Near-infrared 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 having near-infrared emission, characterized by improved physicochemical and biological properties, and to conjugates with their biological ligands. The present invention also relates to the use of these compounds as optical diagnostic agents in the imaging or therapy of solid tumors, their preparation methods and compositions containing them. The compounds have formula (I), formula (I), wherein X is a direct bond or -O-; Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group, which is substituted by at least two hydroxyl groups; R1 and R2 are each independently straight-chain or branched C1-C6 alkyl, which is substituted by a group selected from -SO3H, -COOH, -CONH2 and -COO-C1-C6 alkyl; and R3 is hydrogen, -SO3H or straight-chain or branched C1-C6 alkyl substituted by -COOH or -CONH-Y, wherein Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group substituted by at least two hydroxyl groups.

Description

Field of the Invention

[0001] The present invention relates to the field of optical imaging. More specifically, the present invention relates to compounds of the cyanine family having near-infrared emission, characterized by improved physicochemical and biological properties, and to conjugates thereof with biological ligands. The present invention also relates to the use of these compounds as optical diagnostic agents in the imaging or therapy of solid tumors, to their preparation methods, and to compositions containing them. Background Art

[0002] Dyes are chemical entities that absorb photons of a specific wavelength upon photoexcitation and generally re-emit that energy at a longer wavelength, depending on the quantum efficiency. In particular, cyanine dyes are fluorescent organic molecules characterized by a delocalized electron system spanning a polymethine bridge and confined between two nitrogen atoms. Some of them have favorable optical properties, low toxicity, and good solubility in aqueous media and can be used as contrast agents for biomedical imaging. Cyanine dyes emitting in the near-infrared region (700 - 900 nm) are particularly suitable for biomedical imaging applications due to their higher penetration depth compared to dyes having fluorescence emission in the visible spectrum.

[0003] Among the near-infrared dyes used for biomedical imaging, indocyanine green (ICG) is the only pharmaceutical product currently approved for human use. Due to the strong binding of ICG to plasma proteins (blood pool effect) and the rapid clearance of the unbound fraction by the liver, ICG is routinely used for assessing tissue perfusion and angiography applications (Cherrick et al., J Clin Invest 1960; 39(4): 592 - 600). In addition, ICG has also been tested as an investigational pharmaceutical product for tumor imaging during diagnostic and interventional (fluorescence-guided surgery) procedures. ICG is distributed and accumulated in tumor tissues through a combination of passive diffusion and the enhanced permeability and retention (EPR) effect (Onda N. et al., Int J Cancer 2016; 139, 673 - 682). Due to the non-specific accumulation property, false positives are a common clinical finding associated with tumor imaging using ICG (Tummers Q. et al., PlosOne 2015; 10(6): e0129766).

[0004] Other contrast agents for near-infrared imaging are under development, which utilize the use of dyes conjugated to a carrier moiety (i.e., biomolecule) to target overexpressed tumor epitopes to improve the sensitivity and specificity of detection (Achilefu S. et al., J Med Chem 2002; 45, 2003-2015). For example, ICG and S0456 are examples of near-infrared dyes that have been conjugated to tumor-targeting moieties and are currently being tested in clinical trials for intraoperative tumor detection (Fidel J. et al., Cancer Res. 2015; 15; 75(20): 4283-4291; Hogstins C. et al., Clin Cancer Res 2016; 22(12); 2929-38).

[0005] Despite several efforts to find suitable imaging agents, there is still a need to find improved dyes with optimal solubility in aqueous media and low aggregation, high fluorescence efficiency, and optimal biological properties. The biological properties of dyes, especially once administered to a living organism, the binding affinity for plasma proteins such as albumin may strongly affect distribution and tissue accumulation. For example, dyes with high binding affinity for human albumin are sequestered in the plasma compartment after intravenous administration and have a low tissue extravasation rate, which strongly limits their diagnostic applications. In addition, the biological properties of dyes can affect the tissue distribution of conjugates composed of the dye itself and biomolecules targeting biological epitopes on pathological tissues. Near-infrared dyes with low binding affinity for human serum albumin and non-specific accumulation may be preferably used for applications in living organisms. This need is most important when the dye is conjugated to a biomolecule that specifically binds to an epitope or pathological tissue (e.g., tumor). The present invention addresses these and other needs.

[0006] WO2002 / 024815 under the name of Li-Cor Inc. and WO2007 / 136996 and WO2004 / 065491 under the name of Schering AG report stable cyanine dyes that can be used for optical imaging applications and are characterized by high solubility in aqueous media and functional groups for direct conjugation with biomolecules. However, no teachings on how to obtain dyes with optimal biological properties are provided therein.

[0007] WO2015 / 114171 discloses small molecule targeted drug conjugates for delivering drugs to inhibit cancer cells. In particular, it reports the IRDye 750 conjugate "C6" for flow cytometry analysis and in vivo imaging of tumors.

[0008] Wada H. et al., Chemical Engineering Journal 2018, 340(3): 51-57 discloses a NIR fluorescent nanoprobe using a mannose-conjugated ZW800-1 derivative for intraoperative whole lymph node mapping and real-time optical imaging.

[0009] Vendrell M. et al., Organic&Biomolecular Chemistry 2011, 9(13): 4760-4762 reported a NIR fluorescent deoxyglucose analogue CyNE 2-DG, which showed preferential uptake in cancer cells and was validated as an optical reagent in tumor imaging.

[0010] Despite several efforts to find suitable imaging agents, there is still a need to find improved dyes with optimal stability and fluorescence efficiency, as well as optimal physicochemical and biological properties, and designed for optical imaging in living organisms. This need is of the utmost importance, especially when the dyes are conjugated with biomolecules that specifically bind to molecular epitopes or pathological tissues (such as tumors). The present invention addresses these and other needs. Summary of the Invention

[0012] Generally, the object of the present invention is to provide new cyanine dyes or their corresponding conjugates with a binding moiety, which can be used as contrast agents for optical imaging and are intended to solve the above problems.

[0013] The new cyanine derivatives described herein are surprisingly endowed with remarkable optical properties and high solubility in aqueous media. Surprisingly, it has been found that, compared with the near-infrared dyes known in the prior art, the compounds of the present invention have a very low binding affinity for human albumin, which is particularly advantageous when these compounds are used after intravenous administration; the low affinity prevents the compounds in the plasma compartment from being chelated by large proteins (such as albumin) present in the blood, thereby preventing the reduction of the free dye moiety available for effective extravasation and distribution in the extracellular space.

[0014] The new cyanine dyes can be conveniently conjugated with a suitable targeting moiety through a suitable functional group serving as a binding site, thereby providing a very specific and sensitive contrast agent for molecular imaging. This low albumin binding affinity of the compounds of the present invention is particularly important in the case of dye-conjugates, because only their free portion (not bound to albumin) can effectively interact with the molecular target.

[0015] Another aspect of the present invention relates to such dyes as diagnostic agents, particularly for optical imaging of human or animal organs or tissues, for optical imaging methods, wherein the imaging is tomography of organs, monitoring of organ functions, including angiography, tissue perfusion imaging, urinary tract imaging, bile duct imaging, nerve imaging, intraoperative cancer identification, fluorescence-guided surgery, fluorescence endoscopy, fluorescence laparoscopy, robotic surgery, open field surgery, laser-guided surgery, photodynamic therapy, fluorescence lifetime imaging or photoacoustic or ultrasonic fluorescence methods.

[0016] Furthermore, the present invention relates to a method for preparing the provided dyes, their corresponding conjugates and / or pharmaceutically acceptable salts, and their use in the preparation of diagnostic agents.

[0017] According to another aspect, the present invention relates to a pharmaceutically acceptable composition comprising at least one dye or dye-conjugate compound of the present invention or a pharmaceutically acceptable salt thereof, mixed with one or more physiologically acceptable carriers or excipients. The composition can be particularly used as an optical imaging agent to provide useful imaging of human or animal organs or tissues.

[0018] On the other hand, the present invention relates to a method for optically imaging a body organ, tissue or region by using optical imaging techniques, the method comprising using an effective dose of a compound of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] Therefore, a first object of the present invention is to provide a compound of formula (I),

[0021]

[0022] wherein

[0023] X is a direct bond or -O-;

[0024] Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group, which is substituted by at least two hydroxyl groups;

[0025] R1 and R2 are each independently a straight-chain or branched C1-C6 alkyl, which is substituted by a group selected from -SO3H, -COOH, -CONH2 and -COO-C1-C6 alkyl; and

[0026] R3 is hydrogen, -SO3H or a straight-chain or branched C1-C6 alkyl substituted by -COOH or -CONH-Y, wherein Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group substituted by at least two hydroxyl groups,

[0027] or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0028] Another aspect of the present invention relates to the corresponding conjugated dyes represented by the compounds of formula (II),

[0029]

[0030] wherein

[0031] X is a direct bond or -O-;

[0032] Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group, which is substituted by at least two hydroxyl groups;

[0033] R1 is a linear or branched C1-C6 alkyl, which is substituted by a group selected from -SO3H, -COOH, -CONH2 and -COO-C1-C6 alkyl;

[0034] R4 is a linear or branched C1-C6 alkyl, which is substituted by a group selected from -SO3H, -COOH and -CONH-(S) m -T, wherein

[0035] S is a spacer;

[0036] T is a targeting moiety; and

[0037] m is an integer equal to 0 or 1; and

[0038] R5 is selected from hydrogen, -SO3H, linear or branched C1-C6 alkyl substituted by -COOH or -CONH-Y, and the group CONH-(S) m -T, wherein Y, S, T and m are as defined above;

[0039] and wherein at least one between R4 and R5 is a linear or branched C1-C6 alkyl substituted by CONH-(S) m -T,

[0040] or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0041] The present invention also relates to a method for preparing the compounds of formula (I) or (II) through synthetic transformation steps.

[0042] The present invention also encompasses the compounds of formula (I) or (II) used as fluorescent probes for biomedical optical imaging applications.

[0043] Definitions

[0044] In the present specification, unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings.

[0045] The expression "linear or branched C1-C6 alkyl" refers to an aliphatic hydrocarbon radical group which can be linear or branched and has 1 to 6 carbon atoms in the chain. For example, "C4 alkyl" includes within its meaning linear or branched chains containing 4 carbon atoms. Similarly, "C1-C 20 alkyl" is an alkyl group containing 1 to 20 carbon atoms. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, pentyl, and hexyl. Unless otherwise specified, a linear or branched C1-C6 alkyl is a monovalent group. In some cases, it can be a "divalent" or "polyvalent" group, where two or more hydrogen atoms are removed from the above hydrocarbon radical and replaced, such as methylene, ethylene, isopropylidene, etc.

[0046] As used herein, the term "C3-C7 cycloalkyl" includes within its meaning saturated (i.e., alicyclic) carbocyclic rings containing 3 to 7 carbon atoms. Suitable examples include C5-C7 carbocyclic rings, such as a cyclohexyl ring.

[0047] As used herein, the term "heterocyclic group" includes saturated alicyclic rings, preferably 5-7 membered saturated rings, which also contain heteroatoms selected from N, O, and S in the ring chain. Preferably, it refers to tetrahydropyran.

[0048] The term "hydroxyalkyl" refers to any corresponding alkyl chain in which one or more hydrogen atoms are replaced by a hydroxy group.

[0049] The term "alkoxy" includes within its meaning alkyl chains as defined above which further contain one or more oxygen atoms; examples include, for example, alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy, etc., and alkyl-(poly)oxy in which the alkyl chain is interrupted by one or more oxygen atoms.

[0050] In this specification, the term "protecting group" (Pg) denotes a protecting group suitable for maintaining the function of the group to which it is attached. Specifically, protecting groups are used to retain amino, hydroxy, or carboxy functional groups. Suitable protecting groups can include, for example, benzyl, carbonyl groups (such as formyl, 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), isopropoxycarbonyl, or allyloxycarbonyl (Alloc)), alkyl groups (such as tert-butyl or triphenylmethyl), sulfonyl groups, acetyl groups (such as trifluoroacetyl), benzyl esters, allyl groups, or other substituents well-known to those skilled in the art and commonly used for protecting such functional groups (see, for example, the general reference T.W. Green and P.G.M. Wuts, Protective Groups in Organic Synthesis, Wiley, N.Y. 2007, 4th Edition, Chapter 5).

[0051] In addition, the present invention also encompasses precursor or intermediate compounds suitable for preparing the desired compound of formula (I) or a salt thereof. In such derivatives, the functional groups of R1-R5, such as carboxylic acid or formamide, can be protected with appropriate protecting groups (Pg) as defined above, preferably with an alkyl or ester group. If necessary, during the preparation of the compound of formula (I) or (II), the hydroxyl group of the Y group can also be protected with an appropriate protecting group (Pg), thereby forming, for example, acetoxy, alkoxy or ester group.

[0052] The expression "coupling reagent" refers to a reagent used, for example, to form an amide bond between a carboxyl moiety and an amino moiety. This reaction can consist of two consecutive steps: activation of the carboxyl moiety and then acylation of the amino group with the activated carboxylic acid. Non-limiting examples of such coupling agents are selected from: carbodiimides, such as N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC); phosphonium reagents, such as (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyAOP), [ethyl cyano(hydroxyimino)acetato-O2]tris-1-pyrrolidinophosphonium hexafluorophosphate (PyOxim), bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP) and 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT); and ammonium / uronium-iminium reagents, such as N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1-[1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU) and fluoro-N,N,N',N'-tetramethylformamidinium (TFFH) or other compounds well known to those skilled in the art.

[0053] The term "small molecule" is widely used herein and refers to an organic, inorganic, or organometallic compound having a molecular weight of less than about 5000 daltons and capable of modulating a biological process or eliciting a biological effect when administered to an animal (including a human). The term "small molecule" may also be used interchangeably with the terms "drug" or "bioactive moiety". For example, it may include any agent, active molecule, or compound that provides a beneficial effect against a tumor and produces a local or systemic effect in a patient by binding to a specific biological target. A small molecule may also refer to a part or residue of a parent drug that is activated or chemically modified and covalently linked to a conjugated dye of the present invention.

[0054] The expression "activated carboxylic acid" refers to a carboxyl derivative that is more susceptible to nucleophilic attack than a free carboxyl group; suitable derivatives may include, for example, acid anhydrides, thioesters, acyl halides, NHS esters, and sulfo-NHS esters.

[0055] Furthermore, the terms "portion" or "residue" are herein intended to define the remaining part of a designated molecule once it is appropriately linked or conjugated, either directly or through a suitable linker and / or spacer, to the remainder of the molecule.

[0056] Targeting moiety (T)

[0057] According to the present invention, the targeting moiety (T) is a molecule that specifically and selectively binds to a biological target and promotes the accumulation of a contrast agent in a specific tissue or part of the body. Generally, it is represented by a natural or synthetic molecule for use in biological systems.

[0058] This specific binding can be achieved through the interaction of a ligand (such as a small molecule, protein, peptide, peptidomimetic, enzyme substrate, antibody or fragment thereof, or aptamer) with a specific biological target expressed on the surface of a tissue or cell of interest.

[0059] Suitable biological targets for the compounds of the present invention may be, for example, epidermal growth factor (EGF) receptors, such as EGFR or HER2; vascular endothelial growth factor (VEGF) receptors, such as VEGFR1 or VEGFR2; carbonic anhydrase (CA), such as CAIX, CAII or CAXII; mucin glycoproteins, such as MUC1; glucose transporters, such as GLUT-1; sodium-hydrogen antiporter, such as NHE1; carcinoembryonic glycoprotein, such as carcinoembryonic antigen (CEA); chemokine receptors, such as chemokine receptor type 4 (CXCR4); cell adhesion molecules, such as ICAM, EPCAM, VCAM, E-selectin, P-selectin; hepatocyte growth factor HGFR (c-met); transferrin receptor; ephrin receptor, such as EPHA2; folate receptor, such as FR-α; glycoprotein binding to iduronic acid, such as CD44; bombesin receptor, such as BB1, BB2, BB3; N-acetyl-L-aspartyl-L-glutamate (NAAG) peptidase, such as prostate-specific membrane antigen (PSMA); and especially integrin receptors, such as α v β3, α v β5, α v β6 or α5β1 integrin receptor.

[0060] For example, the integrin receptor targeting moiety is represented by a linear or cyclic peptide containing the sequence Arg-Gly-Asp (RGD). This tripeptide has high binding specificity for the receptor and is recognized as a ligand by the integrin receptor family located in the cell membrane. In fact, it has been identified in some extracellular matrix glycoproteins, such as fibronectin or vitronectin, which utilize this RGD motif to mediate cell adhesion.

[0061] Thus, linear and cyclic peptides and peptidomimetics containing the sequence Arg-Gly-Asp (RGD), such as cRGD, cRGDfK, cRGDyK, cRGDfC, RGD-4C, RGD-2C, AH111585, NC100692, RGD-K5 (Kapp et al., Sci Rep, 2017, 7:3905) or their analogs and derivatives are well-known examples of binding motifs that target cancer tissues with upregulated cell membrane integrins compared to healthy tissues.

[0062] In one embodiment, the compounds of the present invention can be conjugated to carriers known to target prostate-specific membrane antigen (PSMA), thereby allowing the detection and imaging of prostate cancer. These ligands are represented, for example, by the carrier glutamate-urea-lysine (EuK) or other PSMA-binding carriers of the formula "EuX" as described in EP3636635A1, i.e., glutamate is linked through a bridging urea to another amino acid or analogue, such as EuFA (glutamate-urea-3-(2-furyl)-alanine), EuPG (glutamate-urea-2-(2'-propynyl)-alanine), EuE (glutamate-urea-glutamate) or other urea-based peptidomimetics, such as EuK-(3-(2-naphthyl)-alanine)-tranexamic acid, as described by B et al., J Nucl Med 2015, 56:914-920.

[0063] In another embodiment, the compounds of the present invention can be conjugated to other small molecules, peptides, proteins or antibodies, such as monoclonal antibodies that have already been used in therapy. Small molecules containing the drug acetazolamide, such as compounds 4a, 5a, 6a, 7a and 8a (Wichert et al., Nat Chem 2015, 7:241-249) or their analogues and derivatives are examples of small molecules that target the enzyme CAIX. Linear and cyclic peptides and peptidomimetics, such as the peptide GE11 (described by Li et al., FASEB J 2005, 19:1978-85) and / or the peptide L1 (described by Williams et al., Chem Biol Drug Des 2018, 91:605-619) or their analogues and derivatives are examples of peptides that target the epidermal growth factor receptor (EGFR). Among proteins, derivatives of epidermal growth factor (EGF) are examples of small proteins that target the epidermal growth factor receptor (EGFR). Among antibodies, pembrolizumab and cetuximab are examples of monoclonal antibodies that target the epidermal growth factor receptor (EGFR).

[0064] Preferably, the targeting ligands of the present invention are capable of selectively binding to tumor cells or tissues. In particular, they are capable of binding to tumors selected from brain cancer, breast cancer, head and neck cancer, ovarian cancer, prostate cancer, esophageal cancer, skin cancer, gastric cancer, pancreatic cancer, bladder cancer, oral cancer, lung cancer, kidney cancer, uterine cancer, thyroid cancer, liver cancer and colorectal cancer. In addition, the targeting ligands are capable of linking the metastatic spread of cancers in tissues and organs different from the original source. In addition, the targeting ligands are capable of linking pre-tumor lesions and dysplasia in different tissues and organs.

[0065] Spacer S

[0066] According to the present invention, S is an optionally present spacer that separates the targeting moiety from the dye. The presence of a spacer is particularly relevant for some embodiments where there is a risk of adverse interaction between the targeting moiety and the dye with each other. In addition, when the dye is relatively large and may interfere with the binding of the targeting moiety to the target site, the presence of a spacer may be necessary.

[0067] The spacer can be flexible (e.g., a simple alkyl chain) or rigid (e.g., a cycloalkyl or aryl chain) such that the dye is oriented away from the target. The spacer can also alter the pharmacokinetics and metabolism of the conjugate of formula (I) used as an imaging agent in a living organism.

[0068] A hydrophilic spacer can reduce the interaction with plasma proteins, reduce the blood circulation time and promote excretion. For example, if the spacer is a polyethylene glycol (PEG) moiety, the pharmacokinetics and blood clearance rate of the imaging agent in the body can be altered. In such embodiments, the spacer can improve the clearance of the imaging agent from background tissues (i.e., muscle, blood), thereby obtaining a better diagnostic image due to the high target-to-background contrast. In addition, the introduction of a specific hydrophilic spacer can shift the elimination of the contrast agent from the liver to the kidney, thereby reducing the systemic retention.

[0069] Thus, in a preferred embodiment, the spacer is a hydrophilic moiety comprising C1-C 20 alkyl, C3-C7 cycloalkyl or aryl. Preferably, the spacer is selected from -(CH2) p COO-, -(CH2CH2O) p CH2CH2COO- and -(CH2CH2O) p CH2CH2NH-, where p is an integer from 0 to 20. Preferably, p is 2, 6 or 12.

[0070] When not needed, the spacer is preferably absent, i.e., m is 0 and S represents a direct bond.

[0071] The spacer, or the targeting moiety when the spacer is absent, can be attached in the compound of formula (II), or at the R4 and / or R5 residues.

[0072] The linking group of R4-R5 is a reactive functional group suitable for conjugating the dye to the targeting moiety by forming a chemical bond, such as a carboxylic acid or formylamino residue.

[0073] For example, when an amine-containing targeting moiety (T) is conjugated to a compound of formula (II) in which R4 and / or R5 is an alkyl group substituted with a carboxylic acid, the carboxylic acid can optionally be activated by conversion to a more reactive form using an activating reagent, forming, for example, an N-hydroxysuccinimide (NHS) ester or a mixed anhydride. The amine-containing targeting moiety is then treated with the resulting activated acid to form an amide bond in order to obtain the corresponding compound of formula (II). Typically, the reaction is carried out in an aqueous buffer at pH 8 to 9, an optional co-solvent and DMSO or DMF, or in an organic solvent with an organic base such as DIPEA, TEA or NMM.

[0074] Alternatively, "unactivated" carboxylic acids can be used for direct conjugation.

[0075] Similarly, when the linking group of R4 and / or R5 is a formylamino group, the method for linking a suitable targeting moiety is similar, but generally does not require an activation step of the linker, and the dye and the targeting moiety are directly treated.

[0076] The compounds of formula (I) or (II) above can have one or more asymmetric carbon atoms, also known as chiral carbon atoms, and thus can give rise to diastereoisomers and optical isomers. Unless otherwise provided, the present invention also includes all these possible diastereoisomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers and their pharmaceutically acceptable salts.

[0077] As a non-limiting example, when the dye of formula (I) or (II) is substituted with a D-glucamine group (Y is a C6 alkyl group substituted with five hydroxyl groups), the present invention encompasses the corresponding enantiomerically pure compounds and any of their stereoisomers, such as compounds bearing an L-glucamine group or any possible mixtures of their D- / L-enantiomers.

[0078] The present invention also relates to the compounds of formula (I) or (II) above, wherein the functional groups of R1, R2 / R4 and / or R3 / R5, such as a sulfonyl group, a carboxylamino group or a carboxylic acid group, can be in the form of a pharmaceutically acceptable salt.

[0079] In one embodiment, the present invention relates to a compound of formula (I) or (II), wherein Y is selected from a straight-chain or branched C1-C6 alkyl group, a cycloalkyl group and a heterocyclic group substituted with 2 to 5 hydroxyl groups.

[0080] In a preferred embodiment, the present invention relates to a compound of formula (I) or (II), wherein Y is selected from:

[0081]

[0082] More preferably, the present invention relates to a compound of formula (I) or (II), wherein Y is a group of formula (ii) as defined above. Preferably, the group (ii) has the following stereochemical configuration obtained by using D-glucosamine in the preparation of the compound:

[0083]

[0084] Another embodiment of the present invention relates to a compound of formula (II), wherein m is 0 and the spacer S is represented by a direct bond, or m is 1 and the spacer is a hydrophilic moiety comprising a C1-C 20 alkyl, C3-C7 cycloalkyl or aryl. Preferably, the spacer is selected from -(CH2) p COO-, -(CH2CH2O) p CH2CH2COO- and -(CH2CH2O) p CH2CH2NH-, wherein p is an integer from 0 to 20. Preferably, p is 2, 6 or 12.

[0085] In another embodiment, T is a targeting moiety selected from small molecules, proteins, peptides, peptidomimetics, enzyme substrates, antibodies or any fragment thereof and aptamers.

[0086] Preferably, T is represented by a peptide, and in particular by a moiety that interacts with integrin receptors such as α v β3, α v β5, α v β6, α5β1, etc., preferably with α v β3 integrin receptor.

[0087] In a preferred embodiment, R1 is a straight-chain C4 alkyl substituted with -SO3H.

[0088] In another preferred embodiment, the present invention relates to a compound of formula (I) or (II), wherein Y represents the group (ii) as defined above, and is additionally represented by formula (Ia) or (IIa) respectively:

[0089]

[0090] wherein R1, R2, R3, R4, R5 and X are as defined above.

[0091] Particularly preferred are the compounds of formula (I) listed in Table Ia and the compounds of formula (II) listed in Table Ib.

[0092] Table Ia - Preferred compounds of formula (I)

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Table Ib - Preferred Conjugated Dyes of Formula (II)

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] The present invention also relates to a method for synthesizing a compound of formula (I) or (II), the compound of formula (I) or (II) being prepared as shown in the following description, the compound of formula (I) or (II) being a near-infrared dye optionally conjugated to a targeting moiety via a linking group.

[0106] Accordingly, the present invention provides a compound of formula (I) or (II) as defined above, which is used as an optical imaging agent for diagnostic biomedical applications in mammals (humans and animals). Preferably, the mammalian subject to be imaged is a human.

[0107] In a preferred embodiment, the compounds of the present invention are used as imaging agents for detecting normal (healthy) tissue or abnormal (pathological) tissue, especially tumors.

[0108] Preferably, the compound of formula (I) or (II) as defined above is used to detect normal (healthy) tissue by imaging techniques including, for example, angiography, perfusion imaging, cholangiography, and neuroimaging.

[0109] In another preferred embodiment, the present invention provides a compound of formula (I) or (II) as defined above for detecting abnormal (pathological) tissues, such as primary tumor lesions, local or distant metastases or pre-tumor lesions, in particular dysplasia and hyperplasia. In particular, the compound of formula (II) as defined above is preferably used for detecting and demarcating tumor margins in guided surgery of individual patients. Preferred uses are those in which the tumor is a tumor showing overexpression of a biological epitope, said biological epitope being selected, for example, from receptors, enzymes, glycoproteins, lipid rafts, transmembrane proteins located on the cell surface and soluble factors present in serum, plasma or the interstitial space. Preferably, the biological epitope is an integrin receptor for vitronectin, fibronectin and / or transforming growth factor-β (TGF-β).

[0110] The present invention also provides a compound of formula (I) or (II) as a fluorescent probe as defined above, wherein the detection and demarcation of the tumor is carried out under NIR radiation. Preferably, such detection and demarcation of the tumor is carried out before, during or after a surgical procedure to remove such tumor tissue. Fluorescence-guided surgical procedures are an example of such uses.

[0111] In addition, the present invention provides a compound of formula (I) or (II) as defined above for detecting inflamed tissue, fibrotic tissue, ischemic tissue or tissue with an abnormal metabolic rate.

[0112] The present invention also provides a method for imaging tissues and cells, the method comprising the steps of:

[0113] i) contacting the cells or tissue with a compound of formula (I) and (II);

[0114] ii) irradiating the tissue or cells at a wavelength absorbed by the imaging agent;

[0115] iii) detecting near-infrared emission using a fluorescence camera.

[0116] Preferably, the contacting of the cells or tissue with the imaging agent of formula (I) or (II) is effected by topical or local administration (e.g., by spraying, dipping or applying an ointment, foam or cream) or by systemic administration (enteral or parenteral administration).

[0117] The present invention also relates to a pharmaceutical diagnostic composition comprising a compound of formula (I) as defined above or a conjugate of formula (II) and at least one pharmaceutically acceptable carrier or excipient.

[0118] In particular, the present invention relates to a pharmaceutical composition comprising a dye of formula (I) or a salt thereof and one or more pharmaceutically acceptable adjuvants, excipients or diluents.

[0119] Alternatively, the present invention relates to a pharmaceutical composition comprising a conjugate of formula (II) as defined above or a salt thereof, wherein R4 and / or R5 is a C1-C6 alkyl group substituted with CONH-(S) m -T, and one or more pharmaceutically acceptable excipients, adjuvants or diluents.

[0120] Another aspect of the present invention relates to a diagnostic kit comprising a compound of formula (I) or (II) as defined above. In addition, the kit may contain additional excipients for performing optical imaging. These excipients are, for example, suitable buffers, containers, detection reagents or instructions for use. The kit preferably contains all the materials for intravenous administration of the compounds of the present invention.

[0121] The compounds of the present invention can be administered systemically or locally to the organ or tissue to be imaged before the imaging procedure. For example, the compound can be administered intravenously. In another embodiment, they can be administered parenterally or enterally.

[0122] The composition is administered in a dose effective to achieve the desired optical image of the tumor, tissue or organ, and the dose can vary widely depending on the compound used, the tissue in which imaging is to be performed, the imaging device used, etc.

[0123] The exact concentration of the imaging agent depends on the experimental conditions and the desired results, but typically can be in the range of 0.000001 mM to 0.1 mM. The optimal concentration is determined by systematic variation until a satisfactory result with minimal background fluorescence is obtained.

[0124] Once administered, the imaging agent of the present invention is exposed to light or other forms of energy that can penetrate tissue layers. Preferably, the radiation wavelength or band matches the excitation wavelength or band of the photosensitizer, and non-target cells and the rest of the subject (including blood proteins) have low absorption.

[0125] Typically, the optical signal can be detected by observation or instrumentation, and its response is related to fluorescence or light intensity, distribution and lifetime.

[0126] Synthesis description

[0127] The preparation of the compounds of formula (I) or (II) themselves or in the form of physiologically acceptable salts represents another 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 the experimental section.

[0128] General teachings on the preparation of cyanine dyes can be found in Mujumdar R.B. et al., Bioconjugate Chem. 1993, 4(2): 105-111, which relates to the synthesis and labeling of sulfoindocyanine dyes. However, the cyanines of the present invention are characterized by a specific functionalization pattern not present in compounds of the art, for which an appropriate synthetic method needs to be established. In fact, unlike other known cyanines, the compounds of the present invention even carry three functional moieties (carboxylic acid or amido group) to be derivatized in different ways, such that in most cases protecting groups need to be used to direct the reactions on the desired functional groups.

[0129] It is known that difficulties may arise when operating on cyanines under the strong pH and temperature conditions essential for removing the protecting groups, since in some cases the stability of the cyclohexenyl-polymethine backbone may be compromised, where the dye degrades violently.

[0130] Furthermore, when deprotecting the carboxyl groups of R1-R5, further obstacles may be encountered due to possible hydrolysis and degradation of the amido -CONH-Y (typically, amide derivatives can be hydrolyzed in a concentrated basic medium, see for example Yamana et al., Chem. Pharm. Bull., 1972, 20(5), 881-891).

[0131] In a preferred embodiment, the protecting group of the R4 or R5 moiety is an ester group. More preferably, an ethyl ester group can be advantageously used.

[0132] Preparation of the cyanine dye of formula (I)

[0133] According to the present invention, the compounds of formula (I) can be prepared by a general sequence of synthetic steps as reported in Scheme 1 below.

[0134]

[0135] Scheme 1

[0136] In Scheme 1 above, R1, R2, R3, X and Y are as defined above, and Pg is absent or is a suitable protecting group.

[0137] Thus, the method of the present invention comprises the following steps:

[0138] a) Treating an appropriate amount of 5-carboxy-2,3,3-trimethylpseudoisoindole of formula (III) and (IV) with a polyhydroxylated amine, such as glucamine, glucosamine, aminoglucose, tromethamine, serinol or isoserinol, which bears a suitable protecting group on the hydroxyl moiety;

[0139] b) React the intermediate (V) and intermediate (VI) obtained in step a) with 2-chloro-1-formyl-3-(hydroxy-methylene)-1-cyclohexene to obtain a cyanine intermediate of formula (VII), wherein R1, R2, Y and Pg are as defined;

[0140] c) Optionally remove the protecting group (Pg) of the Y group from the intermediate (VII);

[0141] d) Substitute the chlorine atom on the intermediate (VII) with a suitable nucleophile to obtain the final product of formula (I) or its salt.

[0142] According to step a), the reaction of derivatives (III) and (IV) with a polyhydroxylated amine can be carried out by activating the carboxylic acid ester group with a coupling agent (such as selected from HATU, TBTU, HBTU, PyBOP, DCC, DSC and DCC-NHS) and an organic base (such as TEA, DIPEA, NMM or pyridine) in a solvent (such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetonitrile, etc.) at room temperature for a suitable time from 30 minutes to several hours. This derivatization of the carboxylic acid can be carried out using an alkylated pseudindole or indole prior to quaternization. In this case, it is important to protect the hydroxyl groups of the polyhydroxylated amine with a suitable protecting group such as acetyl before alkylation with a sultone or alkyl bromoacetate. This alkylation can be carried out without solvent or in a high-boiling solvent such as butyronitrile, sulfolane, 1,2-dichlorobenzene, dimethylacetamide, dimethylformamide or dimethyl sulfoxide, and the solution is stirred at a high temperature such as 90 °C - 180 °C for several hours, typically from 12 hours to 5 days.

[0143] According to step b), the reaction can be carried out using a Vilsmeier reagent in the form of a bisanilino form or a bisaldehyde form (as reported in Scheme 1). This reaction can be carried out in several solvents, such as ethanol, methanol, acetic anhydride or acetic acid, with or without different bases, such as trimethylamine, pyridine, sodium acetate, potassium acetate, etc., and the mixture is stirred at different temperatures from 45 °C to 120 °C for several hours (typically 2 - 24 hours).

[0144] According to step c), any protecting group of the intermediate (VII) is removed from the Y moiety according to known methods described, for example, in T.W.Green and P.G.M.Wuts, Protective Groups in Organic Synthesis, Wiley, N.Y. 2007, 4th Edition, Chapter 5. Different from other classes of cyanines, these dyes show higher stability at acidic pH, even at higher temperatures and for several hours.

[0145] According to step d), depending on the X-R3 substituent, the reaction can be carried out using several protocols. When introducing phenol or its derivatives, such as phenol-SO3H, the dye can be heated in DMSO in the presence of an inorganic base such as sodium carbonate or potassium carbonate. While when chlorine is replaced by phenyl or its derivatives, the reaction can be carried out in degassed water or a mixture of degassed water and a co-solvent (such as methanol, ethanol, etc.), heating for a shorter time in the presence of a Pd catalyst (such as palladium acetate or Pd tetrakys) and an optional base (such as sodium carbonate or potassium carbonate).

[0146] When R1 has the same meaning as R2, only one reaction a) is carried out, and subsequent step b) is carried out with units of two intermediates (V) or (VI) instead of one unit of intermediate (V) and one unit of intermediate (VI).

[0147] Alternatively, when R1 has the same meaning as R2 and is a straight-chain or branched C1-C6 alkyl group substituted with -SO3H, the compound of formula (I) can also be prepared according to the following Scheme 2:

[0148]

[0149] Scheme 2

[0150] In the above Scheme 2, R1 is a straight-chain or branched C1-C6 alkyl group substituted with -SO3H, and X, Y and R3 are as defined above.

[0151] Therefore, another method of the present invention comprises the following steps:

[0152] f) Reacting at least two equivalents of a pseudoindole intermediate (III) (wherein R1 is a straight-chain or branched C1-C6 alkyl group substituted with -SO3H) with a Vilsmeier reagent (in the form of a dialdehyde or a dianilino form) to obtain a corresponding cyanine intermediate of formula (IX);

[0153] g) Substituting the chlorine atom on the intermediate (IX) with a suitable nucleophile to obtain an intermediate (X);

[0154] h) Treating an appropriate amount of the intermediate of formula (X) with a polyhydroxylated amine, such as glucosamine, N-methylglucamine, glucosamine hydrochloride, tromethamine, serinol or isoserinol, to obtain the end product of formula (I) or its salt.

[0155] According to step f), the reaction can be carried out in several solvents, such as ethanol, methanol, acetic anhydride or acetic acid, with or without different bases, such as trimethylamine, pyridine, sodium acetate, potassium acetate, etc., stirring the mixture at different temperatures from 45 °C to 120 °C for several hours (typically 2 - 24 hours).

[0156] According to step g), the reaction can be carried out using several protocols, depending on the X-R3 substituent. When introducing phenol or its derivatives such as phenol-SO3H, the dye can be heated in DMSO in the presence of an inorganic base such as sodium carbonate or potassium carbonate. While when the chlorine is replaced by a phenyl or its derivatives, the reaction can be carried out in degassed water or a mixture of degassed water and a co-solvent (such as methanol, ethanol, etc.), heating for a shorter time in the presence of a Pd catalyst (such as palladium acetate or Pd tetrakys) and an optional base (such as sodium carbonate or potassium carbonate).

[0157] According to step h), the reaction of the derivative (X) with the polyhydroxylated amine can be carried out by activating the carboxylic acid ester group with a coupling agent (such as selected from HATU, TBTU, HBTU, PyBOP, DCC, DSC and DCC-NHS) and an organic base (such as TEA, DIPEA, NMM or pyridine) in a solvent (such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetonitrile, etc.) at room temperature for a suitable time from 30 minutes to several hours.

[0158] In another embodiment, the compound of formula (I) prepared by the method according to the invention can be conveniently converted into another compound of formula (I) by operating according to well-known synthesis conditions. The following are examples of possible conversions:

[0159] e) Converting the compound of formula (I) in which R2 is -COOH, i.e., the compound of formula (Ib), into the corresponding compound of formula (I) in which R2 is -CONH2, i.e., the compound of formula (Ic):

[0160]

[0161] According to step e), the conversion of the carboxylic acid of formula (Ib) to the carboxamide of the corresponding formula (Ic) can be accomplished in a variety of ways and experimental conditions well known in the art for preparing carboxamides. As an example, the carboxylic acid can first be converted to a suitable activated ester and then preferably reacted with an ammonium salt such as NH4Cl in the presence of a coupling agent such as HBTU.

[0162] Preparation of the conjugate compound of formula (II)

[0163] The cyanine derivative of formula (I) or its salt can be conjugated with a suitable targeting moiety, optionally inserting a spacer, to obtain the corresponding compound of formula (II). The conjugation can be carried out according to different methods known in the art, for example, by directly coupling the carboxylic acid group of the compound with the nucleophilic residue of the targeting moiety or optionally with a spacer, or by pre-activation, in which the carboxylic acid group is converted to a more reactive group, such as an ester, such as NHS, before coupling.

[0164] In one embodiment, in the case of activating a carboxylic acid by forming an NHS ester, the present invention provides a method of labeling a targeting moiety using a dye of formula (XIa)

[0165]

[0166] or a dye of formula (XIb)

[0167]

[0168] wherein

[0169] X is a direct bond or -O-;

[0170] Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl, and heterocyclic group, which is substituted by at least two hydroxyl groups;

[0171] Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl, and heterocyclic group, which is substituted by at least two hydroxyl groups;

[0172] R1 is straight-chain or branched C1-C6 alkyl, which is substituted by a group selected from -SO3H, -COOH, -CONH2, and -COO-C1-C6 alkyl; and

[0173] R3 is hydrogen, -SO3H, or straight-chain or branched C1-C6 alkyl substituted by -COOH or -CONH-Y, where Y is a group selected from straight-chain or branched C1-C6 alkyl, C3-C7 cycloalkyl, and heterocyclic group substituted by at least two hydroxyl groups,

[0174] the method comprising reacting a dye of formula (XIa) or (XIb) with the targeting moiety.

[0175] If the compounds of formula (I) or (II) prepared according to the above method are obtained in the form of an isomer mixture, separating them into the respective single isomers of formula (I) or (II) using conventional techniques is within the scope of the present invention.

[0176] The final compounds can be separated and purified using conventional methods, such as chromatography and / or crystallization and salt formation.

[0177] 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 subsequently be formulated with a pharmaceutically acceptable carrier or diluent to obtain a pharmaceutical composition.

[0178] Experimental Section

[0179] The present invention and its specific embodiments described in the following sections are merely exemplary and should not be considered as limiting the present invention: they show how to implement the present invention and are intended to be illustrative rather than limiting the scope of the present invention.

[0180] Materials and Equipment

[0181] All chemicals and solvents used for the reactions were of reagent grade. Analytical grade solvents were used for chromatographic purification. Most of the reagents were commercially available unless otherwise stated, including the targeting moieties (e.g., Panitumumab (Vectibix, Amgen; CAS Nr: 339177-26-3); c(RGDfK) (cyclo(Arg-Gly-Asp-D-Phe-Lys), Bachem; CAS Nr: 161552-03-0).

[0182] All synthesized compounds were purified by reverse-phase chromatography (RP-HPLC) and characterized by mass spectrometry using an LC / MS instrument equipped with a UV-Vis detector and an ESI source. An analytical run was performed using a Waters Atlantis dC18 5μm, 4.6×150mm column applying a gradient of A phase CH3COONH4 10mM and B phase acetonitrile. The measured mass-to-charge ratios of each compound are listed.

[0183] The absorbance (Abs) of the compounds of the present invention was measured using a double-beam UV-VIS spectrophotometer (Lambda 40, Perkin Elmer). Emission / excitation (Em / Ex) spectra and absolute fluorescence quantum yield (φ) measurements were performed on a fluorescence spectrophotometer (FluoroLog-3 1IHR-320, Horiba Jobin Yvon) equipped with an F-3018 integrating sphere attachment. Measurements were carried out using the excitation wavelength at the maximum absorbance of different dyes and the samples were excited with a 450W xenon light source. Detection was performed either by a photomultiplier tube (PMT-NIR) cooled detector or by a TBX-04 detector. Dye solutions with an absorbance below 0.1 (optical density) were carefully prepared to minimize the reabsorption phenomenon.

[0184] In vivo imaging experiments were performed using an IVIS Spectrum in vivo imaging system (Perkin Elmer Inc.). The system is equipped with 10 narrowband excitation filters (30nm bandwidth) spanning 430 - 850nm and 18 narrowband emission filters (20nm bandwidth).

[0185] List of Abbreviations

[0186] DCC N,N'-Dicyclohexylcarbodiimide

[0187] DIPEA N,N-Diisopropylethylamine

[0188] DMF Dimethylformamide

[0189] DMSO Dimethyl sulfoxide

[0190] DSC N,N'-Disuccinimidyl carbonate

[0191] EuK Glutamic acid-urea-lysine

[0192] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0193] HBTU O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0194] HPLC High performance liquid chromatography

[0195] PBS Phosphate buffered saline

[0196] NHS N-Hydroxysuccinimide

[0197] NMM N-Methylmorpholine

[0198] RT Room temperature

[0199] PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0200] TEA Triethylamine

[0201] TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0202] TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0203] μL Microliter

[0204] μM Micromole per liter

[0205] t R Retention time (HPLC)

[0206] cRGDfK Cyclo-(Arg-Gly-Asp-D-Phe-Lys)

[0207] The abbreviations for individual amino acid residues are conventional: for example, Asp or D is aspartic acid, Gly or G is glycine, Arg or R is arginine. Unless otherwise stated, amino acids referred to herein are to be understood as being in the L-isomer configuration (e.g., the "f" in Phe or c(RGDfK) is in the D-isomer form, as shown in the list of abbreviations).

[0208] Example 1: Synthesis of Compound 11

[0209] Preparation of Intermediate (Va)

[0210] 5-Carboxy-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (10.8 g, 31.9 mmol) was suspended in dry DMF (100 mL) under N2 atmosphere: D-glucosamine (6.9 g, 38.2 mmol), DIPEA (11 mL, 63.7 mmol) and HATU (14.5 g, 38.2 mmol) were added. The solution was stirred at RT for 16 h, then cold ether (200 mL) was added. The mixture was filtered and the solid was washed with ethyl acetate (2 x 50 mL). The solid was dissolved in water and purified by flash chromatography using a pre-packed C18 silica column with a gradient of 0.1% ammonium acetate / acetonitrile. The fractions containing the pure product were combined, vacuum distilled and freeze-dried three times to give a pale pink solid (13.6 g, 80% yield). HPLC purity at 270 nm: 94%. MS: [M+H] + 504.2。

[0211] Preparation of Intermediate (VIIa)

[0212] Acetic acid (8.65 mL) was added to a suspension of Intermediate (Va) (2.0 g, 3.98 mmol) in acetic anhydride (10 mL). The mixture was heated at 45 °C, 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene (377.8 mg, 2.19 mmol) was added to give a yellow solution. The temperature was raised to 75 °C (the solution turned green-brown), sodium acetate (408 mg, 4.97 mmol) was added and immediately a green solution was obtained. The solution was stirred at 100 °C for 3 h, then cooled to RT and the solvent was removed under reduced pressure. The crude product was dissolved in water-acetonitrile and purified by flash chromatography using a pre-packed C18 silica column with a gradient of water-acetonitrile. The fractions containing the pure product were combined, vacuum distilled to give a green solid (2.5 g, 40% yield). HPLC purity at 780 nm: 90%. MS: [M+H] + 1560.5。

[0213] Preparation of Intermediate (VIIIa)

[0214] Intermediate (VIIa) (2.5 g, 1.59 mmol) was dissolved in water / acetonitrile 1 / 1 (20 mL) and the pH was adjusted from 2.2 to 1.5 with 1N HCl. The solution was stirred at 80 °C for 16 h. The crude product was purified by flash chromatography using a pre-packed C18 silica column with a gradient of water-acetonitrile. The fractions containing the pure product were combined, vacuum distilled and freeze-dried to give a green solid (1.09 g, 60% yield). HPLC purity at 780 nm: 95%. MS: [M+H]+1140.7。

[0215] Synthesis of Compound 11

[0216] To a suspension of intermediate (VIIIa) (1.09 g, 0.95 mmol) in degassed water (20 mL) was added 4-(2-carboxyethyl)phenylboronic acid (332 mg, 1.71 mmol), Pd(PPh3)4 (165 mg, 0.14 mmol), and sodium carbonate (181 mg, 1.71 mmol). The mixture was stirred at 80 °C under a nitrogen atmosphere for 16 h. Then, after cooling to RT, the pH was adjusted to 6.5 with 2 N HCl. The crude mixture was purified by flash chromatography using a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the pure product were combined, distilled in vacuo, and lyophilized to give a green solid (0.714 g, 60% yield). HPLC purity at 780 nm: 99%. MS: [M+H]+ 1253.5.

[0217] Example 2: Synthesis of Compound 12

[0218] Compound 11 (56 mg, 0.0434 mmol) prepared as in Example 1 was suspended in dry DMSO (5 mL) under a nitrogen atmosphere. NMM (19 μL, 0.174 mmol), HATU (66 mg, 0.174 mmol), and D-glucosamine (39 mg, 0.217 mmol) were added, and after stirring at RT for 2 h, the reaction mixture was precipitated in cold ethyl acetate (30 mL). The green solid was dissolved in water and purified by flash chromatography using a pre-packed C18 silica column with a 0.1% ammonium acetate-acetonitrile gradient. The fractions containing the product were combined, distilled in vacuo, and lyophilized three times to give a green solid as the ammonium salt (69.88 mg). To remove the ammonium counterion, the solid was dissolved in water, loaded onto a C18 column, washed with water (2 CV), 0.1% HCOOH (2 CV), water (5 CV), and eluted with water / acetonitrile 1 / 1. The solvent was distilled in vacuo and lyophilized to give a green solid (37 mg, 60% yield). HPLC purity at 780 nm: 100%. MS: [M+H] + 1419.4.

[0219] Example 3: Synthesis of Compound 13

[0220] Preparation of Intermediate (VIb)

[0221] In a dry round-bottom flask, 2,3,3-trimethyl-3H-indole-5-carboxylic acid (711 mg, 3.5 mmol) was dissolved in dry DMF (4 mL) under a nitrogen atmosphere, and then DIPEA (380 μL, 4.90 mmol) was added. After stirring for 30 minutes at RT, a solution of TBTU (603 mg, 4.20 mmol) in dry DMF (2 mL) was added. After stirring for 1 hour at RT, a suspension of D-glucosamine (312 mg, 3.85 mmol) in dry DMF (2 mL) was added. After overnight, the reaction was not complete. Therefore, the same amounts of TBTU, DIPEA, and D-glucosamine were added and stirred for another 2 hours. The mixture was dried in vacuo and purified using a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the pure product were combined, distilled in vacuo, and freeze-dried to give a white-brown powder (888.2 mg, 70% yield). HPLC purity at 270 nm: 93% purity, MS: [M+H] + 502.57。

[0222] Acetic anhydride (3 mL) and pyridine (0.5 mL) were added to this intermediate (888.2 mg, 2.42 mmol) to give a suspension that gradually dissolved over time. The mixture was kept under stirring in a nitrogen atmosphere at RT for 4 hours. The solution was concentrated in vacuo and purified using a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the pure product were collected, concentrated in vacuo, and freeze-dried to give a white-brown solid (861.4 mg, 62% yield). HPLC purity at 270 nm: 97.5%, MS: [M+H] + 204。

[0223] In a round-bottom flask, such obtained product (1.103 g, 1.91 mmol) and 1-bromohexanoic acid (933 mg, 4.77 mmol) were dissolved in 1,2-dichlorobenzene (6 mL). The mixture was heated at 130 °C in a nitrogen atmosphere for 6 hours, then 1-bromohexanoic acid (933 mg, 4.77 mmol) was added again, and the reaction system was kept under the same conditions overnight. The crude product was washed with ether, the solvent was decanted, the solid was dissolved in acetonitrile, and purified using a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the product were collected, concentrated in vacuo, to give a red oil (861.4 mg, 62% yield). HPLC purity at 270 nm: 97.5%, MS: [M+H] + 691.0。

[0224] Preparation of Intermediate (VIIb)

[0225] In a dry round-bottom flask, the intermediate (Va) (1.170 g, 2.33 mmol) prepared as in Example 1 and (VIb) (1.79 g, 2.33 mmol) were suspended in acetic anhydride (20 mL) and acetic acid (5 mL). The mixture was heated at 45 °C until the two powders were completely dissolved. Then 2-chloro-3-(hydroxy-methylene)-1-cyclohexene-1-carbaldehyde (430 mg, 2.49 mmol) was added and the mixture was heated to 50 °C. Potassium acetate (237 mg, 2.89 mmol) was added and the mixture was heated at 100 °C for 2 hours. The solvent was removed in vacuo and the crude green solid was purified by a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the product were collected, concentrated in vacuo and freeze-dried to give a green powder (1.221 g, 35% yield). HPLC purity at 780 nm: 72%, MS: [M+H] + 1540.6.

[0226] Preparation of intermediate (VIIIb)

[0227] The intermediate (VIIb) (701 mg, 0.33 mmol) was dissolved in acetonitrile (3 mL) and water (15 mL) was added. The solution was acidified to pH 1.6 with 1N HCl, heated at 80 °C for 4 hours and then at 55 °C overnight. The organic solvent was removed under reduced pressure and the aqueous solution was purified by a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the product were collected, concentrated in vacuo and freeze-dried to give a green powder (179 mg, 50% yield). HPLC purity at 780 nm: 95%, MS: [M+H] + 1120.3.

[0228] Synthesis of compound 13

[0229] In a dry round-bottom flask, the intermediate (VIIIb) (115 mg, 1.03 mmol) was dissolved in degassed water (3 mL), then phenylboronic acid (22.7 mg, 1.85 mmol), sodium carbonate (19.6 mg, 1.85 mmol) and palladiumtetrakis (17.8 mg, 0.15 mmol) were added. The mixture was heated at 80 °C in a nitrogen atmosphere for 2 hours. The solution was adjusted to pH 7.15 with 0.1N HCl and purified by a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the product were collected, concentrated in vacuo and freeze-dried to give a green powder (99 mg, 83% yield). HPLC purity at 780 nm: 98%, MS: [M+H] + 1162.2.

[0230] Example 4: Synthesis of compound 14

[0231] A solution of intermediate (VIIIb) (120 mg, 0.107 mmol) prepared as in Example 2 in dry DMSO (5 mL) was added dropwise under a nitrogen atmosphere to a suspension of phenol (101 mg, 1.07 mmol) and anhydrous potassium carbonate (148 mg, 1.07 mmol) in dry DMSO (7 mL). The mixture was stirred at 50 °C for 4 h. After cooling to RT, cold ether (30 mL) was added, the solid was filtered and washed twice with cold ether. It was dissolved in water and the pH was adjusted from 11.5 to 6 with 0.5 N HCl. The crude solid was purified by flash chromatography using a pre-packed C18 silica column with a gradient of 0.1% ammonium acetate - acetonitrile. The fractions containing the pure product were combined and evaporated in vacuo. To remove the ammonium counterion, the pH was adjusted to 1.6, the product was loaded onto a C18 silica column, washed with water and eluted with water - acetonitrile 1:1. The solvent was evaporated in vacuo and the aqueous solution was freeze-dried to give a green solid (42 mg, 33% yield). HPLC purity at 780 nm: 99.3%. MS: [M+H] + 1178.3。

[0232] Example 5: Synthesis of Compound 15

[0233] A solution of intermediate (VIIIb) (20 mg, 0.018 mmol) prepared as in Example 2 in dry DMSO (3 mL) was added dropwise under a nitrogen atmosphere to a suspension of sodium 4-hydroxybenzenesulfonate (35 mg, 0.18 mmol) and anhydrous potassium carbonate (25 mg, 0.18 mmol) in dry DMSO (5 mL). The mixture was stirred at 50 °C for 4 days. Cold ether (30 mL) was added to the brown mixture, the solid was filtered and washed twice with cold ether. The solid was dissolved in water and the pH was adjusted from 11.5 to 6 with 0.5 N HCl: the solution turned green again. The crude product was purified by flash chromatography using a pre-packed C18 silica column with a water - acetonitrile gradient. The fractions containing the pure product were collected, concentrated in vacuo and freeze-dried to give a green solid (15 mg, 65% yield). HPLC purity at 780 nm: 100%. MS: [M+H] + 1257.3。

[0234] Example 6: Synthesis of Compound 16

[0235] A solution of the intermediate (VIIIa) (30 mg, 0.026 mmol) prepared as in Example 1 in dry DMSO (3 mL) was added dropwise under a nitrogen atmosphere to a suspension of sodium 4-hydroxybenzenesulfonate (14 mg, 0.08 mmol) and anhydrous potassium carbonate (10 mg, 0.08 mmol) in dry DMSO (3 mL). The mixture was stirred at 80 °C for 6 hours. Cold ethyl acetate (20 mL) was added to the brown mixture, the solid was filtered, dissolved in water, and the pH was adjusted from 11.5 to 3 with 0.5 N HCl: the solution turned green again. The crude product was purified by flash chromatography using a pre-packed C18 silica column with a water-acetonitrile gradient. The fractions containing the pure product were collected, concentrated in vacuo, and lyophilized to give a green solid (14 mg, 42% yield). HPLC purity at 780 nm: 97.8%. MS: [M+H] + 1277.3

[0236] Example 7: Synthesis of Compound 17

[0237] In a dry round-bottom flask, the intermediate (VIIIa) (9 mg, 0.008 mmol) prepared as in Example 1 was dissolved in degassed water (2 mL), then phenylboronic acid (2.2 mg, 0.018 mmol) and palladium acetate (0.15 mg, 0.0006 mmol) were added. The mixture was refluxed for 2 hours under a nitrogen atmosphere. The solution was purified using a pre-packed C18 column with a water-acetonitrile gradient. The fractions containing the product were collected, concentrated in vacuo, and lyophilized to give a green powder (7 mg, 75% yield). HPLC purity at 780 nm: 98%, MS: [M+H] + 1184.3

[0238] Example 8: Synthesis of Compound 19

[0239] Preparation of Intermediate (IXa)

[0240] Compound (IIIa) (180 mg, 0.53 mmol), N-[(3-(phenyliminoethylidene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride (101 mg, 0.265 mmol), and sodium acetate (109 mg, 1.32 mmol) were dissolved in absolute ethanol (55 mL), and the solution was refluxed for 26 hours. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography using a pre-packed C18 silica column with a water / acetonitrile gradient. The fractions containing the pure product were combined, distilled in vacuo, and lyophilized 3 times to give a green solid (154 mg, 72% yield). HPLC purity at 780 nm: 97%. MS: [M+H] + 815.2

[0241] Preparation of Intermediate (Xa)

[0242] A solution of intermediate (IXa) (40 mg, 0.05 mmol) in dry DMSO (4 mL) was added dropwise under a nitrogen atmosphere to a suspension of phenol (45 mg, 0.49 mmol) and anhydrous potassium carbonate (68 mg, 0.49 mmol) in dry DMSO (8 mL). The mixture was stirred at 50 °C for 8 hours. After cooling to RT, cold ether (30 mL) was added, the solid was filtered, washed twice with cold ether, dissolved in water, and the pH was adjusted from 12 to 6 with 0.5 N HCl. The crude solid was purified by flash chromatography using a pre-packed C18 column with a water / acetonitrile gradient. The fractions containing the pure product were combined and distilled in vacuo. The solvent was distilled off under reduced pressure, and the aqueous solution was freeze-dried to give a green solid (26 mg, 61% yield). HPLC purity at 780 nm: 98%. MS: [M+H] + 872.1

[0243] Synthesis of Compound 19

[0244] TBTU (5.1 mg, 0.015 mmol) was added to a solution of intermediate (Xa) (3.3 mg, 0.0038 mmol) and DIPEA (6 μL, 0.031 mmol) in anhydrous DMF (5 mL). The solution was stirred at RT for 30 minutes, then serine alcohol (1 mg, 0.011 mmol) was added, and the solution was stirred for 90 minutes. The solvent was removed under reduced pressure, and the crude product was purified by flash chromatography using a pre-packed C18 column, eluting with a water-acetonitrile gradient. The fractions containing the pure product were combined, concentrated under reduced pressure, and freeze-dried to give a dark green solid (2 mg, 53% yield). HPLC purity at 780 nm: 98.5%. MS: [M+H] + 1019.1

[0245] Similarly, compounds 18, 20, 21, 22, and 23 were prepared by the following similar methods, but using different hydroxylated amines (as the Y group) as described above or starting from a skeleton with a phenyl group instead of phenol.

[0246] Example 9: Synthesis of Compound 1

[0247] Compound 11 (5 mg, 0.004 mmol) was dissolved in dry DMF (3 mL) under an inert gas atmosphere. DIPEA (6.8 μL, 0.04 mmol) and TSTU (12 mg, 0.04 mmol) were added, and the solution was stirred at RT for 48 h. Then cold diethyl ether (20 mL) was added, the precipitate was filtered, and washed twice with cold diethyl ether to obtain a green solid. HPLC purity at 780 nm: 90%. MS: [M+H] + 1351.

[0248] The NHS ester was dissolved in a solution of c(RGDfK) (2.7 mg, 0.004 mmol) in 1 mL of borate buffer at pH 9. The reaction system was stirred at RT for 24 h, then the product was precipitated in cold diethyl ether and washed twice with cold diethyl ether to obtain a green solid. The crude solid was purified by HPLC chromatography using a Kromasil C8 column with a linear gradient of 0.1% ammonium acetate / acetonitrile. The fractions containing the pure product were combined, distilled in vacuo, and freeze-dried three times to obtain a green solid (3.9 mg, 50% yield). HPLC purity at 780 nm: 97.6%. MS: [M+H] + 1840.6.

[0249] Example 10: Synthesis of Compound 3

[0250] Method A by direct coupling

[0251] In a dry round-bottom flask, compound 13 (2 mg, 0.0017 mmol) was dissolved in DMF (1 mL) containing DIPEA (0.6 μl, 0.003 mmol) under a nitrogen stream atmosphere at RT. After stirring at RT for 30 min, a solution of TBTU (0.6 mg, 0.002 mmol) in dry DMF (1 mL) was added. After stirring at RT for 1 h, a solution of c(RGDfK) (1.4 mg, 0.002 mmol) in dry DMF (1 mL) was added dropwise to the stirred solution. The reaction system was stirred overnight at RT, then the solvent was removed in vacuo, and the crude product was purified by an analytical HPLC C18 silica column with a 0.1% ammonium acetate-acetonitrile gradient. The fractions containing the pure product were combined, concentrated in vacuo, and freeze-dried three times to obtain a green powder (0.7 mg, 23% yield). HPLC purity at 780 nm: 98%, MS: [M / 2] + 872.8.

[0252] Method B via NHS ester

[0253] In a dry round-bottom flask, dissolve compound 13 (5 mg, 0.0043 mmol) in dry DMF (2 mL) containing DIPEA (2.2 μL, 0.0129 mmol). Add a solution of TSTU (3.78 mg, 0.0129 mmol) in dry DMF (1 mL) under a nitrogen stream atmosphere at room temperature. Stir the green solution overnight at RT, then precipitate the product by adding cold ether. Centrifuge the solid and wash it twice with ether, and use it for the next step without further purification. Dissolve the NHS ester in a solution of c(RGDfK) (2.2 mg, 0.0043 mmol) in pH 9 borate buffer (1 mL), and stir this solution overnight at room temperature. Then adjust the pH to 7 with 0.1 N HCl, and purify the crude product using an analytical HPLC C18 silica column with a 0.1% ammonium acetate - acetonitrile gradient. Combine the fractions containing the pure product, concentrate in vacuo, and lyophilize three times to obtain a green powder (3.9 mg, 52% yield). HPLC purity at 780 nm: 97.7%, MS: [M / 2] + 872.8

[0254] Example 11: Synthesis of Compound 2

[0255] Suspend compound 15 (6 mg, 0.0048 mmol) in dry DMF (6 mL), then add NMM (2.6 μL, 0.024 mmol) and TSTU (7.2 mg, 0.024 mmol). Stir the solution for 16 hours at RT. Add cold ether (30 mL), filter the precipitate, and wash it twice with cold ether. Use the green solid for the next step without any further purification. HPLC at 780 nm: 88%, MS: [M + H] + 1354.3

[0256] Dissolve the NHS ester in borate buffer at pH 9 (1 mL), and add a solution of c(RGDfK) (3.4 mg, 0.0048 mmol) in borate buffer at pH 9 (1 mL). Stir the solution for 5 hours at RT, then adjust the pH to 6.5 with 0.1 N HCl, and purify the crude product by HPLC chromatography using a phenyl - C18 column with a linear gradient of 0.1% ammonium acetate - acetonitrile. Combine the fractions containing the pure product, distill in vacuo, and lyophilize three times to obtain a green solid (6.4 mg, 71% yield). HPLC purity at 780 nm: 99.6%. MS: [M + H] + 1845.5

[0257] Example 12: Synthesis of Compound 4

[0258] Compound 14 (12 mg, 0.0102 mmol) was dissolved in dry DMF (1 mL) under a nitrogen atmosphere. DIPEA (4.8 μL, 0.0286 mmol) and TBTU (4.6 mg, 0.0143 mmol) were added. After stirring for 1 hour at RT, c(RGDfK) (6.15 mg, 0.0102 mmol) was added. The reaction system was stirred at RT for 16 hours, then cold ether (25 mL) was added, the precipitate was filtered and washed twice with cold ether. The crude solid was dissolved in water and purified by HPLC chromatography using a phenyl-C18 column with a linear gradient of 0.1% ammonium acetate - acetonitrile. The fractions containing the pure product were combined, vacuum distilled, and freeze-dried three times to give a green solid (12.3 mg, 67% yield). HPLC purity at 780 nm: 98.3%, MS: [M+H] + 1763.6.

[0259] Example 13: Synthesis of Compound 5

[0260] The monoclonal antibody EGFR ligand pamumab (6 mg) was diluted to 5 mg / mL with PBS, and the pH was adjusted to 9 by adding 120 μL of 1.0 M potassium phosphate. The compound 15-NHS ester (prepared as described for compound 2 in Example 11) was dissolved in DMSO at a concentration of 10 mg / ml; then the dye and the antibody were immediately mixed at a molar ratio of 2.5:1 and kept at room temperature in the dark for 3 h. After 3 h, the conjugation reaction mixture was layered on a PBS-equilibrated Zeba spin column and centrifuged at 1500 g for 2 min to separate the conjugate from the dye. After filtration through a 0.22-μm polyethersulfone (PES) membrane, the conjugated pamumab in PBS at pH 7.4 was analyzed by SE-HPLC, RP-HPLC, and UV / VIS spectrophotometry to determine the concentration and purity. The molar conjugation ratio (number of dye molecules conjugated per antibody) was 1.53.

[0261] Example 14: Synthesis of Compound 6

[0262] The small molecule CAIX ligand 4a described in Wichert et al., Nat Chem 2015, 7, 241-249 was prepared according to the method disclosed therein and conjugated with compound 15. 11 mg of compound 15 (8.7 μmol) was dissolved in 1 mL of DMF, and then 4.5 mg of PyBOP (8.7 μmol) and 6 μL of DIPEA (35.0 μmol) were added under continuous stirring. After 20 minutes, 8 mg of small molecule 4a (13.0 μmol) was dissolved in 1 mL of DMF and added to the reaction mixture, and the mixture was stirred for another 30 minutes at room temperature. Purification was carried out by preparative HPLC with a yield of 50%. The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI(+) using a Waters Atlantis dC18 column (μm, 4.6x150 mm). HPLC purity at 779 nm: 99%; MS: [M / 2] + 929.7.

[0263] Example 15: Synthesis of Compound 7

[0264] The small molecule CAIX ligand 8a described in Wichert et al., Nat Chem 2015, 7, 241-249 was prepared according to the method disclosed therein and conjugated with compound 15. 9 mg of compound 15 (7.1 μmol) was dissolved in 1 mL of DMF, and then 3.7 mg of PyBOP (7.1 μmol) and 5 μL of DIPEA (28.0 μmol) were added under continuous stirring. After 20 minutes, 11 mg of molecule 8a (11.0 μmol) was dissolved in 1 mL of DMF and added to the reaction mixture, and the mixture was stirred for another 30 minutes at RT. Purification was carried out by preparative HPLC with a yield of 50%. The isolated pure product was characterized by HPLC-UV-VIS-MS-ESI(+) using a Waters Atlantis dC18 column (μm, 4.6x 150 mm).

[0265] HPLC purity at 780 nm: 99%; MS: [M / 2]+1157.8.

[0266] Example 16: Synthesis of Compound 8

[0267] 9.9 mg of compound 15 (7.9 μmol) was dissolved in 3 mL of dry DMF, then 2 μL of NMM (18.2 μmol) and 7.11 mg of TSTU (23.6 μmol) were added, and the mixture was stirred at RT for 2 h. The NHS ester of compound 15 (HPLC conversion rate 85.9%) was precipitated in 25 mL of ice-cold ethyl acetate. The precipitate was washed with ethyl acetate and dried in a stream of N2.

[0268] Dissolve the NHS ester of compound 15 in 1 mL of dry DMF. Add dropwise the solution prepared by dissolving 5.74 mg of EuK TFA salt (13.43 μmol) in 1 mL of DMF. Then add dropwise the solution of 13.72 μL of DIPEA (7.8 μmol) in 1 mL of DMF. Stir the solution overnight at RT under a N2 atmosphere. Precipitate the product (HPLC conversion rate 84%) in 25 mL of ice-cold diethyl ether and purify it using a pre-packed silica C18 column ( SNAP ULTRA 26 g) with a water / acetonitrile gradient elution. Combine the fractions containing the desired pure product, concentrate in vacuo, freeze-dry, and recover 6.65 mg of a green solid (HPLC purity: area %: 98.7% at 785 nm; and 100% at 254 nm; [M-H] + 1558.7). The yield from compound 15 is 54.0%.

[0269] Example 17: Synthesis of compound 9

[0270] Dissolve 9.9 mg of compound 15 (7.9 μmol) in 3 mL of dry DMF. Add 2 μL of NMM (18.2 μmol) and 7.11 mg of TSTU (23.6 μmol), and stir the mixture at RT for 2 h. Precipitate the NHS ester of compound 15 (HPLC conversion rate 85.9%) in 25 mL of ice-cold ethyl acetate. Wash the precipitate with ethyl acetate and dry it under a N2 stream.

[0271] Dissolve the NHS ester of compound 15 in dry DMF (1 mL). Add dropwise the solution of EuK-(3-(2-naphthyl)-alanine)-tranexamic acid TFA salt prepared as described by et al., J Nucl Med 2015, 56: 914-920, (7.23 mg, 0.00945 mmol) in DMF (1 mL). Then add dropwise the solution of DIPEA (6.86 μL, 0.039 mmol) in DMF (1 mL). Stir the solution overnight at RT under a N2 atmosphere. Precipitate the product (HPLC conversion rate 93%) in 25 mL of ice-cold diethyl ether and purify it using a pre-packed silica C18 column ( SNAP ULTRA 26 g) with a water / acetonitrile gradient elution. Combine the fractions containing the desired pure product, concentrate in vacuo, freeze-dry, and recover 4.25 mg of a green solid (HPLC purity: area %: 99.6% at 785 nm; and 98% at 254 nm; [M-H] +1894.6). The yield from Compound 15 was 28.0%.

[0272] Example 18: Optical Properties

[0273] The compounds of the present invention were characterized in terms of their in vitro optical properties in an aqueous medium (i.e., water / PBS pH 7.4) and in clinical chemistry control serum (Seronorm, Sero SA), which mimics the chemical composition and optical properties of human serum. All dye or dye-conjugate solutions were freshly prepared. ICG and S0456 were used as commercial references.

[0274] In particular, the excitation and emission maxima and the absolute fluorescence quantum yield (Φ) of representative compounds of formula (I) and conjugates of formula (II) are shown in Table II.

[0275] Table II - Excitation / Emission Maxima and Absolute Fluorescence Quantum Yield of Compounds of Formula (I) and (II)

[0276]

[0277] n / a: Not available

[0278] The compounds of the present invention are characterized in that the absorption maxima are included in the range of about 760 nm - 810 nm. Even when conjugated to a targeting moiety, the dyes are endowed with fluorescence emission in the near-infrared region and a high fluorescence quantum yield. In summary, the dyes and conjugates show a higher fluorescence quantum yield than ICG and S0456.

[0279] Example 19: Affinity for Human Albumin (HSA)

[0280] The binding affinity of the compounds of the present invention for human albumin was analyzed, and the results were compared with those of ICG and S0456 used as references. Depending on the binding affinity level of the compounds, two methods were used to measure the binding affinity for human serum albumin (HSA; Sigma Aldrich, A9511).

[0281] The first method, which is optimal for compounds that interact strongly with HSA, is based on the analysis of the absorption spectral peak shift after incubating the dye in a solution containing HSA. Briefly, before measurement, the sample was diluted with HSA diluent (1x10 -6 to 4x10 -4 M) at a fixed concentration (1 μM) in phosphate buffer and incubated in a spectrophotometer at 25 °C for 5 min. The measurement was carried out at the maximum absorption wavelength of the shifted peak.

[0282] The second method, which is optimal for compounds with low affinity for HSA, is based on measuring the change in absorbance of solutions containing the dye and different concentrations of HSA after ultrafiltration. Briefly, each compound was incubated at a fixed concentration (2 μM) with HSA dilutions (1 x 10 -6 to 4 x 10 -4 M). Samples were centrifuged (10,000 g, 30 min at 25 °C) in a Microcon device (10 kDa MWCO, Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane, Millipore), and absorbance measurements of the filtrate were obtained using a spectrophotometer at the maximum absorption wavelength of the fluorophore.

[0283] For both methods, the affinity constant (K A , M -1 ) was calculated by fitting the raw data to the following equation:

[0284]

[0285] where

[0286] ΔA / b = measured absorbance (b = 1 cm)

[0287] K RL = K calculated by regression analysis (curve fitting) A

[0288] Δε·Rt calculated by regression analysis (curve fitting)

[0289] [L] = albumin concentration

[0290] In the first method, ΔA / b corresponds to the absorbance measured for each sample, while in the second method, ΔA / b is obtained by subtracting the absorbance of each other sample from the absorbance of the control sample (dye without HSA).

[0291] As shown by parallel experiments performed on the commercial cyanine dye IRDye 800CW carboxylate (LI-COR Inc., Lincoln, USA) using the first method (HSA K A = 215,000 M -1 ) and the second method (HSA K A = 216,000 M -1 ), both methods have been shown to provide comparable results. However, the measured values of the affinity constant are more precise when using the appropriate method as a function of the affinity level of the compound.

[0292] The results of the binding affinities measured for representative compounds of the present invention by one of two methods are reported in Table III and compared with the results obtained with the cyanine dye IRDye 800CW carboxylate dye as a reference compound.

[0293] Table III - Binding Affinity for Human Serum Albumin (HSA)

[0294] <![CDATA[HSA affinity (K A , M -1 )]]> ICG (reference substance) 347,000 S0456 (reference substance) 350,000 Compound 11 6,500 Compound 14 23,800 Compound 15 90,600 Compound 17 12,262 Compound 18 14,600 Compound 19 28,300 Compound 20 13,800 Compound 21 32,400 Compound 22 8,360 Compound 2 26,200 Compound 4 10,000

[0295] As shown in Table III, both the dyes and the dye-conjugates of the present invention show significantly lower binding affinities for human albumin compared to the known near-infrared dyes ICG and S0456, with the affinity constants being one or two orders of magnitude lower.

[0296] Even when conjugated to targeting moieties (such as Compounds 2 and 4), the dyes of the present invention retain this favorable feature, as the conjugation of the dye to the targeting moiety does not affect the affinity for human albumin.

[0297] Example 20: Receptor Binding Affinity

[0298] The binding affinities of the conjugates of formula (II) for specific receptors were determined to evaluate whether the targeting efficacy of the molecular carriers was retained after labeling with the dyes of the present invention.

[0299] As examples of small molecule and peptide / peptide mimetic conjugates, their IC 50 (half maximal inhibitory concentration) was calculated using an enzyme-linked immunosorbent assay (ELISA) to evaluate the receptor affinity of representative integrin-binding conjugates, as previously reported (Kapp et al., Sci. Rep. 2017, 7, 39805).

[0300] Briefly, a 96-well ELISA plate was coated overnight at 4 °C with extracellular matrix (ECM) protein vitronectin in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Each well was then washed with PBS-T buffer (phosphate buffered saline / Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween20, pH 7.4) and blocked for 1 h at RT 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). Meanwhile, serial dilutions of the compound and internal standard were prepared in an additional plate. After washing the assay plate 3 times with PBS-T, 50 μL of the dilution series was transferred to each well. 50 μL of a solution of human recombinant integrin α v β3 (R&D Systems, 1 μg / mL) in TS-B buffer was transferred to the wells and incubated for 1 h. The plate was washed 3 times with PBS-T buffer and then the primary antibody anti-α v β3 was added to the plate. After incubation and washing 3 times with PBS-T, the secondary antibody anti-IgG peroxidase-labeled antibody was added to the plate and incubated for 1 h. After washing the plate 3 times with PBS-T, the plate was developed by rapid addition of 3,3',5,5'-tetramethylbenzidine (TMB) and incubated for 5 min in the dark. The reaction was terminated with 3 M H2SO4 and the absorbance was measured at 450 nm using a plate reader (Victor3, Perkin Elmer).

[0301] The IC 50 s of representative compounds 1, 2, and 4 were tested in duplicate and the resulting inhibition curves were analyzed using GraphPad Prism version 4.0 for Windows (GraphPad Software). The inflection point defined the IC 50 value. All experiments were performed using c(RGDfK) as the internal standard.

[0302] The tested molecular probes conjugated to c(RGDfK) showed comparable affinity for the human α v β3 receptor and, as reported in Table IV, had similar affinity for the unconjugated reference peptide mimetic c(RGDfK).

[0303] Table IV - Binding affinity of the compounds of formula (II) for the human α v β3 integrin receptor compared to the peptide mimetic c(RGDfK).

[0304] <![CDATA[Receptor affinity (IC 50 , nM ± St.Dev.)]]> c(RGDfK) 2.69±0.70 Compound 1 2.73±0.50 Compound 2 1.64±0.41 Compound 4 1.84±0.38

[0305] Example 21: Cellular Uptake

[0306] Based on the high expression of integrin receptors, particularly α v β3, the human melanoma cell line WM-266-4 (ATCC, CRL-1676) was used as an in vitro model to evaluate the cellular uptake of representative integrin-binding compounds 1, 2, and 4 on the membranes of these cells (Capasso et al., PlosOne 2014).

[0307] In the presence of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin, adherent cells at approximately 70% confluence were incubated with compound 1 or 3 (1 μM) at 37 °C (5% CO2) for 2 hours. After two washes with PBS, the cells were detached using 0.1 mM EDTA in PBS, centrifuged, and resuspended in buffer (PBS, 0.5% BSA, 0.1% NaN3) for flow cytometry experiments. Fluorescence-activated cell sorting (FACS) was used to detect the fluorescence signal inside the cells as a measure of cellular uptake. The samples were excited with an argon laser, and the emission was detected using a 670 nm long-pass filter. Fluorescence intensity values were obtained from the histograms generated by the instrument software.

[0308] To evaluate the specificity of receptor-mediated cellular uptake, experiments were conducted by incubating the cells with the molecular probe in the presence of a high concentration (100 μM) of the unlabeled molecular carrier c(RGDfK) as a competitor. The residual internalization rate was calculated by considering the fluorescence intensity value in the absence of the competitor as 100%.

[0309] Furthermore, to evaluate the effect of biological fluids on cellular uptake, parallel experiments were conducted by incubating the compounds of the present invention with the cells in the presence of human serum (SigmaAldrich, H4522) from male AB plasma. The residual internalization rate was calculated by considering the fluorescence intensity value in the absence of serum as 100%. This uptake assessment also represents an indication of the percentage of the compound sequestered by plasma proteins when the compound diffuses through the vascular compartment before reaching the tissue of interest and the specific target receptor. In Table V, the cellular uptake performance of representative compounds 2 and 4 of the present invention is shown.

[0310] This compound shows a high cellular uptake rate in the presence of human serum. Thus, for this compound, internalization in cells was observed to be receptor-mediated and was only slightly affected by binding to human serum proteins, especially albumin (about 10 - 20% residual uptake), confirming a medium to low binding affinity (K A = approximately 1 - 6 x 10 3 M -1 , as shown in Example 10). Additionally, the compounds of the present invention were compared to the reference compound ICG-RGD (Capozza et al., Photoacoustic 2018, 11, 36 - 45) and ICG-c(RGDfK) prepared by the same method as ICG-RGD. These results show that it has surprisingly been found that once incubated in the presence of human serum, the compounds of the present invention have a higher efficacy in cellular internalization relative to similar compounds known in the art.

[0311] Table V - Uptake of integrin-binding fluorescent probes of the present invention in WM-266-4 human melanoma cells.

[0312] Residual cellular uptake in the presence of human serum ICG-RGD 12％ ICG-c(RGDfK) 8％ Compound 2 90％ Compound 4 85％

[0313] n / a: Not available.

[0314] Notably, neither the interaction of the compounds of the present invention with receptors on the cell surface nor the internalization of the receptor-probe complex within the cell is impaired by the structure of the conjugated dye, and in particular not by the presence of strongly hydrophilic and highly sterically hindered moieties at the Y-position of the compound. Thus, even in the presence of plasma proteins, the presence of hydrophilic moieties on the conjugated dye provides efficient and specific receptor binding and probe internalization, as the presence of plasma proteins can chelate conjugates lacking hydrophilic moieties and have a negative impact on binding efficiency.

[0315] Example 22: Tumor Uptake in Animal Models

[0316] Human Glioblastoma

[0317] In glioblastoma overexpressing integrin receptors, especially α vTumor uptake experiments were conducted in an animal model of human glioblastoma (subcutaneous) of 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) at 4 - 6 weeks of age were subjected to subcutaneous implantation (right flank) of approximately 10 million cells suspended in 0.1 ml of EMEM. Four mice were housed per cage with ad libitum access to food and water. The animals were fed with VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the adaptation period (5 days). Then, irradiated AIN-76A rodent diet (Research Diets), a special diet that reduces autofluorescence, was used until the end of the experiment. Tumor growth was monitored by longitudinal assessment using calipers with a target size of up to 300 - 600 mm 3 (3 - 4 weeks after cell implantation). Imaging experiments were performed using a preclinical optical system, IVIS Spectrum (Perkin Elmer).

[0318] In vivo imaging was performed under gas anesthesia (6 - 8% sevoflurane in oxygen). The animals were injected intravenously (lateral tail vein) with the compound and euthanized 24 h after administration. Regions of interest (ROIs) were drawn on the excised tumor and healthy muscle tissue to evaluate the signal intensity (expressed as mean radiant efficiency). Then, the ratio of the fluorescence signal in the tumor and muscle (background tissue) was calculated to evaluate the contrast.

[0319] The tumor - to - background ratios of representative compounds 1 and 4 are shown in Table VI. These results showed significantly high tumor uptake rates, suggesting tumor - specific accumulation.

[0320] Table VI - Ex vivo tumor and excretory organ - to - muscle ratios 24 h after administration of compounds 1 and 4 in mice bearing glioblastoma tumors

[0321] Tumor-to-background ratio (n = 5 / group), (mean ± St.Dev) Compound 1 6.92±0.64 Compound 4 7.64±0.86

[0322] Human head and neck cancer

[0323] Specifically, overexpression of integrin receptor α vDetroit-562 cells of β6 were used for tumor uptake experiments in an animal model of human head and neck cancer (orthotopic). Briefly, human pharyngeal cancer 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) at 4 - 6 weeks of age were orthotopically implanted with approximately 2.5 million cells suspended in 0.03 mL of EMEM in the anterior part of the tongue. Four mice were housed per cage with free access to food and water. The animals were fed with VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the adaptation period (5 days). Then, irradiated AIN-76A rodent diet (Research Diets), a special diet that reduces autofluorescence, was used until the end of the experiment. Tumor growth was monitored by longitudinal assessment using calipers with a target size of up to 10 - 20 mm 3 (7 - 10 days after cell implantation).

[0324] 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 overdose anesthesia 24 hours after administration, and the tongue was excised for ex vivo optical imaging. Regions of interest (ROIs) were drawn on the anterior part (tumor cell implantation site) and posterior region (healthy tissue) of the tongue to derive the tumor - to - background ratio.

[0325] Ex vivo imaging performed 24 h after administration of Compounds 1 and 2 revealed bright regions in the tongue site of tumor cell implantation. In contrast, the healthy regions in the posterior part of the tongue showed low signal, indicating low retention in healthy tissue. As shown in Table VII, administration of the compounds of the present invention revealed the location of tumors with moderate (TBR ~ 2) tumor - to - background contrast.

[0326] Table VII - Ex vivo mean tumor - to - background ratio (TBR) 24 h after administration of Compounds 1 and 2 in H&N tumor - bearing mice.

[0327] Tumor-to-background ratio (n = 5 / group), (mean ± St.Dev) Compound 1 2.27±0.50 Compound 2 2.26±0.10

[0328] Human colorectal cancer

[0329] Tumor uptake experiments were conducted 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 (Envigo) at 4-6 weeks of age were subjected to subcutaneous implantation (right flank) of approximately 5 million cells suspended in 0.1 ml of serum-free medium. Four mice were housed per cage with ad libitum access to food and water. The animals were fed with a VRF1(P) sterile diet (Special Diets Services Ltd) until the end of the adaptation period (5 days). Then, irradiated AIN-76A rodent diet (Research Diets), a special diet that reduces autofluorescence, was used until the end of the experiment. Tumor growth was monitored by longitudinal assessment using calipers of a target size of up to 300 - 600 mm 3 (3 - 4 weeks after cell implantation). Imaging experiments were performed using a preclinical optical system, IVIS Spectrum (Perkin Elmer).

[0330] In vivo imaging was performed under gas anesthesia (6 - 8% sevoflurane in oxygen). The compound of interest was injected intravenously (lateral tail vein) into the animals, and euthanasia was performed 24 h after administration. Regions of interest (ROIs) were drawn on the excised tumors and healthy reference tissue (muscle) to evaluate the signal intensity (expressed as mean radiant efficiency). Then, the ratio of the fluorescence signal in the tumor and muscle (background tissue) was calculated to evaluate the tumor-to-background ratio (TBR).

[0331] As shown in Table VIII, representative compounds 1 and 2 showed moderate tumor-to-background ratios (TBR ~ 4), enabling clear delineation of tumor tissue against a healthy background.

[0332] Table VIII - Ex vivo tumor-to-background ratios (mean, SD, n = 5) 24 h after administration of compounds 1 and 2 in mice bearing colorectal cancer.

[0333] Tumor-to-background ratio (n = 5 / group), (mean ± St.Dev) Compound 1 4.30±1.50 Compound 2 4.20±0.80

[0334] References:

[0335] 1. Cherrick et al., J Clin Invest 1960; 39(4): 592 - 600

[0336] 2. Onda N. et al., Int J Cancer 2016; 139: 673 - 682

[0337] 3. Tummers Q. et al., PlosOne 2015; 10(6): e0129766

[0338] 4. Achilefu S. et al., J Med Chem 2002; 45: 2003 - 2015

[0339] 5. Fidel J. et al., Cancer Res. 2015; 15; 75(20): 4283 - 4291

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Claims

1. A compound of formula (I), wherein X is a direct bond or -O-; Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group, which is substituted by at least two hydroxyl groups; R1 is a linear or branched C1-C6 alkyl substituted by -SO3H; R2 is a linear or branched C1-C6 alkyl substituted by a group selected from -COOH and -CONH2; and R3 is hydrogen or -SO3H, or a stereoisomer or a pharmaceutically acceptable salt thereof.

2. The compound of formula (I) according to claim 1, wherein Y is selected from 3. The compound of formula (I) according to claim 1, which is represented by formula (Ia) wherein X, R1, R2 and R3 are as defined in claim 1.

4. The compound of formula (I) according to claim 3, which is 5. A conjugate of the compound of formula (I) as defined in claim 1, which is represented by a compound of formula (II) wherein X is a direct bond or -O-; Y is a group selected from linear or branched C1-C6 alkyl, C3-C7 cycloalkyl and heterocyclic group, which is substituted by at least two hydroxyl groups; R1 is a linear or branched C1-C6 alkyl substituted by -SO3H; R4 is a straight-chain or branched C1-C6 alkyl group substituted by the group -CONH-(S) m -T, wherein S is a spacer group selected from -(CH2) p COO-, -(CH2CH2O) p CH2CH2COO- and -(CH2CH2O) p CH2CH2NH-, where p is an integer from 0 to 20; T is a bioactive moiety having a molecular weight of less than 5000 daltons; and m is an integer equal to 0 or 1; and R5 is selected from hydrogen and -SO3H, or a stereoisomer or a pharmaceutically acceptable salt thereof.

6. The conjugate of the compound of formula (I) according to claim 5, wherein T is a moiety that interacts with integrin receptors.

7. The conjugate of the compound of formula (I) according to claim 5, which is represented by formula (IIa) wherein R1, R4, R5 and X are as defined in claim 5.

8. The conjugate of the compound of formula (I) according to claim 7, which is 9. The compound or its conjugate as defined in any one of the preceding claims 1 to 8, for use as a fluorescent probe in biomedical optical imaging applications in mammals.

10. The compound or its conjugate according to claim 9, wherein the imaging application involves the detection of normal tissues and includes angiography, perfusion imaging, cholangiography and neuroimaging.

11. The compound or its conjugate according to claim 9, wherein the imaging application involves the detection of abnormal tissues, including primary tumor lesions, local or distant metastases or pre-tumor lesions, and is performed under NIR radiation.

12. A pharmaceutical diagnostic composition comprising the compound or its conjugate as defined in any one of the preceding claims 1 to 8 and at least one pharmaceutically acceptable carrier or excipient.

13. A diagnostic kit comprising at least one compound or its conjugate as defined in any one of the preceding claims 1 to 8 and additional accessories for performing biomedical optical imaging applications.

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