than HF-1.

[0217] Table II - Binding affinity of representative compounds of formula (I) to CAII receptors compared with unlabeled acetazolamide and with the known probe HF-1.

[0218]

[0219] Cell binding assessment

[0220] 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 Cells were detached with cell dissociation reagent (Life Technologies), collected in DPBS and counted. 5 Place 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 (Compound 1: range 1-5 μM; Compounds 2 and 3: range 0-20 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 a suitable 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 ):

[0221]

[0222] Among them B max is the maximum specific binding, with the same units as Y.

[0223] K D is the equilibrium dissociation constant and has the same units as X.

[0224] NS is the slope of nonspecific binding, expressed as Y units divided by X units.

[0225] Background is the amount of nonspecific binding in the absence of added compound.

[0226] All compounds were analyzed in at least three independent experiments. The calculated K D The K values ​​obtained for HF-1 are summarized in Table III and compared with those obtained for HF-1. D The binding curves are shown in Figure 2. Figure 1 shown.

[0227] Table III - Binding affinity of representative compounds of Formula (I) to HT-29 cells expressing CA-IX compared to HF-1.

[0228]

[0229] This assay demonstrated that compounds of the invention bearing the C8-AZA moiety have high affinity for cells expressing CA-IX, with a K of less than 70 nM. D , and exhibits significantly improved binding characteristics compared to HF-1, which carries the same moiety but is located in the middle (center) position of the cyanine backbone.

[0230] Cell Specificity

[0231] For all compounds, additional in vitro experiments were performed to evaluate the specificity of targeting CA-IX by using the fully human recombinant MSC8 antibody, which has been shown to selectively recognize isoform IX of human CA and block CA-IX enzymatic activity (WO2011 / 139375). HT-29 cells were co-incubated on ice in suspension with compounds 1 / 2 / 3, which had a defined concentration of 0.1 μM, alone or in combination with different concentrations of MSC8 (0.1-fold, 1-fold and 10-fold relative to the compound). The binding results of compounds 1 / 2 / 3 were then recorded as cell-associated fluorescence intensity and expressed as a percentage relative to the cell-associated fluorescence generated by the sample incubated with compounds 1 / 2 / 3 in the absence of MSC8 (taken as 100%). Indeed, MSC8 reduced the binding of all compounds to HT-29 cells in a dose-dependent manner, indicating that it prevented them from accessing the catalytic pocket of CA-IX, thus confirming their specificity of CA-IX targeting. The results are shown in Figure 2 middle.

[0232] Example 6: Cellular Uptake

[0233] HT-29 cells were used as an in vitro model to evaluate the cellular uptake of the compounds targeting CA-IX of the present invention, more specifically, whether cellular internalization of the compounds occurred after targeting CA-IX expressed on the cell surface. Cellular uptake was evaluated by flow cytometry. In brief, the compounds were diluted to a working concentration of 1 μM in serum-free medium and supplemented with 25 mM HEPES pH 7.4; in competition experiments, 100-fold excess of acetazolamide (Sigma-Aldrich A6011, dissolved in DMSO at a stock concentration of 10 mg / mL and stored in aliquots at -20°C) was added to the same treatment medium. Nonspecific cellular uptake not related to CA-IX was estimated using unconjugated dye treatment. Cell-associated fluorescence (37°C) from internalized compounds and cell-surface-associated compounds was distinguished from cell-associated fluorescence (on ice) from individual cell-surface-bound compounds using cell incubation on ice. HT-29 cells grown in 24-well plates were treated by rinsing them in DPBS and then incubating them in the dark with the treatment solutions for the indicated times and temperatures. At the end of the treatment, the cells were rinsed three times with cold DPBS and The cell dissociation reagent was removed from the plate, collected in a 1.5 mL tube after dilution in DPBS and centrifuged (5 minutes, 350 RCF, 4°C). The supernatant was then discarded and the cell pellet was resuspended in 300 μL of cold FACS buffer or in a suitable volume to obtain no more than 1,000 recorded events per μL during FACS analysis. Using a 640 nm excitation laser and a 780 / 60 nm emission filter, the flow cytometer Accuri TM C6 (BD Biosciences) analyzes samples. At least 15,000 valid events were collected for each sample. At the end of the analysis, each sample was plotted into a histogram, where X represents the fluorescence intensity (FL4-A) on a logarithmic scale and Y represents the count. Typically, a Gaussian distribution of cell populations was observed. The data obtained is the average fluorescence of each population, which is considered to be the fluorescence intensity value of the corresponding sample. Each experimental condition was independently repeated. The normalized cell-associated fluorescence was calculated using the following formula:

[0234]

[0235] In experiments where the effect of excess acetazolamide had to be compared with standard uptake conditions (ie in serum-free medium), the data were expressed as a percentage of the uptake under standard conditions.

[0236] For evaluation Time-dependent uptake, on ice (blocking internalization) and at 37 ° C (temperature allowing internalization) with 1 μ M compound concentration for 1 to 6 hours of cell treatment experiments, to distinguish the cell-related fluorescence from the internalized compound from the fluorescence generated by the cell surface-related compound. The cell surface-related fluorescence remains roughly constant over time, while the binding of the corresponding unconjugated dye is undetectable. In contrast, for compounds 1, 2 and 3 of the present invention, the total cell-related fluorescence increases over time, indicating that they are gradually internalized. The internalization potential is calculated as the percentage of internalized compounds after 2 hours of cell treatment (arbitrary selection) at 37 ° C.

[0237] These results are reported in Table IV together with the slopes of the fitted lines relating to the increase in cell-associated fluorescence, which can be considered to represent the accumulation trends of the different compounds.

[0238] Table IV - Internalization Potential of Representative Compounds of Formula (I)

[0239]

[0240]

[0241] also, Figure 3 and 4 The time-dependent uptake of compounds 1, 2 and 3 and the corresponding unconjugated dye at 37°C and after incubation on ice are reported, respectively ( Figure 3 ) and a linear schematic diagram of the cellular uptake of compounds 1, 2, and 3 at 37°C ( Figure 4 ).

[0242] For analysis Receptor-mediated uptake To investigate whether the uptake of the probes was mediated by binding to the target receptor CA-IX, competition experiments were performed. Compounds were administered to cells (1 μM, 2 h at 37°C) in the presence or absence of a 100-fold excess of acetazolamide, which competed with the co-administered compounds for CA-IX binding sites. Under the above conditions, the cell-associated fluorescence of each probe decreased to the values ​​reported in Table V, indicating that the majority of the recorded cellular uptake was dependent on CA-IX targeting.

[0243] Table V - Residual cellular uptake of representative compounds of formula (I)

[0244] % Uptake in the presence of a 100-fold excess of acetazolamide Compound 1 16.4±1.9 Compound 2 28.4±2.2 Compound 3 18.1±11.5

[0245] Example 7: Tumor Imaging Efficacy

[0246] 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.

[0247] When the tumor is at least 200 mm 3 Optical imaging experiments were performed by IVIS Spectrum system on HT-29 tumor-bearing animals that had previously been administered HF-1 (as a reference compound) and compounds 1, 2 and 3, 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.

[0248] 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.

[0249] The results are shown in Figure 5 In and Table VI, the in vivo TBR values ​​for samples collected 24 hours after administration are reported. All compounds of the invention and HF-1 were tested at a dose of 1 nmol / mouse.

[0250] The compounds of the present invention showed selective accumulation in tumors, and at the same dose of 1 nmol / mouse, the TBR value was 1.2 to 3.2 times that of the reference compound HF-1.

[0251] Table VI - In vivo TBR values ​​24 hours after administration (mean ± SD)

[0252] TBR (mean ± SD) HF-1(1nmol) 1.8±0.3 Compound 1 5.9±1.2 Compound 2 5.1±1.9 Compound 3 2.2±0.6

[0253] Example 8: Biodistribution and elimination

[0254] At the end of the in vivo OI course of 24 hours after intravenous administration, the in vitro biodistribution of representative compounds 1, 2 and 3 and reference compound HF-1 (at 1 nmol / mouse) was evaluated in HT-29 excised tumors and tissues. At the end of the in vivo experiment, the animals were euthanized by overdose of gas anesthesia and subsequent cervical dislocation. Tumor tissue, muscle and other organs of interest were removed 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 signal intensity in tissues was evaluated (expressed as mean radiant efficiency). The ratio between the fluorescence signals in organs and tumors was then calculated to evaluate the contrast with hindlimb muscle (representing background).

[0255] The results are shown in Figure 6 (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), with the ratio of tumor to background value being higher than about 6.

[0256] In contrast, it should be noted that the reference compound HF-1 at the same dose of 1 nmol / mouse showed a much smaller difference between the fluorescence signals in the tumor and in the muscle (background).

[0257] Table VII - Ex vivo TBR values ​​(mean ± SD) for samples collected 24 hours after administration of compounds 1, 2 and 3 and reference compound HF-1.

[0258]

[0259] also, Figure 6 (b) and Table VIII show the accumulation and distribution of the fluorescent probes in dissected organs, reporting the ex vivo organ to muscle ratio values ​​for samples collected 24 hours after administration of representative compounds 1, 2, and 3 and reference compound HF-1 (1 nmol / mouse).

[0260] Compound fluorescence showed very low concentrations in most tissues except the kidney (probably due to renal excretion of unbound dye) and liver. Some uptake was also seen in the stomach, which is known to express CA-IX.

[0261] Table VIII - Organ to muscle ratio values ​​(mean ± SD) 24 hours after administration of compounds 1, 2 and 3 and reference compound HF-1.

[0262]

[0263] References:

[0264] 1. Onda N. et al., Int J Cancer 2016, 139: 673-682

[0265] 2. Tummers Q. et al., PlosOne 2015 (doi.org / 10.1371 / journal.pone.0129766)

[0266] 3. Gioux S. et al., Mol Imaging 2010, 9(5): 237-255

[0267] 4. Bunschoten A. et al., Bioconjugate Chem. 2016, 27: 1253-1258

[0268] 5. Hilvo M. et al., J Biol Chem 2008; 283(41): 27799-27809

[0269] 6. Wykoff et al., Cancer Res. 2000, 60: 7075-7083

[0270] 7. Muz et al., Hypoxia (Auckl) 2015; 3: 83-92

[0271] 8. WO2014 / 128258

[0272] 9. McDonald et al., Am J Clin Oncol 2020; 43(7): 484-490

[0273] 10. Wichert et al., Nat Chem 2015; 7(3): 241-249

[0274] 11.Groves et al., Bioorg Med Chem Lett 2012;22:653-657

[0275] 12. WO2017 / 161195

[0276] 13. Mahalingam SM et al., Bioconjugate Chem 2018, 29: 3320-3331

[0277] 14.Aggarwal M. et al., J Enzyme Inhib Med Chem 2013, 28(2):267-277

[0278] 15. Wang SC et al., Biochem Mol Biol Educ. 2006, 34(5): 364-368

[0279] 16. WO2011 / 139375.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, in Each R 1 is independently a straight or branched C1-C 10 alkyl; R 2 Selected from -SO3H; a group -CONH-Y, wherein Y is a straight or branched C1-C 10 Alkyl; and group (II) R 3 is selected from hydrogen and a group optionally substituted by -SO3H, phenyl or -O-phenyl; L is a key or linker; n is an integer equal to 0 or 1.

2. A 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 a group -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 R 2 It is -SO3H.

5. A compound of formula (I) according to claims 1 to 3, wherein R 2 is a group -CONH-Y, wherein Y is selected from:

6. A compound of formula (I) according to claim 5, wherein Y is a group 7. A compound of formula (I) according to any preceding claim, wherein both R 1 It is the group -(CH2)4-SO3H.

8. The compound of formula (I) according to claim 1, which is represented by formula (Ia): Where R 1 , R 2 , R 3 and n is as defined in claim 1.

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

10. The compound of formula (I) according to claim 1, which is represented by formula (Ic): Where R 1 , R 2 and L is 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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