Modified cyanine dyes and their conjugates

Abstract

The present invention relates to the field of optical imaging. More specifically, the present invention relates to compounds of the cyanine family, characterized by improved physicochemical and biological properties, and their conjugates with biological ligands. The present invention also relates to the use of these compounds as optical diagnostic agents in the imaging or treatment of solid tumors, methods for preparing the compounds, and compositions containing the compounds.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of optical imaging. More specifically, the present invention relates to compounds of the cyanine family, characterized by improved physicochemical and biological properties, and their conjugates with biological ligands. The present invention also relates to the use of these compounds as optical diagnostic agents in imaging or treating solid tumors, to methods for producing these compounds, and to compositions containing these compounds. [Background technology]

[0002] Background technology Dyes are chemicals that, upon excitation with light, absorb photons of a certain wavelength and re-emit part of their energy, usually at a longer wavelength, depending on the quantum efficiency. In particular, cyanine dyes are fluorescent organic molecules characterized by a delocalized electron system confined between two nitrogen atoms, spanning a polymethine bridge. Some of them have favorable optical properties, low toxicity and good solubility in aqueous media, allowing them to be used as contrast agents for biomedical imaging.

[0003] Fluorescent molecules currently used in biomedical imaging, such as indocyanine green (ICG), fluorescein, methylene blue, and 5-aminolevulinic acid (5-ALA), can passively diffuse in tissues and accumulate in pathological areas by various homing mechanisms. Fluorophores such as fluorescein and ICG distribute to 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). Metabolic intermediates such as 5-ALA accumulate in tissues with increased metabolism (Stummer W. et al., Neurosurgery 2015, 77(5), 663-673). Other fluorophores, not specific to a particular molecular target, exploit the physiological properties of tumors (i.e., enhanced perfusion and permeability) to generate image contrast, following the principles of contrast-enhanced X-ray or magnetic resonance imaging (Licha et al., Photochemistry and Photobiology, 2000, 72(3), 392-398). In general, these fluorescent contrast agents require relatively large doses for adequate visualization. Moreover, false positives and false negatives are common clinical findings associated with their use (Tummers Q. et al., PlosOne 2015).

[0004] Additional contrast agents for optical imaging are under development that utilize dyes conjugated to carrier moieties that target overexpressed tumor receptors to improve the sensitivity and specificity of detection (Achilefu S. et al, J Med Chem 2002, 45, 2003-2015).

[0005] However, the in vivo behavior of dye-biomolecule conjugates can be strongly influenced by the biological properties of the fluorescent moiety. For example, small structural changes in the cyanine Cy5 strongly modulate the accumulation of related bioconjugates in tumors and off-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 biological applications.

[0006] US 6,913,743 B2 (Institut fur Diagnostikforschung GmbH) for example discloses an in vivo diagnostic process using novel water-soluble dyes and their biomolecular adducts as contrast agents.

[0007] WO2018 / 189136 and WO2016 / 097317 (both to Bracco Imaging SpA) disclose the compound DA364, which is an aza-bicycloalkane-based cyclic RGD peptide conjugated with a sulfo-Cy5.5 dye, for use in demarcating tumor margins in intraoperative imaging.

[0008] An example of a conjugate of ICG bound to a cRGD peptide is disclosed in Capozza et al, Photoacoustics, 2018, 11, 36-45, which is used for photoacoustic imaging of tumors.

[0009] US 6,329,531 B1 (Schering AG) relates to optical diagnostic agents for the diagnosis of neurodegenerative diseases by NIR radiation, in particular heptamethine cyanine dyes are disclosed as fluorescent labels. Some of these cyanine dyes are disclosed, for example, in Licha et al., Photochemistry and Photobiology, 2000, 72(3), 392-398 and Ebert et al., J. Biomed. Optics, 2011, 16(6), 066003, and compared with the clinically approved dye ICG. In particular, the latter discloses a compound called SIDAG, which shows similar physicochemical properties as MRI contrast agents and, unlike ICG, is characterized by an extravascular distribution with a distribution volume in the range of extracellular water. SIDAG is a hydrophilic alternative to ICG, designed to solve the technical problems of its negligible extravasation and rapid liver uptake. However, there is no mention of the possible use of such compounds, especially SIDAG, as fluorescent probes for molecular imaging, and of any synthetic approaches for functional derivatization or further conjugation of such symmetric cyanine derivatives. For example, SIDAG was proposed as a biologically inert dye with an extravascular distribution pattern that mimics the behavior of MRI contrast agents such as gadopentetinic acid (Gd-DTPA). Although SIDAG can reach deeper regions of tumor tissues by passive diffusion, as it rapidly diffuses through the pores of altered tumor blood vessels, it lacks the necessary features for molecular imaging applications, such as the ability to internalize and accumulate with high efficiency and specificity in diseased cells and tissues.

[0010] WO2005 / 123768 (Amersham Health AS) describes novel peptide-based compounds labelled with at least one cyanine dye reporter suitable as optical imaging contrast agents for the diagnosis of angiogenesis-related diseases.

[0011] WO2012 / 088007 (Pierce Biotechnology Inc. and Dyomics Gmbh) discloses cyanine dyes having 2 to 4 sulfonic acid functional groups on an indole heterocyclic skeleton. Summary of the Invention [Problem to be solved by the invention]

[0012] Although some efforts have been made to find suitable contrast agents, there remains a need to find improved dyes with optimal stability and fluorescence efficiency, as well as optimal physicochemical and biological properties, designed for optical imaging of living organisms. This need is particularly important for molecular imaging applications, which require highly specific tools with the ability to image subtle molecular changes and accumulate in target cells with high efficiency, such as when the dyes are conjugated to molecular epitopes or biomolecules that specifically bind to diseased tissues (e.g., tumors). The present invention addresses these and other needs. [Means for solving the problem]

[0013] In general, the object of the present invention is to provide novel cyanine dyes, in particular their corresponding conjugates to binding moieties, useful as contrast agents for optical imaging and aimed at solving the above-mentioned problems. In particular, the present invention addresses the technical problem of obtaining optical imaging agents with optimal properties for molecular imaging applications. For example, it has been found that the novel cyanine derivatives of the present invention exhibit particularly suitable properties for interacting with biological substrates and accumulating in pathological cells and tissues.

[0014] The novel cyanine derivatives described herein surprisingly possess remarkable optical properties that lead to improved imaging efficiency at low doses and improved signal-to-noise ratios. Furthermore, the inventors have discovered suitable procedures for synthesizing novel cyanine dyes and for conjugating them with various targeting moieties via suitable functional groups acting as binding sites, providing highly specific and sensitive contrast agents for optical imaging. The novel combinations of dyes, spacers and targeting moieties provided in the present invention surprisingly resulted in compounds with optimal cellular internalization efficiency for optical imaging agents and overall properties for molecular imaging.

[0015] In detail, among the several advantages that can be achieved by the compounds of the invention, in particular for conjugates with binding moieties, the following features can be highlighted, for example: low interaction with plasma proteins, selectivity for the target tissue with low accumulation due to non-specific interactions with other tissues, high solubility in water, negligible or no observed side effects after systemic administration, good chemical and optical stability in plasma after administration.

[0016] In particular, the compounds of the invention were found to have a very low binding affinity for human albumin, which is particularly advantageous when these compounds are used for imaging in humans: this low affinity prevents the compounds from being sequestered in the plasma compartment by large proteins present in the blood, such as albumin, and thus reducing the proportion of free dye available for accumulation in the tissue of interest. In fact, the high molecular weight of albumin determines the behavior of the bound dye, so that the albumin-bound fraction of the dye accumulates in the tissue with the transport rate of macromolecules, causing undesirable non-specific accumulation in healthy tissues and organs. This biological property is particularly important in the case of dye conjugates, since only their free fraction (not bound to albumin) can properly interact with molecular targets and be efficiently internalized by cells expressing a specific antigen.

[0017] A further aspect of the invention relates to such dye conjugates as diagnostic agents, in particular for use in optical imaging methods for human or animal organs or tissues, where the imaging is tomographic imaging of an organ, monitoring of organ function including angiography, urography, cholangiography, 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 ultrasound fluorescence methods.

[0018] Furthermore, the present invention relates to manufacturing processes for the preparation of the provided dyes, corresponding conjugates and / or pharma- ceutically acceptable salts thereof, and to their use in the manufacture of diagnostic agents.

[0019] According to a further aspect, the present invention relates to a pharma- ceutically acceptable composition comprising at least one dye or dye conjugate compound of the present invention, or a pharma- ceutically acceptable salt thereof, in admixture with one or more physiologically acceptable carriers or excipients, said composition being particularly useful as an optical imaging agent for providing useful imaging of human or animal organs or tissues.

[0020] In another aspect, the present invention relates to a method for optical imaging of an organ, tissue or region of the body by use of optical imaging techniques, comprising the use of an effective amount of a compound of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description of the Invention Accordingly, a first object of the present invention is to provide a compound of formula (I) or a pharma- ceutically acceptable salt thereof. [ka] During the ceremony, R1 and R2 are independently -CO-NH-Y groups, where Y is selected from linear or branched alkyl, cycloalkyl and heterocyclyl substituted with at least two hydroxyl groups; R3 is a straight or branched chain alkyl substituted with a group selected from -NH2, -SO3H, -COOH and -CONH2; R4 is a straight or branched chain divalent alkyl; R5 is selected from -COO- and -CONH-; S is a spacer; T is a targeting moiety; n is an integer equal to 1, 2, or 3; m is an integer equal to 0 or 1.

[0022] The present invention further provides the corresponding functionalized dye represented by the intermediate compound of formula (II), or a pharma- ceutically acceptable salt thereof. [ka] During the ceremony, R1, R2, R3, R4, and n are as defined above; R5' is selected from -COOH, -CONH2, -COO-Pg, and -CONH-Pg, where Pg is a protecting group.

[0023] The present invention also relates to processes for preparing compounds of formula (I) or (II) by synthetic transformation processes.

[0024] The present invention also includes compounds of formula (I) for use as fluorescent probes for detecting tumor margins in guided surgery.

[0025] definition As used herein, and unless otherwise specified, the following terms used herein are intended to have the following meanings.

[0026] The term "straight or branched chain alkyl" refers to an aliphatic hydrocarbon group, which may be a straight or branched chain having 1 to 8 carbon atoms in the chain. For example, "C4 alkyl" includes within its meaning straight or branched chains containing 4 carbon atoms. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, pentyl and hexyl. Unless otherwise specified, straight or branched chain alkyl is a monovalent group. In some cases, it may be a "divalent" or "polyvalent" group (e.g., methylene, ethylene, isopropylene groups, etc.) in which two or more hydrogen atoms have been removed and replaced from the above-mentioned hydrocarbon group.

[0027] The term "cycloalkyl" as used herein includes within its meaning saturated (i.e., alicyclic) carbocyclic rings containing from 3 to 7 carbon atoms. Suitable examples include C5-C7 carbocyclic rings (e.g., cyclohexyl rings).

[0028] The term "heterocyclyl" as used herein includes saturated alicyclic rings, preferably 5- to 7-membered saturated rings, further containing a heteroatom selected from N, O and S in the cyclic chain. Preferably, it refers to tetrahydropyran.

[0029] The term "hydroxyalkyl" refers to any corresponding alkyl chain in which one or more hydrogen atoms have been replaced with a hydroxyl group.

[0030] The term "alkoxy" includes within its meaning an alkyl chain as defined above which further comprises one or more oxygen atoms, and includes, for example, alkyl-oxy groups such as methoxy, ethoxy, n-propoxy, iso-propoxy, and alkyl-(poly)oxy groups in which the alkyl chain is interrupted by one or more oxygen atoms.

[0031] As used herein, the term "protecting group" (Pg) refers to a protecting group suitable for preserving the functionality of the group to which it is attached. Specifically, protecting groups are used to preserve amino, hydroxyl, or carboxyl functionality. Suitable protecting groups include, for example, benzyl, carbonyl (such as formyl, 9-fluoromethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc), isopropyloxycarbonyl, or allyloxycarbonyl (Alloc)), alkyl (e.g., tert-butyl or triphenylmethyl), sulfonyl, acetyl groups such as trifluoroacetyl, benzyl esters, allyl, or other substituents commonly used for the protection of such functionality that are well known to those skilled in the art (see, for example, TW Green and PGM Huts, Protective Groups in Organic Synthesis, Wiley, NY 2007, 4 th Ed., Ch. 5).

[0032] More specifically, the present invention includes compounds of formula (II) in which the R5' functional group, i.e., carboxylic acid or carboxamide, is protected with a suitable protecting group (Pg) as defined above, preferably an alkyl group. Such derivatives are used, for example, as suitable precursor or intermediate compounds in the preparation of the desired compounds of formula (I) or salts thereof.

[0033] If necessary, in the preparation of the compounds of formula (I) or (II), the hydroxyl groups of R1 and R2 and / or the functional groups of R3 may be protected with a suitable protecting group (Pg) to form, for example, an acetoxy, alkoxy, ester or amide group.

[0034] The expression "coupling reagent" refers, for example, to a reagent used for the formation of an amide bond between a carboxyl moiety and an amino moiety. The reaction can consist of two successive steps: activation of the carboxyl moiety, followed by acylation of the amino group with an activated carboxylic acid. Non-limiting examples of such coupling agents are selected from the group consisting of carbodiimides (e.g., N,N-diisopropylcarbodiimide (DIC), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC)); phosphonium reagents (e.g., (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate (PyAOP), [ethylcyano(hydroxyimino)acetato-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP) and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) etc.);and aminium / uronium-imonium reagents (e.g., O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), N,N,N',N'-tetramethyl-O-(benzotriazole-1 -yl)uronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (ΗBTU), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (ΗATU), 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) and fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TFFH), etc., or other compounds well known to those skilled in the art.;

[0035] The term "activated carboxylic acid" refers to a derivative of the carboxyl group that is more susceptible to nucleophilic attack than the free carboxyl group; suitable derivatives include, for example, acid anhydrides, thioesters, acyl halides, NHS esters and sulfo-NHS esters.

[0036] The term "moiety" or "residue" is intended herein to define the remaining portion of a given molecule when suitably attached or conjugated to the remainder of the molecule, either directly or via a suitable linker and / or spacer.

[0037] The term "imaging agent", when used in connection with an appropriate diagnostic imaging technique, refers to a detectable entity that can be used to visualize or detect in vitro or in vivo biological components including cells, biological fluids and biological tissues derived from a living mammalian patient, preferably a human patient, as well as organs, regions or tissues of the human body.

[0038] Targeting moiety (T) According to the present invention, a targeting moiety (T) is a molecule that binds to a biological target with specific selectivity and promotes the accumulation of the contrast agent in a specific tissue or part of the body. In general, a targeting moiety is represented as a natural or synthetic molecule for use in biological systems. Such specific binding is achieved through a ligand, such as, for example, a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or a fragment of an antibody, or an aptamer, which interacts with a specific biological target expressed on the surface of the tissue or cell of interest.

[0039] Suitable biological targets of the compounds of the invention include, for example, epidermal growth factor (EGF) receptors (such as EGFR or HER2); vascular endothelial growth factor (VEGF) receptors (such as VEGFR1 or VEGFR2); carbonic anhydrases (CA) (such as CAIX, CAII or CAXII); mucin glycoproteins (such as MUC1); glucose transporters (such as GLUT-1); sodium hydrogen antiporters (such as NHE1); carcinoembryonic glycoproteins (such as carcinoembryonic antigen (CEA)); chemokine receptors (such as chemokine receptor type 4 (C XCR4); cell adhesion molecules (ICAM, EPCAM, VCAM, E-selectin, P-selectin, etc.); hepatocyte growth factor HGFR (c-met); receptor for transferrin; ephrin receptors (EPHA2, etc.); receptor for folate (FR-alpha, etc.); glycoprotein-bound ialuronate (CD44, etc.); bombesin receptors (BB1, BB2, BB3, etc.); N-acetyl-L-aspartyl-L-glutamate (NAAG) peptidases (prostate-specific membrane antigen (PSMA), etc.); and especially integrin receptors (α v β3, α v β5, α v β6 or α5β1 integrin receptors, etc.

[0040] For example, integrin receptor targeting moieties are represented by linear or cyclic peptides containing the sequence Arg-Gly-Asp (RGD). This tripeptide has high binding specificity for the receptor and is recognized as a ligand by the family of integrin receptors present in the cell membrane. Indeed, it has been identified in extracellular matrix glycoproteins, such as fibronectin and vitronectin, which utilize this RGD motif to mediate cell adhesion.

[0041] Thus, linear and cyclic peptides and peptidomimetics containing the sequence Arg-Gly-Asp (RGD), such as, for example, cRGD, cRGDfK, cRGDyK, cRGDfC, RGD-4C, RGD-2C, AH111585, NC100692, RGD-K5 (Kapp et al., Sci Rep, 2017, 7:3905), or their analogues and derivatives, are well-known examples of binding motifs that target cancer tissues, where cell membrane integrins are upregulated compared to healthy tissues.

[0042] In one embodiment, the compounds of the invention can be conjugated to other small molecules, peptides, proteins or antibodies (such as monoclonal antibodies already used in therapy). Small molecules including the drug acetazolamide (e.g. compounds 4a, 5a, 6a, 7a and 8a (Wichert et al., Nat Chem 2015, 7: 241-249), or analogs and derivatives thereof, are examples of small molecules that target the enzyme CAIX. Linear and cyclic peptides and peptidomimetics (peptide GE11 (described in Li et al., FASEB J 2005, 19:1978-85) and / or peptide L1 (Williams et al., Chem Biol Drug Des 2018, 91:605-619), or their analogs and derivatives, are examples of peptides that target the epidermal growth factor receptor (EGFR). In proteins, derivatives of epidermal growth factor (EGF) are examples of small proteins that target the epidermal growth factor receptor (EGFR). In antibodies, panitumumab and cetuximab are examples of monoclonal antibodies that target the epidermal growth factor receptor (EGFR).

[0043] Preferably, the targeting ligand of the present invention can selectively bind to tumor cells or tissues.In particular, it can bind to tumors 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.Furthermore, the targeting ligand can bind to the metastatic spread of the above cancer in various tissues and organs different from the origin of the cancer.Furthermore, the targeting ligand can bind to preneoplastic lesions and dysplasia in various tissues and organs.

[0044] In one embodiment, the targeting moiety can also be a chelator that complexes and tightly binds to metals. Examples of such metal chelators are known to those skilled in the art and have been reported in the prior art, and include molecules such as DOTA, NODAGA, TETA, CB-TE2A, EDTA, DTPA, deferoxamine, NOTA, and AAZTA. These chelators are capable of binding Gd 3+ Or Mn 2+ and the like, and can be used as contrast agents in magnetic resonance imaging. In a preferred embodiment, the chelating agent is used to form a complex with a radioactive metal (such as technetium, indium, aluminum fluoride, zirconium, yttrium, lutitium, scandium, attenuium, gallium, or copper) and can be used as a contrast agent in, for example, positron emission tomography, single photon emission computed tomography, and gamma counting analysis.

[0045] Spacer S According to the present invention, S is an optional spacer that separates the targeting moiety and the dye.The presence of a spacer is particularly relevant in embodiments where there is a risk that the targeting moiety and the dye will interact unfavorably with each other.Furthermore, the presence of a spacer may be necessary when the dye is relatively large and may interfere with the binding of the targeting moiety to the target site.

[0046] The spacer can be flexible (e.g., a simple alkyl chain) or rigid (e.g., a cycloalkyl or aryl chain) to position the dye away from the target. The spacer can also modify the pharmacokinetics and metabolism of the conjugates of formula (I) used as imaging agents in vivo.

[0047] A hydrophilic spacer can reduce interactions with plasma proteins, reduce blood circulation time, and facilitate excretion. For example, when the spacer is a polyethylene glycol (PEG) moiety, the pharmacokinetics and blood clearance rate of the imaging agent in vivo can be altered. In such an embodiment, the spacer can improve the excretion of the imaging agent from background tissues (i.e., muscle, blood), thus allowing for better diagnostic images with higher target-to-background contrast. Furthermore, the introduction of certain hydrophilic spacers can shift the excretion of the imaging agent from the liver to the kidney, thus reducing systemic retention.

[0048] Thus, in a preferred embodiment, the spacer is -NH(CH2) p COO-, -NH(CH2CH2O) p CH2CH2COO- and -NH(CH2CH)2O) p Preferably, p is selected from the group consisting of CH2CH2NH- (wherein p is an integer from 0 to 20, and preferably p is 2, 6 or 12).

[0049] If not necessary, the spacer is preferably absent (ie, m is 0 and S represents a direct bond).

[0050] The spacer, or the targeting moiety when the spacer is absent, can be attached in the compound of formula (I) at the R5 residue, represented as the linking moiety R5' in the compound of formula (II).

[0051] The linking group of R5' is a reactive functional group, such as a carboxylic acid or carboxamide residue, suitable for conjugating the dye to a targeting moiety by formation of a chemical bond.

[0052] For example, when amine-containing targeting moiety (T) is conjugated with a compound of formula (II) in which R5' is a carboxylic acid, this carboxylic acid can be optionally activated before carrying out conjugation by conversion to a more reactive form using an activation reagent, for example, forming an N-hydroxysuccinimide (NHS) ester or mixed anhydride.Then, to obtain the corresponding compound of formula (I), the amine-containing targeting moiety is treated with the obtained activated acid to form an amide bond.Usually, this reaction is carried out in an aqueous buffer.

[0053] Alternatively, direct conjugation may be carried out using a "non-activated" carboxylic acid. Similarly, when the R5' linking group is a carboxamide group, the procedure for attachment of the appropriate targeting moiety is similar, although a linker activation step is generally not required, rather the dye and targeting moiety are treated directly.

[0054] The compounds of formula (I) or (II) above may have one or more asymmetric carbon atoms (also referred to as chiral carbon atoms) and may therefore give rise to diastereomers and optical isomers. Unless otherwise specified, the present invention further includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and their pharma- ceutically acceptable salts.

[0055] The present invention further relates to compounds of the above formula (I) or (II), wherein the functional groups of R3 and / or R5', such as sulfonyl, carboxyamino or carboxyl groups, may be in the form of a pharma- ceutically acceptable salt.

[0056] In one embodiment, the present invention relates to a compound of formula (I) or (II) wherein R1 and R2 are independently a group represented by -CO-NH-Y, where Y is the same moiety for R1 and R2 and is selected from linear or branched chain alkyl, cycloalkyl and heterocyclyl, optionally substituted with 2 to 5 hydroxyl groups.

[0057] In a preferred embodiment, the present invention relates to a compound of formula (I) or (II), wherein n is 3, R is alkyl substituted with -SOH, and R and R may be the same or different and are selected from the group consisting of: [ka]

[0058] More preferably, n is 3, R3 is alkyl substituted with -SO3H, and R1 and R2 are the same.

[0059] In another embodiment of the invention, m is 0 and the spacer (S) is represented as a direct bond, or m is 1 and the spacer is a hydrophilic moiety comprising an alkyl, cycloalkyl, or aryl group.

[0060] Preferably, the spacer is -NH(CH2) p COO-, -NH(CH2CH2O) p CH2CH2COO- and -NH(CH2CH2O) p CH2CH2NH- (wherein p is an integer from 0 to 20, preferably p is 2, 6 or 12).

[0061] In a further embodiment, T is a targeting moiety selected from a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or any fragment of an antibody, and an aptamer. Preferably, T is expressed as a peptide, in particular an integrin receptor (α v β3, α v β5, α v β6, α5β1, etc.), preferably α v It is expressed as a moiety that interacts with the β3 integrin receptor.

[0062] In another preferred embodiment, the present invention relates to a compound of formula (I) or (II) or to a compound of formula (Ia) or (IIa), respectively, wherein n is 3, R is C-alkyl substituted with -SOH, and R and R are both group (iv) as defined above. [ka] [ka] wherein R4, R5, R5', S, m and T are as defined above.

[0063] In another embodiment, the present invention relates to a compound of formula (I) or (II) wherein n is 1 or 2.

[0064] In particular, the compounds of formula (I) or (II) set out in Tables Ia and Ib are preferred. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0065] The present invention also relates to a method for synthesizing the compounds of formula (I) and (II) prepared as described in the following description. The compounds of the present invention are useful as imaging agents in the detection of tumors in both humans and animals. Thus, the present invention provides compounds of formula (I) as defined above for use as fluorescent probes for the detection and demarcation of tumor margins in guided surgery of individual patients, particularly those tumors that show reduced or altered overexpression of integrin receptors. Preferably, the subject to be imaged is a human.

[0066] The present invention also provides compounds of formula (I) for use as fluorescent probes as defined above, in which the detection and demarcation of tumour margins is carried out under NIR radiation.

[0067] A further aspect of the present invention relates to a pharmaceutical composition comprising a conjugate of a dye of formula (I) or formula (II) or a salt thereof as defined above and one or more pharma- ceutically acceptable adjuvants, excipients or diluents.

[0068] Another aspect of the present invention relates to a diagnostic kit comprising the conjugate of formula (I) defined above.Furthermore, the kit may comprise additional adjuvants for carrying out optical imaging.These adjuvants are, for example, suitable buffers, containers, detection reagents or instructions for use.The kit preferably comprises all the materials for intravenous administration of the compound of the present invention.

[0069] The compounds of the invention may be administered systemically or locally to the organ or tissue to be imaged prior to the imaging procedure. For example, the compounds may be administered intravenously. In another embodiment, the compounds may be administered parenterally or enterally. The compositions are administered in a dose effective to obtain the desired optical image of the tumor, tissue or organ, which may vary widely depending on the compound used, the tissue to be subjected to the imaging procedure, the imaging device used, etc. The exact concentration of the imaging agent will depend on the experimental conditions and the desired results, but typically may range from 0.000001 mM to 0.1 mM. The optimal concentration is determined by systematically varying it until a satisfactory result is obtained with minimal background fluorescence. After administration, the imaging agent of the invention is exposed to light or other forms of energy capable of passing through the tissue layer. The radiation wavelength or wavelength band is preferably consistent with the excitation wavelength or wavelength band of the photosensitizer, and has low absorption by control non-target cells and others (including blood proteins). Typically, the optical signal is detectable by observation or instrumentation, and the response is related to the intensity, distribution, and lifetime of the fluorescence or light.

[0070] Synthesis Description The preparation of compounds of formula (I) or (II) as such or in the form of a physiologically acceptable salt is a further object of the present invention. The cyanine dyes and dye conjugates of the present invention can be prepared, for example, according to the methods described in the following sections and experimental part. General teachings on the preparation of cyanine dyes are described in Mujumdar RB et al., Bioconjugate Chem. 1993, 4(2), 105-111, on the synthesis and labeling of sulfoindocyanine dyes. However, the cyanines of the present invention are characterized by a specific functionalization pattern that is not present in the compounds of the art, which requires the setting up of a suitable synthetic approach. Indeed, unlike other known cyanines, the compounds of the present invention also have three functional moieties (carboxylic acid or amino / amide groups) that are derivatized in different ways, so that in many cases the use of protecting groups was necessary to react on the desired functional groups.

[0071] It is known that manipulation of cyanines under the harsh pH and temperature conditions required for removal of the protecting groups can be problematic, as the stability of the polymethine backbone can be compromised, leading to severe degradation of the dye.

[0072] Furthermore, when deprotecting the carboxyl group of R5, further problems may be encountered due to possible hydrolysis and decomposition of the amide groups R1 and R2 (usually amide derivatives can be hydrolyzed in concentrated alkaline media, see for example Yamana et al, Chem. Pharm. Bull., 1972, 20(5), 881-891).

[0073] Contrary to expectations, the compounds of the present invention were found to be very stable at basic pH (ie, pH approx. 11), with no or negligible decomposition observed during removal of the protecting groups.

[0074] In a preferred embodiment, the protecting group of the moiety represented by R5' is an ester group. More preferably, an ethyl ester group can be advantageously used.

[0075] Preparation of cyanine dyes of formula (II) According to the present invention, compounds of formula (II) can be prepared by the general synthetic process shown in Scheme 1 below for embodiments in which R1 is the same as R2, or by the general synthetic process shown in Scheme 2 below for embodiments in which R1 and R2 have different meanings. [ka] In the above scheme 1, R1, R2, R3, R4 and n are as defined above, R5'' is -COOPg or -CONHPg, Pg is a protecting group, R5''' is -COOH or -CONH2, and X is a suitable leaving group such as halogen, mesylate, triflate, etc.

[0076] Thus, the manufacturing method of the present invention comprises the steps of: a) treating an amount of 5-carboxy-2,3,3-trimethylindolenine (III) with a nucleophile R3-X (IV) (wherein R3 is as defined above and X is a suitable leaving group such as a halide group or R3-X represents a C1-C6 alkane sulfone molecule) to obtain intermediate (VI); b) treating another amount of 5-carboxy-2,3,3-trimethylindolenine (III) with a nucleophile R5″-R4-X(V), where R5″ and R4 are as defined above and X is a suitable leaving group such as a halide group, to give intermediate (VII); c) reacting intermediate (VI) and intermediate (VII) with reagent (VIII) to obtain cyanine scaffold (IX), where n, R3, R4 and R5″ are as defined above; d) derivatizing the carboxylic acid group on the indolene ring with a polyhydroxylated amine, such as glucamine, meglumine, glucosamine, trometamol, serinol, or isoserinol; e) Optionally, removing a protecting group from the resulting intermediate (X) and isolating the compound of formula (II) or a salt thereof.

[0077] According to steps a) and b), the reaction of derivative (III) with nucleophiles R3-X (IV) or R5″-R4-X (V) can be carried out neat or in high boiling solvents (such as butyronitrile, sulfolane, 1,2-dichlorobenzene, dimethylacetamide, dimethylformamide or dimethylsulfoxide) with stirring at elevated temperature (e.g. 90° C. to 180° C.) for several hours (typically 12 hours to 5 days).

[0078] According to step c), the reaction can be carried out with the Vilsmeier reagent in the bis-anilide form (as shown in scheme 1) or in the bis-aldehyde form. The reaction can be carried out in solvents such as for example ethanol, methanol, acetic anhydride or acetic acid, with or without the addition of various bases such as trimethylamine, pyridine, sodium acetate, potassium acetate, etc., at various temperatures (45° C.-120° C.) while stirring the mixture for several hours (usually 2-24 hours).

[0079] According to step d), the reaction can be carried out using several coupling agents such as TBTU, HBTU, HATU, PyBOP, DCC, DSC, DCC-NHS, and several organic bases such as TEA, DIPEA, NMM, pyridine, in a solvent such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, acetonitrile, and the like at room temperature for an appropriate time from 30 minutes to several hours.

[0080] Optionally, according to step e), the protecting group of intermediate (X) can be converted to a protective group according to, for example, the method described in TW Green and PGM Wets, Protective Groups in Organic Synthesis, Wiley, NY 2007, 4 th It is removed from the part indicated by R5'' according to the known procedure described in Ed., Ch.

[0081] Alternatively, as described above, when R1 is different from R2, compounds of formula (II) can be prepared as shown in Scheme 2 below. [ka] wherein R1, R2, R3, R4 and n are as defined above, R5'' is -COOPg or -CONHPg, Pg is a protecting group, R5''' is -COOH or -CONH2, and X is a suitable leaving group such as halogen, mesylate or triflate.

[0082] According to scheme 2, polyhydroxylated amines are reacted with intermediate (VII) in step b') to form intermediate (XI) bearing residue -R2, which is then subjected to a Vilsmeier reaction in step c) to form cyanine (IX). Another different residue -R1 can be added to another indolenine ring later. Alternatively, -R1 can be introduced into intermediate (VI) in the corresponding step a') before carrying out step c).

[0083] Thus, step b') and / or optionally step a') are carried out as described above for step d). In a further embodiment, the compound of formula (II) prepared according to the process of the invention can be conveniently converted into another compound of formula (II) by operating according to well-known synthetic conditions, examples of possible conversions are as follows: f) converting a compound of formula (II) wherein R5''' is -COOH, i.e., a compound of formula (IIb), into the corresponding compound of formula (II) wherein R5''' is -CONH2, i.e., a compound of formula (IIc). [ka]

[0084] According to step f), the conversion of the carboxylic acid of formula (IIb) to the corresponding carboxamide of formula (IIc) can be carried out by various methods and experimental conditions widely known in the art for the preparation of carboxamides. As an example, the carboxylic acid can be first converted to a suitable activated ester and then reacted with an ammonium salt such as NH4Cl, preferably in the presence of a coupling agent such as HBTU.

[0085] Preparation of the conjugate compounds of formula (I) The cyanine derivative of formula (II) or a salt thereof can then be conjugated, optionally with a spacer, to a suitable targeting moiety to give the corresponding compound of formula (I), as shown in Scheme 3. [ka]

[0086] Conjugation can be carried out by various procedures known in the art, for example, direct coupling, in which the functional group R5''' is reacted directly with a nucleophilic residue of the targeting moiety, optionally with a spacer, or by coupling after activation, in which the functional group represented by R5''' (typically an acid) is converted into a more reactive group (e.g. an ester such as NHS).

[0087] When the compounds of formula (I) or (II) prepared according to the above processes are obtained as a mixture of isomers, it is within the scope of the present invention to separate them into the single corresponding isomers of formula (I) or (II) using conventional techniques.

[0088] The final compounds can be isolated and purified using conventional procedures (eg, chromatography and / or crystallization and salt formation).

[0089] The compounds of formula (I) or (II) as defined above can be converted into pharmaceutically acceptable salts. The compounds of formula (I) or (II) as defined above, or their pharmaceutically acceptable salts, can be formulated together with a pharmaceutically acceptable carrier or diluent to form a pharmaceutical composition. [Brief description of the drawings]

[0090] [Figure 1] Figure 1 shows the decay of the fluorescent signal in healthy muscle tissue after intravenous administration of compound 1 to Balb / c nu / nu mice (n=5 / group) at three different doses (0.3 nmol / mouse ◯, 1 nmol / mouse □, 3 nmol / mouse △). [Diagram 2] Figure 2 shows the fluorescence decay (left y-axis) of tumor (●) and muscle tissue (◯) after intravenous administration of 1 nmol of compound 1 to Balb / c nu / nu mice implanted with U87MG cells (10 million cells), as well as the change in tumor-to-background ratio over time (□, right y-axis). [Diagram 3] FIG. 3 shows the tumor-to-background ratio (TBR) of Compound 1 administered at a dose of 3 nmol / mouse to Balb / c nu / nu mice (n=5) implanted with Detroit-562 cells (2.5 million cells). [Figure 4] Figure 4 shows the fluorescence decay (left y-axis) of tumor (●) and muscle tissue (◯) after administration of 1 nmol of compound 1 to athymic nude mice (n=5) implanted with HT-29 cells (5 million cells), as well as the time course of tumor-to-background ratio (□, right y-axis). Data are presented as all means ± standard deviations.

[0091] Experimental Part The invention and its specific embodiments described in the following parts are merely illustrative and should not be considered as limitations of the invention. They show how the invention can be implemented and are meant to be illustrative, not limiting of the scope of the invention.

[0092] Materials and equipment All chemicals and solvents were of reagent grade for the reactions. Analytical grade solvents were used for chromatographic purification. Most reagents, including the targeting moieties, were commercially available unless otherwise indicated (e.g., panitumumab (Vectibix, Amgen; CAS Registry Number for active substance: 339177-26-3); cetuximab (Erbitux, Merck; CAS Registry Number for active substance: 205923-56-4)).

[0093] All synthesized compounds were purified by reversed-phase chromatography (RP-HPLC) and characterized by mass spectrometry using an LC / MS instrument equipped with a UV-VIS detector and an ESI source. The analysis was performed on a Waters Atlantis dC 18 5 μm, 4.6 × 150 mm column with a gradient of phase A (CH3COONH4 10 mM) and phase B (acetonitrile). The measured mass / charge ratio is given for each compound.

[0094] The absorbance (Ab) of the compounds of the invention was determined using a dual beam UV-VIS spectrophotometer (Lambda 40, Perkin Elmer). Emission / excitation (Em / Ex) spectra and absolute fluorescence quantum yield (Φ) measurements were performed on a spectrofluorometer (FluoroLog-3 1IHR-320, Horiba Jobin Yvon) equipped with an F-3018 integrating sphere accessory. Measurements were performed using excitation wavelengths at the absorbance maxima of the various dyes, and samples were excited with a 450W xenon light source. Detection was performed by a photomultiplier tube (PMT-NIR) cooled detector or a TBX-04 detector. To minimize reabsorption phenomena, the dye solutions were carefully prepared to have an absorbance of less than 0.1 (optical density).

[0095] In vivo imaging experiments were performed using an IVIS Spectrum In Vivo Imaging System (Perkin Elmer Inc.), which is equipped with 10 narrowband excitation filters (30 nm bandwidth) and 18 narrowband emission filters (20 nm bandwidth) spanning 430–850 nm.

[0096] List of abbreviations DCC N,N'-dicyclohexylcarbodiimide DIPEA N,N-Diisopropylethylamine DMF Dimethylformamide DMSO Dimethyl sulfoxide DSC N,N'-disuccinimidyl carbonate HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HBTU O-Benzotriazol-1-yl-N,N,N,N'-tetramethyluronium hexafluorophosphate HPLC High Performance Liquid Chromatography PBS Phosphate Buffered Saline NHS N-hydroxysuccinimide NMM N-Methylmorpholine RT room temperature PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate TEA Triethylamine TBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate μL microliter μM micromolar t R Retention time (HPLC) cRGDfK cyclo-(Arg-Gly-Asp-D-Phe-Lys) aza-cRGD-NH2 aza-cyclo-(Arg-Gly-Asp) EXAMPLES

[0097] Abbreviations for individual amino acid residues are conventional, e.g., Asp or D for aspartic acid, Gly or G for glycine, Arg or R for arginine. Amino acids referred to herein are understood to be in the L-isomer configuration unless otherwise specified.

[0098] Working Example 1: Synthesis of compound 4 [ka] Preparation of intermediate (VIa), step a A round bottom flask was charged with 5-carboxy-2,3,3-trimethylindolenine (1.50 g, 7.38 mmol), 2,4-butanesulfone (1.11 g, 8.15 mmol) and butyronitrile (2 g). The mixture was heated at 120° C. for 2 days. The solid was then treated with dry acetone, filtered under vacuum and washed twice with acetone. The pink solid was dried under vacuum and used without further purification (2.40 g, 96%). HPLC purity 94% at 270 nm, MS: [M + H] +339.2.

[0099] Preparation of intermediate (VIIa), step b To a round bottom flask were added intermediate III (1.5 g, 7.38 mmol), ethyl 6-bromohexanoate (1.97 g, 8.85 mmol) and sulfolane (6.8 g). The suspension was heated at 90 °C for 3 days. Ethyl acetate (25 mL) was then added and the purple dispersion was stirred at room temperature for 15 min and filtered under vacuum. The solid was washed three times with ethyl acetate, then redispersed in fresh ethyl acetate (15 mL), stirred at room temperature for 15 min, filtered under vacuum and washed with ethyl acetate. This operation was repeated two more times. Finally, the pink / purple solid containing both the desired product and starting material was dried under vacuum. It was then dispersed in acetonitrile (4 mL) and the dense dispersion was stirred at room temperature for 15 min and filtered. The solid was washed with several volumes of cold acetonitrile. The mother liquor contained mainly the desired product, while the solid contained the starting material. The acetonitrile was removed in vacuo and the solid was redispersed in acetonitrile (4 mL), stirred for 15 min, filtered, and the solid was washed with cold acetonitrile. The solution was concentrated in vacuo to give a pink / purple solid with 92% HPLC purity at 270 nm (1.25 g, 40% as the bromide salt). MS: [M + H] +346.5.

[0100] Preparation of intermediate (IXa), step c To a round bottom flask were added glutaconaldehyde dianil hydrochloride (0.48 g, 1.4 mmol), acetic anhydride (47 mL) and acetic acid (13 mL). The orange-brown solution was heated at 60° C. for 3 hours. The reaction mixture was then cooled to room temperature and a solution of intermediate (IVa) (0.60 g, 1.4 mmol) in ethanol (10 mL) was added dropwise at room temperature in about 20 minutes. After heating the red solution at 35° C. for 2 hours, the temperature was raised to 50° C. and an ethanolic solution (19 mL) of intermediate (III) (0.48 g, 1.4 mmol) and sodium acetate (0.11 g, 1.3 mmol) was added. Pyridine (2.0 mL) was added immediately after. The reaction mixture was heated at 60° C. for 1.5 hours, after which the solvent was removed in vacuo and the dark oil was precipitated in cold water (100 mL). The dark green solid was filtered and washed with cold water. It was then dissolved in a minimum amount of ethanol and this solution was added dropwise to ethyl acetate (400 mL) with stirring. The solid was filtered, washed with ethyl acetate, dissolved in dichloromethane and purified on silica gel with a slow dichloromethane-methanol gradient. Fractions containing the pure product were combined, evaporated under vacuum and dried to give a blue-green solid (350 mg, 33% yield). HPLC purity 94% at 756 nm, MS: [M + H] +747.3.

[0101] Synthesis of compound 4, steps d and e In a dry round-bottom flask, dry DIPEA (115 μL, 0.56 mmol) was added to a solution of intermediate (IXa) (150 mg, 70% purity, 0.14 mmol) in DMF (2 mL). After stirring for 30 min under nitrogen flow, a solution of TBTU (309 mg, 0.67 mmol) in DMF (2 mL) was added. After 1 h, a suspension of D-glucamine (87 mg, 0.336 mmol) in DMF (2 mL) was added. The mixture was stirred at room temperature for 2 h, then concentrated and purified on a prepacked C18 silica column with a water-acetonitrile gradient. Fractions containing the pure product were collected, concentrated and lyophilized to give a blue-green powder (95.8 mg, 64% yield). HPLC purity 97% at 756 nm, MS: [M + Na] +1095.3.

[0102] The intermediate (Xa) thus obtained (95 mg, 0.089 mmol) was solubilized in water (10 mL, pH 6.35), and then 2N NaOH was added until pH 11.14. The solution was stirred at room temperature and maintained at pH 11 by adding 0.1N NaOH. After 40 h, the reaction was complete, after which the mixture was neutralized with 1N HCl and purified on a prepacked C18 silica column with a water-acetonitrile gradient. The fractions containing the pure product were collected, concentrated and lyophilized to give a blue-green powder (81 mg, 86% yield). HPLC purity 98% at 756 nm, MS: [M + H] +1045.3.

[0103] Example 2 Synthesis of Compound 5 [ka] Step f In a dry round-bottom flask, N-methylmorpholine (10 μL, 0.091 mmol) was added to a solution of compound 4 (10 mg, 0.0096 mmol) prepared in Example 1 in dry DMF (1 mL). After stirring for 30 min under nitrogen flow, a solution of HBTU (13 mg, 0.03 mmol) in DMF (1 mL) was added. After 1 h, a suspension of NH4Cl (10 mg, 0.19 mmol) in DMF (1 mL) was added. The mixture was left stirring for 2 days, then dried under vacuum and purified on a prepacked C18 silica column with a water-acetonitrile gradient. The fractions containing the pure product were combined, concentrated and lyophilized to give a blue solid (2,56 mg, 20% yield). HPLC purity at 756 nm 97.5%, MS: [M + H] + 1043.1.

[0104] Example 3 Synthesis of compound 7 [ka] Preparation of intermediate Xb, step d Intermediate IXb was prepared according to the procedure of Example 1, but using the reagent malonaldehyde dianilide hydrochloride in step c). Intermediate IXb (58 mg, 0.081 mmol) was suspended in dry DMF (10 mL). Trometamol (23.5 mg, 0.194 mmol), DIPEA (61 μL, 0.352 mmol) and HATU (73.77 mg, 0.194 mmol) were added. The reaction was stirred for 2 h at room temperature under inert atmosphere. The solvent was removed under reduced pressure and the crude was purified by flash chromatography (water / methanol gradient) on a prepacked C18 silica column. The fractions containing the pure product were combined, distilled under vacuum and lyophilized to give a light blue solid (60 mg, 80% yield). HPLC purity at 655 nm: 99.6%. MS: [M + H] + 927.3.

[0105] Synthesis of compound 7, step e Intermediate Xb (110 mg, 0.106 mmol) was dissolved in ethanol (2 mL) and water (18 mL) was added. The solution was heated to 40° C. and the pH was kept at 11 with 0.1 N NaOH until hydrolysis was complete (approximately 6 h). The pH was brought to 7 with 0.1 N HCl and the crude was purified by flash chromatography (water / methanol gradient) on a prepacked C18 silica column. Fractions containing the pure product were combined, distilled under vacuum and lyophilized to give a light blue solid (18.7 mg, 19% yield). HPLC purity at 655 nm: 95.7%. MS: [M + H] + 899.3.

[0106] Example 4: Synthesis of Compound 8 [ka] Intermediates VIc and VIIc were prepared following the procedures described above for intermediates VIa and VIIa. Treatment with the corresponding reagent N,N-diphenylformamidine in analogy to Example 1 afforded the trimethine intermediate IXc.

[0107] Preparation of intermediate Xc, step d Intermediate IXc (62 mg, 0.089 mmol) was suspended in dry DMF (15 mL). D-glucamine (33.9 mg, 0.187 mmol), DIPEA (62 μL, 0.356 mmol) and HATU (71.10 mg, 0.187 mmol) were added and the reaction was stirred at room temperature under inert atmosphere for 4 h. The solvent was removed under reduced pressure and the crude was purified by flash chromatography (water / acetonitrile gradient) on a prepacked C18 silica column. Fractions containing the pure product were combined, evaporated under vacuum and lyophilized to give a light pink solid (82.4 mg, 91% yield). HPLC purity at 560 nm: 99.8%. MS: [M + H] + 1021.3.

[0108] Synthesis of compound 8, step e Intermediate Xc (81.4 mg, 0.08 mmol) was dissolved in ethanol (2 mL) and water (18 mL) was added. The solution was stirred at room temperature and the pH was kept at 11 with 0.1 N NaOH until hydrolysis was complete (approximately 46 h). The pH was then adjusted to 7 with 0.1 N HCl and the crude was purified by flash chromatography (water / acetonitrile gradient) on a prepacked C18 silica column. The fractions containing the pure product were combined, distilled under vacuum and lyophilized to give a light pink solid (79 mg, 99.4% yield). HPLC purity at 560 nm: 100%. MS: [M + H] + 993.3.

[0109] Example 5: Synthesis of Compound 1 [ka] Compound 1 was synthesized by conjugation of the dye corresponding to compound 4 with the cyclic pentapeptide c-RGDfK, the conjugation being carried out by two procedures A or B. Compound 4 was prepared as described in Example 1. c-RGDfK, which represents the targeting moiety, is a commercially available reagent.

[0110] Step A - Direct conjugation step In a dry round-bottom flask, N-methylmorpholine (10 μl, 0.09 mmol) was added to a solution of compound 4 (10 mg, 0.009 mmol) prepared as described in Example 1 in dry DMF (1 mL). After stirring for 30 min under nitrogen flow, a solution of TBTU (6 mg, 0.02 mmol) in DMF (1 mL) was added. After 1 h, a solution of c(RGD)fK (6 mg, 0.009 mmol) in DMF (1 mL) was added. The mixture was stirred at room temperature for 20 h, after which additional TBTU (4 mg) and N-methylmorpholine (2 μL) were added, and after 2 h, c(RGD)fK (2 mg) was added. The mixture was stirred for another 6 h, then concentrated and the crude was purified by analytical HPLC (0.1% ammonium acetate-acetonitrile gradient) on a C18 silica column. Fractions containing pure product were collected, concentrated and lyophilized three times to give a blue solid (4.4 mg, 29% yield). HPLC purity at 756 nm 99%, MS: [M / 2] + 841.0.

[0111] Procedure B - Conjugation step with previous activation by NHS ester Compound 4 (4 mg, 0.0038 mmol), prepared as described in Example 1, was suspended in dry DMF (3 mL) under a stream of nitrogen. N-methylmorpholine (0.8 μl, 0.0076 mmol) and TSTU (2.3 mg, 0.0076 mmol) were added and the solution was stirred at room temperature for 20 h. The desired product was precipitated by the addition of cold diethyl ether, centrifuged, and washed twice with diethyl ether.

[0112] The solid was dissolved in a solution of c(RGD)fK (3.0 mg, 0.0042 mmol) in pH 9 borate buffer (2 mL). After stirring the solution overnight, the pH was adjusted to 6 with 1N HCl and the crude was purified by analytical HPLC (0.1% ammonium acetate / acetonitrile gradient) on a C18 silica column. Fractions containing the pure product were collected, concentrated and lyophilized three times to give a blue solid (5.2 mg, 83% yield). HPLC purity 99% at 756 nm, MS: [M / 2]+ 841.0.

[0113] Example 6: Synthesis of Compound 3 [ka] In a dry round-bottom flask, N-methylmorpholine (10 μl, 0.09 mmol) was added to a solution of compound 4 (10 mg, 0.009 mmol) prepared as described in Example 1 in dry DMF (1 mL). After stirring for 15 min under nitrogen flow, a solution of HBTU (3.6 mg, 0.01 mmol) in DMF (1 mL) was added. After 30 min, a solution of aza-cRGD-NH2 (5.7 mg, 0.01 mmol) in DMF (1 mL) was added. The mixture was stirred for 2 days, then concentrated under vacuum and purified by analytical HPLC (0.1% ammonium acetate / acetonitrile gradient) on a C18 silica column. Fractions containing the pure product were collected, concentrated, and lyophilized three times to give a blue powder (3.57 mg, 21% yield). HPLC purity 99% at 756 nm, MS: [M / 2] + 780.8.

[0114] Example 7: Synthesis of Compound 9 Panitumumab (6 mg), a monoclonal antibody EGFR ligand, was diluted to 5 mg / mL in PBS and adjusted to pH 9 by adding 120 μL of 1.0 M potassium phosphate. Compound 4-NHS ester (prepared as described in Example 5, Procedure B) was dissolved in DMSO at a concentration of 10 mg / ml. The dye and antibody were then quickly mixed in a 2.5:1 molar ratio and left in the dark at room temperature for 3 hours. After 3 hours, the conjugation reaction mixture was loaded onto a Zeba Spin column equilibrated with phosphate-buffered saline (PBS) and centrifuged at 1500 g for 2 minutes to separate the conjugate from the free dye. After filtration through a 0.22 μm polyethersulfone (PES) membrane, the conjugated panitumumab solution in PBS at pH 7.4 was analyzed by SE-HPLC, RP-HPLC, and UV / VIS spectrophotometry to determine the concentration and purity. The conjugation molar ratio (dye molecules bound to antibody) was approximately 1.3.

[0115] Example 8: Synthesis of Compound 10 Cetuximab (5 mg / mL), a monoclonal antibody EGFR ligand, was dialyzed against PBS with a 10K DaMWCO membrane. 10 mg of the dialyzed MAb (4.52 mg / mL, 68.8 nmol) was added to 221 μL of 1 M phosphate buffer pH 9. Compound 4-NHS ester (prepared as described in Example 5, Procedure B) was dissolved in DMSO at a concentration of 6.86 mM. 43.3 μL of dye was added to the cetuximab solution (molar ratio 4.3:1) and kept at room temperature in the dark for 3 hours. After this, the conjugation reaction mixture was loaded onto a Zeba Spin column equilibrated with phosphate buffered saline (PBS) and centrifuged at 1000 g for 2 minutes to separate the conjugate from the free dye. After filtration through a 0.22 μm polyethersulfone (PES) membrane, the conjugated cetuximab solution in PBS, pH 7.4, was analyzed by SE-HPLC, RP-HPLC, and UV / VIS spectrophotometry to determine the concentration and purity. The conjugation molar ratio (bound dye molecules to antibody) was approximately 1.1.

[0116] Example 9: Synthesis of Compound 11 [ka] The small molecule CAIX ligand 4a described in Wichert et al., Nat Chem 2015, 7, 241-249 was prepared and conjugated to compound 4 according to the procedure disclosed therein. 11 mg of compound 4 (10.0 μmol) were dissolved in 1 mL of DMF, followed by the addition of 5.5 mg of PyBOP (10,0 μmol) and 7 μL of DIPEA (40,0 μmol) under stirring. After 20 min, 9 mg of small molecule 4a (15.0 μmol) was dissolved in 1 mL of DMF and added to the reaction mixture, which was stirred for another 30 min at room temperature. Purification was performed by preparative HPLC (yield 60%). The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI(+) using a Waters Atlantis dC18 column (μm, 4.6×150 mm). HPLC purity at 758 nm: 98%; MS: [M / 2] + 823.3.

[0117] Example 10: Synthesis of Compound 12 [ka] The small molecule CAIX ligand 8a described in Wichert et al., Nat Chem 2015, 7, 241-249 was prepared and conjugated to compound 4 according to the procedure described in the literature. 15 mg of compound 4 (14.0 μmol) were dissolved in 1 mL of DMF, followed by the addition of 7.3 mg of PyBOP (14,0 μmol) and 10 μL of DIPEA (57,0 μmol) with stirring. After 20 min, 22 mg of the small molecule 8a (21.0 μmol) were dissolved in 1 mL of DMF and added to the reaction mixture, which was stirred for another 30 min at room temperature. Purification was performed by preparative HPLC (yield 34%). The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI(+) using a Waters Atlantis dC18 column (μm, 4.6×150 mm). HPLC purity at 758 nm: 95%; MS: [M / 2] + 1051.8.

[0118] Example 11 :Stability of intermediate (Xa) In order to achieve optimal conditions for the hydrolysis of the ethyl ester, the stability of intermediate (Xa), prepared as described in step d) of Example 1, was evaluated under different conditions. Indeed, it is known that such types of cyanines are rather unstable in acidic and basic media. In contrast to other analogs bearing polysulfonyl groups, such as compound DA364, described for example in WO2018 / 189136, which are more stable in acidic conditions, intermediate (Xa) unexpectedly showed remarkable stability without decomposition of the R1 and R2 groups when hydrolyzed under strong conditions (e.g., at pH 11 and 45 °C for several hours). Surprisingly, good results were also obtained when hydrolyzed with pig and rabbit liver enzyme extracts (1 mg / mL in PBS buffer, working at pH 8 and 38 °C). Complete conversion was obtained after only 20-24 hours without decomposition.

[0119] Example 12 :Optical properties Compounds of the invention were characterized for their in vitro optical properties in aqueous media (i.e., water / PBS pH 7.4) and in clinical chemistry control serum (SeroNorm, Sero SA) that mimics the chemical composition and optical properties of human serum. All dye or dye-conjugate solutions were freshly prepared. In particular, the excitation and emission maxima and absolute fluorescence quantum yields (Φ) of representative compounds of formula (I) and the corresponding dyes of formula (II) are shown in Table II in comparison with the commercially available bisulfonated heptamethine dye indocyanine green (ICG, Sigma), the bisulfonated pentamethine dye sulfo-Cy5 (Lumiprobe), and the bisulfonated trimethine dye sulfo-Cy3 (Lumiprobe). [Table 6] The compounds of the present invention are characterized by an absorption maximum in the range of about 550 nm to 760 nm. The fluorescence quantum yields obtained for the compounds of the present invention are significantly higher than those of the reference compounds, being about 1.5 to 7 times higher than the corresponding reference compounds with the same length of polymethine chain. Notably, the compounds of the present invention exhibited a higher quantum yield in aqueous buffer PBS than the reference compounds with similar solubility properties.

[0120] Example 13 Affinity for human albumin The binding affinity of the compounds of the present invention to human albumin was analyzed and the results were compared with that of the reference, indocyanine green (ICG). Binding affinity to human serum albumin (HSA; Sigma Aldrich, A9511) was measured using two methods, depending on the binding affinity level of the compound. The first method, which is optimal for compounds that strongly interact with HSA, is based on the analysis of the peak shift in the absorbance spectrum after incubating the dye in a solution containing HSA. Briefly, samples are diluted with HSA dilution (1 × 10 -6 ~4×10 -4 The measurements were performed after incubation at a fixed concentration (1 μM) in phosphate buffer at 25° C. for 5 min in a spectrophotometer. Measurements were performed at the maximum absorbance wavelength of the shifted peak. The second method, which is optimal for compounds with low affinity for HSA, is based on measuring the change in absorbance of solutions containing a dye and various concentrations of HSA after ultrafiltration. Briefly, each compound was diluted with HSA dilutions (1 × 10 -6 ~4×10 -4 The samples were incubated with 100 mM glycerol (10 mM NaCl) at a fixed concentration (2 μM) with 10 mM glycerol (10 mM NaCl) and 10 mM glycerol (10 mM NaCl). Samples were centrifuged (10,000 g for 30 min at 25° C.) in a Microcon device (Amicon Ultra-0.5 Centrifugal Filter Units with 10 kDa MWCO, Ultracel-10 membrane, Millipore) and absorbance measurements of the filtrate were obtained spectrophotometrically at the wavelength of maximum absorbance of the fluorophore. Both methods use the affinity constant (KA , M -1 ) was calculated by fitting the raw data with the following equation:

number

[0121] The binding affinity results measured for representative compounds of the invention by either of the two methods are shown in Table III and compared with the results obtained with clinically available ICG dyes, ICG-RGD and DA364 as reference compounds. [Table 7] As shown in Table III, the dyes and dye conjugates of the present invention exhibit significantly lower binding affinity to human albumin, with affinity constants one or two orders of magnitude lower, compared to available ICG and many other known cyanine compounds. This advantageous feature relates to both the dyes of formula (II) (e.g., compound 4) and the corresponding dyes when conjugated with a targeting moiety (e.g., compounds 1 and 3), suggesting that conjugation of the dyes with the targeting moiety does not affect their affinity to human albumin. Rather, RGD conjugates of formula (I), such as representative compounds 1 and 3, can be used to bind ICG-RGD (HSA K A= 219,000 M -1 , Capozza et al., 2018) or DA364 (HSA K A= 28,670 M -1 The dye unexpectedly exhibited a much lower affinity for human albumin than other dyes known in the art when conjugated to targeting moieties such as .alpha.-diaminobenzyl 2-methylpyridine (Pb-1, Pb-1, and Pb-2) (WO2016 / 097317). Furthermore, the reference compound DA364, when analyzed exactly under the same experimental conditions as the present compound, showed a higher affinity for HSA, with an affinity constant (K A ) is 110,000 M -1 It was.

[0122] Example 14 : Receptor binding affinity To assess whether the targeting effect of the molecular vector is maintained after labeling with the dyes of the present invention, the binding affinity of the conjugates of formula (I) to specific receptors was measured. Peptide / peptidomimetic molecular conjugates As an example of peptide / peptidomimetic conjugates, the receptor affinity of a representative integrin-binding conjugate was determined using an enzyme-linked immunosorbent assay (ELISA) as previously reported (Kapp et al., Sci. Rep. 2017, 7, 39805) to determine the IC 50 The activity was evaluated by calculation of the half-maximal inhibitory concentration. Briefly, 96-well ELISA plates were coated with the extracellular matrix (ECM) protein vitronectin in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) overnight at 4 °C. Then, each well was washed with PBS-T buffer (phosphate-buffered saline / Tween 20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween 20, pH 7.4). They were blocked with TS-B buffer (Tris-saline / BSA buffer; 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA) for 1 h at room temperature. Meanwhile, dilution series of compounds and internal standards were prepared in an additional plate. After washing the assay plate three times with PBS-T, 50 μL of the dilution series was transferred to each well. Human recombinant integrin α in TS-B buffer v 50 μL of a solution of β3 (R&D Systems, 1 μg / mL) was transferred to the wells and incubated for 1 h. The plate was washed three times with PBS-T buffer, and then the primary antibody anti-α v β3 was added to the plate. After incubation and washing three times with PBS-T, a secondary anti-IgG peroxidase-labeled antibody was added to the plate and incubated for 1 h. After washing the plate three times with PBS-T, the plate was developed by quickly adding 3,3',5,5'-tetramethylbenzidine (TMB) and incubated for 5 min in the dark. The reaction was stopped with 3M H2SO4 and the absorbance was measured at 450 nm using a plate reader (Victor3, PerkinElmer). Representative ICs of compounds 1 and 3 50 were tested in duplicate and the resulting inhibition curves were analyzed using GraphPad Prism version 4.0 for Windows (GraphPad Software). The inflection point was the IC 50 The value of c(RGDfK) is defined as the internal standard. All experiments were performed using c(RGDfK) as the internal standard.

[0123] Test molecular probes conjugated to either c(RGDfK) (i.e., Compound 1) or c(RGD)aza (i.e., Compound 3) were synthesized from human α v It showed comparable affinity to the β3 receptor and similar affinity to the non-binding reference, the peptidomimetic c(RGDfK), reported in Table IV. [Table 8] Small Molecule Conjugates As an example of small molecule conjugates, the inhibitory activity of representative conjugates against the CAIX enzyme was evaluated using a commercially available colorimetric kit K473 (BioVision). The protocol was performed according to the manufacturer's instructions, and the inhibitory potency of the conjugates (half maximal inhibitory concentration, IC 50 ) was compared to unconjugated acetazolamide. Briefly, the kit utilizes the esterase activity of active carbonic anhydrase on an ester substrate to release a colored product. The released product is quantified using an absorbance microplate reader (absorbance: 405 nm). For acetazolamide, and the conjugate of formula (I), in the presence of a carbonic anhydrase-specific inhibitor, the enzyme loses its activity, resulting in a decrease in absorbance. The small molecule conjugate of formula (I) shows a high inhibitory potency against the enzyme carbonic anhydrase, with IC 2.0, compared to unconjugated acetazolamide. 50 Values ​​were in the low nanomolar range (acetazolamide: 3.7 nM; compound 11: 6.5 nM; compound 12: 1.2 nM).

[0124] Protein Molecular Conjugates As an example of protein-molecule conjugates, the receptor affinity of a representative EGFR-binding conjugate was evaluated using the AlphaLISA kit AL366H (Perkin Elmer). In this AlphaLISA assay, biotinylated EGF binds to streptavidin-coated Alpha Donor beads, and EGFR-Fc is captured by Anti-Human IgG Fc-specific AlphaLISA Acceptor beads. When EGF binds to EGFR, the donor and acceptor beads are brought into close proximity. Excitation of the donor bead causes the release of a singlet oxygen molecule that triggers a cascade of energy transfer in the acceptor bead, resulting in a sharp peak of emission at 615 nm. The experiment was performed by comparing the affinity of the conjugate with the relative unlabeled molecular vector. The unlabeled antibody trastuzumab, which does not bind to the EGFR receptor, was used as a negative control. Sample serial dilutions (compounds 9 and 10, and the corresponding unlabeled molecular vectors panitumumab and cetuximab) were incubated with EGFR acceptor beads for 30 min, followed by incubation with biotinylated EGF for 30 min. Streptavidin-conjugated donor beads were added to the plate and incubated for 30 min. Absorbance was measured at 615 nm using a plate reader (EnSight, Perkin Elmer). No specific binding was observed for the trastuzumab antibody. In contrast, compound 9 (IC 50 , 0.2 nM) and compound 10 (IC 50 Specific receptor binding was observed at IC 0.4 nM and not at IC 0.4 nM with the unlabeled parent antibody panitumumab (IC 50 , 0.5 nM) and cetuximab (IC 50 , 0.4 nM), respectively. Unexpectedly, it was found that large molecules such as cetuximab and panitumumab retain their native antigen-binding properties even after conjugation with the dyes of the present invention. Overall, these results indicate that the attachment of either small molecules, peptides / peptidomimetics, or protein moieties to the dye compounds of formula (II) does not compromise the binding affinity of the final compounds (I) to their targets.

[0125] Example 15 :Cell uptake Using the human melanoma cell line WM-266-4 (ATCC, CRL-1676) as an in vitro model, we investigated the expression of integrin receptors, especially α, on the membrane of these cells. v Based on the high expression of β3 (Capasso et al., PlosOne 2014), the cellular uptake of representative integrin-binding compounds 1 and 3 was evaluated. Adherent cells at approximately 70% confluence were incubated for 2 h at 3 °C (5% CO2) with compounds 1 or 3 (1 μM) in the presence of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 IU / mL penicillin and 100 μg / mL streptomycin. After washing twice with PBS, cells were detached using 0.1 mM EDTA in PBS, centrifuged and suspended in buffer (PBS, 0.5% BSA, 0.1% NaN3) for flow cytometry experiments. Fluorescence-activated cell sorting (FACS) was used to detect intracellular fluorescent signals as a measure of cellular uptake. Samples were excited with an argon laser and emission was detected using a 670 nm long-pass filter. Fluorescence intensity values ​​were determined from histogram statistics generated by the instrument software.

[0126] To evaluate the specificity of receptor-mediated cellular uptake, experiments were performed by incubating cells with the molecular probe in the presence of a high concentration (100 μM) of unlabeled molecular vector c(RGDfK) as a competitor. Residual internalization was calculated by considering the fluorescence intensity value in the absence of competitor as 100%. Furthermore, to evaluate the influence of body fluids on cellular uptake, parallel experiments were performed by incubating cells with the compounds of the invention in the presence of human serum from male AB plasma (Sigma Aldrich, H4522) or human albumin (Sigma Aldrich, A9511). Residual internalization was calculated considering the value of fluorescence intensity in the absence of serum as 100%. Such uptake assessment also represents an indication of the percentage of the compound that is sequestered by plasma proteins, especially albumin, as it diffuses through the vascular compartment before reaching the tissue of interest or a specific target receptor.

[0127] Table V shows the cellular uptake potential of representative compounds 1 and 3 of the present invention. The compounds showed high cellular uptake in the presence of either human serum or 4% HSA (compound 1, residual uptake 70%), but low residual uptake in the presence of vector c(RGDfK) as a competitor (compound 1, residual uptake 10%; compound 3, residual uptake 15%). Thus, for the compounds of the present invention, cellular internalization was observed to be receptor-mediated, with only minor effects of binding to human serum proteins, especially albumin (approximately 10-20% of residual uptake), and the binding affinity of the medium-compounds to human albumin was confirmed to be moderate to low (as shown in Example 10, K A = about 1~6×10 3 M -1 ). Furthermore, Table V shows a comparison of the residual cellular uptake in the presence of human serum of compounds 1 and 3 with reference compounds DA364, ICG-RGD (Capozza et al., Photoacoustic 2018, 11, 36-45) and ICG-c(RGDfK) prepared in the same manner as ICG-RGD. These results show that the compounds of the present invention were surprisingly found to have higher potency in cellular internalization compared to similar compounds known in the art. [Table 9] Notably, neither the interaction of the compounds with receptors on the cell surface nor the internalization of the receptor-probe complexes within the cells was compromised by the structure of the conjugate dye, particularly the highly hydrophilic and sterically hindered moieties present in the compounds at positions R1 and R2. Thus, the presence of hydrophilic moieties on the conjugate dye results in highly efficient and specific receptor binding and probe internalization, even in the presence of plasma proteins and albumin, which would sequester conjugates lacking hydrophilic moieties and adversely affect binding efficiency.

[0128] Example 16 Biodistribution and excretion The biodistribution and excretion of the fluorescent agents of the present invention were assessed by optical imaging after intravenous administration to 6-10 week old male Balb / c nu / nu mice (Charles River Laboratories). Briefly, mice were housed 4 per cage and fed a sterile VRF1(P) diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). Afterwards, mice were fed AIN-76a rodent chow (Research Diets), a special diet that reduces autofluorescence, until the end of the experiment. Imaging experiments were performed using the preclinical optical system IVIS Spectrum (Perkin Elmer). In vivo imaging was performed under gas anesthesia (6–8% sevofluorane in oxygen). Animals were injected intravenously with the compounds and imaged longitudinally at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h after administration. Briefly, animals were euthanized by anesthesia overdose, and the main tissues and organs of interest were excised for ex vivo optical imaging. Regions of interest (ROIs) were drawn on the tissues of interest at all time points for each fluorescent image, and the signal intensity (expressed as mean radiant efficiency) was evaluated. The ratio of the fluorescent signal between muscle (background tissue) and excretory organs (kidneys, liver) was then calculated to assess the contrast. The half-life in tissues was calculated by fitting the decay of the fluorescent signal in muscle to a bi-exponential decay equation model using GraphPad Prism software (version 5 for Windows).

[0129] The tissue kinetics of compound 1 are shown in Figure 1. Here, the decay of the fluorescent signal in muscle tissue was plotted against time to assess the washout of the product at various doses. Notably, compound 1 showed very favorable elimination kinetics with fast tissue washout and improved elimination from the body, in particular a very short (<1 h) tissue half-life at all doses tested (0.3, 1, 3 nmol / mouse). The compound was rapidly distributed in the body and accumulated in the bladder, suggesting renal excretion. Furthermore, it showed rapid elimination from the body and low retention, advantageously allowing imaging early after administration.

[0130] Table VI shows the ratio of fluorescent signals detected by optical imaging (excised excretory organs (liver and kidney) vs. muscle) 24 hours after intravenous (iv) administration of 1 nmol / mouse of compounds 1 or 3. Ex vivo imaging of excised organs and tissues revealed very low retention of compounds 1 and 3 in the excretory organs kidney and liver, associated with faster elimination from the body and reduced non-specific accumulation. [Table 10]

[0131] Example 17 :Tumor uptake Tumor uptake of the compounds of the invention was assessed by optical imaging after intravenous administration to mice. Tumor uptake experiments have demonstrated that integrin receptors, particularly α v The study was performed in an animal model of human glioblastoma (subcutaneous) overexpressing β3. Briefly, human glioblastoma U87MG cells (ATCC, HTB-14) were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Male Balb / c nu / nu mice (Charles River Laboratories), 4–6 weeks of age, were implanted subcutaneously (right flank) with cells (approximately 10 million cells) suspended in 0.1 mL of EMEM. Mice were housed four per cage and had free access to food and water. Animals were fed VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). Afterwards, the mice were fed AIN-76a rodent diet (ResearchDiets), a specialized diet that reduces autofluorescence, until the end of the experiment. Tumor growth was monitored to a target size of 300–600 mm. 3 The results were monitored by longitudinal evaluation using calipers until 3–4 weeks after cell transplantation. Imaging experiments were performed using the preclinical optical system IVIS Spectrum (Perkin Elmer). In vivo imaging was performed under gas anesthesia (6-8% sevofluorane in oxygen). Animals were injected intravenously with compounds (lateral tail vein) and imaged longitudinally at 30 min, 1 h, 2 h, 3 h, 4 h, and 24 h after administration. Regions of interest (ROIs) were drawn on the tissue of interest at all time points for each fluorescent image, and signal intensity (expressed as mean radiant efficiency) was assessed. Contrast was then assessed by calculating the ratio of fluorescent signals in tumor and muscle (background tissue).

[0132] Fluorescence decay values ​​of representative compound 1 in tumor and muscle tissues, as well as tumor-to-background ratios, are shown in Figure 2. These results show significantly higher tumor uptake of representative compound 1 already at early time points (1-2 hours after administration) and a very high tumor-to-background contrast (TBR approx. 2-2.5), suggesting tumor-specific accumulation with low retention in healthy tissues. Indeed, the compounds of the present invention are rapidly excreted from the body, especially from healthy tissues (muscle), while a significant target-mediated accumulation is observed for the conjugated dyes of formula (I) in target tissues. In particular, integrin receptor α v Parallel experiments were performed in an animal model of human head and neck cancer (orthotopic) using Detroit-562 cells overexpressing β6. Briefly, human pharyngeal carcinoma cells Detroit-562 (ATCC, CCL-138) were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Male Balb / c nu / nu mice (Charles River Laboratories), 4–6 weeks old, were orthotopically implanted with cells (approximately 2.5 million cells) suspended in 0.03 mL di EMEM in the anterior part of the tongue. Mice were housed four per cage and had free access to food and water. Animals were fed VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). The mice were then fed AIN-76a rodent diet (ResearchDiets), a specialized diet that reduces autofluorescence, until the end of the experiment. Tumor growth was monitored at a target size of 10–20 mm. 3 The cells were monitored by longitudinal assessment using calipers until 7–10 days after cell transplantation.

[0133] Imaging experiments were performed using a preclinical optical system IVIS Spectrum (Perkin Elmer). Animals were injected intravenously (lateral tail vein) with 3 nmol / mouse, euthanized by anesthesia overdose 24 h after dosing, and tongues were excised for ex vivo optical imaging. Regions of interest (ROIs) were drawn for the anterior (site of tumor cell implantation) and posterior (healthy tissue) parts of the tongue to derive tumor-to-background ratios. One healthy animal (no tumor implantation) was administered 3 nmol / mouse of compound 1 and imaged as above to serve as a negative control. Ex vivo imaging performed 24 hours after administration of compound 1 showed bright areas at the tongue site of tumor cell implantation. In contrast, healthy areas at the back of the tongue showed low signal, suggesting low retention in healthy tissue, similar to that detected in healthy mice. Administration of the compound of the present invention revealed tumor location with high tumor-to-background contrast (as shown in FIG. 3).

[0134] Parallel experiments were performed in an animal model of human colorectal cancer (subcutaneous) using HT-29 cells expressing low levels of integrin receptors. Briefly, human colorectal adenocarcinoma cells HT-29 (ATCC, HTB-38) were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Male athymic nude mice aged 4–6 weeks (Envigo) were implanted subcutaneously (right flank) with cells (approximately 5 million cells) suspended in 0.1 mL of serum-free medium. Mice were housed 4 per cage and had free access to food and water. Animals were fed VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the acclimation period (5 days). Afterwards, the mice were fed AIN-76a rodent diet (ResearchDiets), a specialized diet that reduces autofluorescence, until the end of the experiment. Tumor growth was monitored to a target size of 300–600 mm. 3The results were monitored by longitudinal evaluation using calipers until 3–4 weeks after cell transplantation. Imaging experiments were performed using a preclinical optical system, IVIS Spectrum (Perkin Elmer).

[0135] In vivo imaging was performed under gas anesthesia (6-8% sevofluorane in oxygen). Animals were injected intravenously (lateral tail vein) with the compound of interest and imaged longitudinally at 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h after administration. Regions of interest (ROIs) were drawn on the tissue of interest at all time points for each fluorescent image, and signal intensity (expressed as mean radiant efficiency) was assessed. Contrast was then assessed by calculating the ratio of fluorescent signals in tumor and muscle (background tissue). Fluorescence decay values ​​and tumor-to-background ratios for tumor and muscle tissue are shown for compound 1 in FIG. These results show high tumor uptake of representative compound 1 already at early time points and rapid clearance from healthy tissues, yielding moderate tumor-to-background ratios (TBR ~1.2-1.5) sufficient to clearly delineate tumor tissue from the healthy background.

[0136] As described above, the target specificity of representative compound 1 was compared to that of reference compound DA364 in an animal model of human cancer overexpressing the human integrin receptor αVβ3. Both compounds were administered at a dose of 1 nmol / mouse with (blocking group) or without (control group) an excess of unlabeled cRGD peptide mimetic (200 nmol / mouse). Mice were euthanized 24 hours after injection, and tumor tissues were excised and imaged using an IVIS Spectrum fluorescence system (PerkinElmer). The blocking protocol promoted a 10% decrease in fluorescence signal intensity (imaging surrogate for uptake) in tumor tissues of animals administered DA364. In contrast, the blocking protocol promoted a 75% decrease in fluorescence in tumors of animals administered compound 1, indicating higher in vivo receptor specificity for the compounds of the present invention (see Table VII).

Table 11

[0137] References 1. Onda N. et al., Int J Cancer, 2016, 139, 673-682 2. Stummer W. et al., Neurosurgery, 2015, 77(5), 663-673 3. Licha et al., Photochemistry and Photobiology, 2000, 72(3), 392-398 4. Tummers Q. et al., PlosOne 2015 5. Achilefu S. et al., J Med Chem, 2002, 45, 2003-2015 6. Bunschoten A. et al., Bioconjugate Chem., 2016, 27, 1253-1258 7. US 6,913,743 B2 8. WO2018 / 189136 9. WO2016 / 097317 10. Capozza et al., Photoacoustics, 2018, 11, 36-45 11. US 6,329,531 B1 12. Ebert et al., J. Biomed. Optics, 2011, 16(6), 066003 13. WO2005 / 123768 14. WO2012 / 088007 15. T.W. Green and P.G.M.Wuts, Protective Groups in Organic Synthesis, Wiley, N.Y. 2007, 4 th Ed., Ch. 5 16. Kapp et al., Sci Rep, 2017, 7, 3905 17. Wichert et al., Nat Chem 2015, 7, 241-249 18. Li et al., FASEB J 2005, 19, 1978-85 19. Williams et al., Chem Biol Drug Des 2018, 91, 605-619 20. Mujumdar RB et al., Bioconjugate Chem. 1993, 4(2), 105-111 21. Yamana et al, Chem. Pharm. Bull., 1972, 20(5), 881-891

Claims

1. Formula (I) 【Chemistry 1】 [In the formula, R1 and R2 are independently -CO-NH-Y groups, where Y is selected from linear or branched alkyl, cycloalkyl, and heterocyclyl substituted with at least two hydroxyl groups; R3 is -NH ２ , -SO ３ H, -COOH and -CONH ２ is a straight or branched chain alkyl substituted with a group selected from R4 is a linear or branched divalent alkyl; R5 is selected from -COO- and -CONH-; S is -NH(CH ２ ) ｐ COO-, -NH(CH ２ CH ２ O) ｐ CH ２ CH ２ COO- and -NH(CH ２ CH ２ O) ｐ CH ２ CH ２ NH-, where p is an integer from 0 to 20; T is a targeting moiety selected from the group consisting of small molecules, proteins, peptides, peptidomimetics containing the sequence Arg-Gly-Asp (RGD), enzyme substrates, antibodies or fragments thereof, and aptamers that target CAIX; n is an integer equal to 1, 2, or 3; m is an integer equal to 1. or a pharma- ceutically acceptable salt thereof.

2. n is 3 and R3 is -SO ３ H; R and R are independently 【Chemistry 2】 2. The compound of formula (I) according to claim 1, selected from the group consisting of:

3. Formula (Ia): 【Chemistry 3】 [In the formula, R4 is a linear or branched divalent alkyl; R5 is selected from -COO- and -CONH-; S is a spacer selected from -NH(CH 2 ) p COO-, -NH(CH 2 CH 2 O) p CH 2 CH 2 COO-, and -NH(CH 2 CH 2 O) p CH 2 CH 2 NH-, where p is an integer from 0 to 20; T is a targeting moiety selected from the group consisting of small molecules, proteins, peptides, peptidomimetics containing the sequence Arg-Gly-Asp (RGD), enzyme substrates, antibodies or fragments thereof, and aptamers that target CAIX; m is an integer equal to 0 or 1. A compound represented by the formula:

4. 2. The compound of formula (I) according to claim 1, wherein T is a moiety that interacts with an integrin receptor.

5. 2. A compound of formula (I) according to claim 1 for use as a fluorescent contrast agent for the detection and demarcation of tumor margins in individual patient guided surgery.

6. 6. A compound of formula (I) for use according to claim 5, wherein the detection and demarcation of the tumor margins is carried out under NIR radiation.

7. The compound of formula (I) for use according to claim 6, 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, colorectal cancer.

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

9. A diagnostic kit comprising a compound of formula (I) as defined in claim 1 together with a further adjuvant for carrying out optical imaging.

10. A compound selected from the group consisting of: 【Chemistry 4】

11. Formula (II) 【Chemistry 5】 [In the formula, R1, R2, R3, R4, and n are as defined in claim 1; R5' is -COOH, -CONH ２ , -COO-Pg, and -CONH-Pg, where Pg is a protecting group selected from benzyl, formyl, 9-fluoromethyloxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), t-butoxycarbonyl (Boc), isopropyloxycarbonyl or allyloxycarbonyl (Alloc), alkyl, triphenylmethyl, acetyl, trifluoroacetyl, and allyl. or a pharma- ceutically acceptable salt thereof.

12. n is 3 and R3 is -SO ３ H; R and R are independently 【Chemistry 6】 12. The intermediate compound of formula (II) according to claim 11, selected from the group consisting of:

13. 13. An intermediate compound of formula (II) according to claim 11 or 12, represented by formula (IIa) 【Chemistry 7】 wherein R4 and R5' are as defined in claim 11.

14. The compound of claim 10 for use as a fluorescent contrast agent for the detection and demarcation of tumor margins in guided surgery of an individual patient.

15. The compound of claim 14, wherein the detection and demarcation of tumor margins is performed under NIR radiation.

16. The compound of claim 14, 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.

17. A diagnostic pharmaceutical composition comprising a compound as defined in claim 10 and at least one pharma- ceutically acceptable carrier or excipient.

18. A diagnostic kit comprising a compound as defined in claim 10 together with a further adjuvant for performing optical imaging.

Images