pH-responsive cyanine dyes and their conjugates

Abstract

The present invention relates to the field of optical imaging. More specifically, the present invention relates to a class of monoalkylated cyanine dyes having red to near-infrared (650-900 nm) emission, characterized by pH responsiveness, 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 methods for their preparation and to compositions containing them. The monoalkylated cyanine dyes have the general formula (I), in which R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y; and R2 and R4 are hydrogen, or R1 together with R2 and R3 together with R4 both form two aryl rings optionally substituted by 1 to 4 -SO3H groups together with the atoms to which they are bonded; R5 is an alkyl group optionally substituted by a group selected from -SO3H, -COOH and -CONH2. #imgabs0#

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 family of monoalkylated cyanine dyes with red to near-infrared emission (650-900 nm), whose fluorescence is responsive to pH, and to conjugates thereof with biological ligands. The present invention further relates to the use of these compounds as diagnostic agents, methods for their preparation and compositions comprising them. Background Art

[0002] Dye is a chemical entity that absorbs photons of a specific wavelength when excited by light, and according to the quantum efficiency, usually at a longer wavelength, re-emits a portion of this energy. In particular, cyanine dyes are fluorescent organic molecules, characterized in that they span multiple methyl (polymethine) bridges and are confined to a delocalized electronic system between two nitrogen atoms. Some of them have good optical properties, low toxicity and good solubility in aqueous media and can be used as contrast agents for biomedical imaging. Due to the higher penetration depth compared with dyes with fluorescent emission in the visible spectrum, cyanine dyes with red to near-infrared emission (650-900nm) are particularly useful for biomedical imaging applications.

[0003] Among the near-infrared dyes used for biomedical imaging, indocyanine green (ICG) is currently the only pharmaceutical product approved for human use. Due to its strong binding to plasma proteins (blood pool effect) and rapid clearance of the unbound fraction by the liver, ICG is commonly used to assess tissue perfusion and angiography applications (Cherrick et al., J Clin Invest 1960; 39(4): 592–600). In addition, ICG is also tested as a research drug product for tumor imaging during diagnostic and interventional (fluorescence-guided surgery) procedures (Tummers Q. et al., Plos One 2015; 10(6): e0129766).

[0004] Additional contrast agents for fluorescent imaging are being developed that utilize the use of dyes conjugated to carrier moieties (i.e., biomolecules) 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 has been conjugated to a tumor targeting moiety and is currently being evaluated in clinical trials for intraoperative tumor detection (Fidel J. et al., Cancer Res. 2015; 15; 75 (20): 4283–4291). ICG is an example of a near-infrared cyanine dye with intrinsic fluorescence emission. After administration in the bloodstream, ICG already emits fluorescence upon excitation, and the detected fluorescence intensity is a function of the accumulation rate of the dye in a given tissue. This results in fluorescence emission from the target tissue, i.e., a tissue affected by a specific physiological or pathological feature of diagnostic value.

[0005] However, a limitation of this method is the relatively high fluorescence intensity originating from non-target (background) tissues, where the dye may be distributed and accumulated due to nonspecific retention. The high fluorescence intensity generated by the background area may negatively affect the quality of the diagnostic optical imaging procedure by reducing the image contrast (i.e., the ratio between the signal in the target tissue and the signal in the background area), resulting in low sensitivity and false negatives of the detection.

[0006] In order to overcome the limitations of intrinsic fluorescence emission, new classes of cyanine dyes are being developed that display red to near-infrared (650-900nm) fluorescence emission that is dependent on pH. In particular, these dyes are fully activated and emit fluorescence (ON) only at acidic pH (e.g., <6.2-6.8), while only minimal fluorescence emission (OFF) is displayed at physiological pH (7.2-7.4). This feature is particularly relevant to diagnostic optical imaging of pathological tissues (e.g., tumor tissues) because it is well known that cancer tissues have a lower pH (6.2-6.8) in the extracellular space compared to healthy tissues and blood (7.2-7.4) due to high glycolytic metabolism (known as the "Warburg effect") and subsequent expulsion of high concentrations of protons by membrane pumps (Damaghi M. et al., Front Physiol. 2013; 4: 370). Furthermore, activation of these dyes, especially when the dyes are conjugated to moieties that bind epitopes expressed on the surface of tumor cells, can occur following internalization in cells and trafficking within acidic organelles such as endosomes (pH 6.5-5.5) and lysosomes (pH 5.5-4.5).

[0007] This specific activation of the dye triggered by acidic pH can increase the tumor to background ratio and improve the visualization of tumors, because only in the tumor microenvironment or only after internalization into tumor cells driven by overexpression of specific receptors, the probe becomes fluorescent and the signal can be detected. Ideally, in blood, where the pH is 7.4, and in healthy tissue, where the cellular internalization mediated by the same receptor is much lower, the fluorescence of the probe is almost turned off, resulting in a very low background signal.

[0008] For the above reasons, such "activatable" (on-off) dyes may allow better detection of pathological areas and improve the quality of biomedical optical imaging procedures.

[0009] Some examples of pH-sensitive cyanines are reported in the prior art.

[0010] For example, WO 00 / 75237 describes some examples of pH-sensitive cyanine dyes and discloses novel compounds having a linear trimethine (Cy3), pentamethylene (Cy5) or heptamethylene (Cy7) backbone, non-alkylated or mono-alkylated on the indolenine.

[0011] Briggs M. et al., Chem. Commun. 2000, 2323-2324 reported an example of a pentamethine cyanine dye that is sensitive to proton concentration in the pH range of 6-9, showing a pKa of 7.5.

[0012] WO2004 / 039894 relates to the preparation of different cyanine dyes having functional groups at different positions of the indole base. In particular, it describes pH-dependent Cy5 dyes, including the commercial dye CypHer5E (GE Healthcare) and other derivatives thereof. Beletskii A. et al., BioTechniques 2005, 39: 894-897 also disclose the use of the monoalkylated pentamethine dye CypHer5E with a pKa of about 7.3 to label particles and assess cellular internalization.

[0013] The chemical structures of the compounds disclosed in Briggs et al. (left) and CypHer5E (right) are reported below:

[0014]

[0015] Lee H. et al., Bioconjug Chem. 2011, 22(4):777–784 and Gilson R. et al., Mol. Pharmaceutics 2015, 12:4237-4246 disclose non-alkylated cyclohexenyl Cy7 pH-responsive dyes and their applications in vitro and in vivo. For example, the first reference reports the following chloro and benzoic acid derivatives, which have pKa of 4.7 and 5.2, respectively:

[0016]

[0017] The pKa value of the compound is of critical relevance, because when the pH of the medium is equal to the pKa value (pH=pKa), only half of the dye molecules are protonated and fluoresce, while the other half are deprotonated and quenched. Therefore, pH-sensitive dyes with pKa>7.0 already show high fluorescence emission (>50%) at physiological pH (7.2-7.4), which may lead to high background fluorescence. The compounds disclosed in the "Briggs et al." paper and CypHer5E are examples of such pH-sensitive cyanine dyes, which show suboptimal fluorescence emission characteristics (i.e., high emission at physiological pH), which may lead to high background fluorescence. On the other hand, pH-sensitive dyes with pKa<5.5 will only show high fluorescence emission (>50%) at highly acidic pH values. For example, compounds A and B described above will only achieve 95% protonation at pHs of 3.7 and 4.2, respectively, and such conditions of extreme acidosis are unlikely to exist in tissues and cells under pathological conditions. For this reason, it would be preferable to develop pH-sensitive dyes characterized by having a pKa in the range of 5.5-7.0, such that the fluorescence emission is minimal at physiological pH, but high at weakly acidic pH.

[0018] Other disadvantages associated with known dyes reported in Lee H. et al., Bioconjug Chem. 2011, 22(4):777–784 and Gilson R. et al., Mol. Pharmaceutics 2015, 12:4237-4246 are manifested in a certain instability (e.g., as mentioned in the above papers, it is preferred to use freshly prepared dye solutions in each study), and the fact that in several cases it is difficult to derivatize these dyes with functional groups that can be used for possible conjugation to biological moieties (i.e., molecular carriers). For example, the absence of N-alkylated groups strongly limits possible conjugation to only one coupling site on benzoic acid (a functional group that is not easily incorporated into the cyanine backbone) (Gilson R. et al., Mol. Pharmaceutics 2015, 12:4237-4246).

[0019] Therefore, despite several efforts to find suitable imaging agents, there is still a need to find improved dyes that have optimal fluorescence efficiency as well as optimal physicochemical and biological properties and are designed for optical imaging of living organisms. Dyes with pKa characteristics that allow suitable activation and red to near-infrared (650-900 nm) fluorescence emission only under the acidic pH conditions of the body (e.g., in a tumor environment) would be preferred for application in living organisms.

[0020] This need is critical, especially when dyes are conjugated to biomolecules that specifically bind to molecular epitopes or pathological tissues (eg, tumors).The present invention addresses these and other needs. SUMMARY OF THE INVENTION

[0022] The technical field of the invention is biomedical optical imaging. In particular, the invention relates to new monoalkylated cyanine dyes with red to near infrared (650-900 nm) emission, whose fluorescence depends on the pH of the medium in which they are dissolved, and to conjugates with their biological ligands. Furthermore, the invention relates to the use of these compounds as diagnostic agents, methods for their preparation and compositions comprising them.

[0023] Generally, the object of the present invention is to provide new pH-responsive monoalkylated cyanine dyes, or their corresponding conjugates with binding moieties, useful as contrast agents for optical imaging and aiming at solving the above mentioned problems.

[0024] Specifically, the novel monoalkylated cyanine derivatives described herein have minimal fluorescence at physiological pH and are activated by weakly acidic pH, generating fluorescent signals only in biological regions characterized by greater acidity relative to healthy tissue or blood, thereby significantly reducing background signals.

[0025] Furthermore, the monoalkylated cyanine dyes and conjugates thereof of the present invention are unexpectedly characterized by pKa values ​​in the range of 5.5-7.0, which are more suitable for biomedical imaging applications, and have very good responsiveness to small changes in pH, and have better stability relative to the non-alkylated cyanine dyes of the prior art.

[0026] The new cyanine dyes can be conveniently conjugated to suitable targeting moieties via suitable functional groups that serve as binding sites, thus providing very specific and sensitive contrast agents for molecular imaging.

[0027] Further aspects of the invention relate to such dyes as diagnostic agents, particularly for use in optical imaging of human or animal organs or tissues, in methods of optical imaging where the imaging is cross-sectional imaging of organs, monitoring organ function, including angiography, tissue perfusion imaging, urinary tract imaging, bile duct imaging, neuroimaging, 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 sonofluorescent methods.

[0028] Furthermore, the present invention relates to methods for preparing the provided dyes, their corresponding conjugates and / or pharmaceutically acceptable salts, and their use in the preparation of diagnostic agents.

[0029] 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 is particularly useful as an optical imaging agent to provide usable imaging of human or animal organs or tissues.

[0030] In another aspect, the invention relates to a method for optically imaging a body organ, tissue or region by using an optical imaging technique comprising the use of an effective amount of a compound of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Features of the present invention may be better understood with reference to the following detailed description and accompanying drawings, in which:

[0032] Figure 1 Shown in the graph plotted against the pH of each buffered phosphate solution prepared at pH 4.0 to pH 8.0 is the maximum absorbance value at 780 nm of the compound of the present invention (Compound 5) so as to calculate pK from the inflection point. DETAILED DESCRIPTION OF THE INVENTION

[0034] Therefore, the first object of the present invention is to provide a compound of formula (I), or a pharmaceutically acceptable salt thereof,

[0035]

[0036] in

[0037] W is a group or -CH=C(R6)-CH= or -C(Cl)=, wherein

[0038] R6 is hydrogen or chlorine;

[0039] R7 is chloro or phenyl optionally substituted by a group selected from -SO3H, -COOH, -CONH-Y, -alkyl-COOH and -alkyl-CONH-Y, wherein

[0040] Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups, and

[0041] * indicates bonding position;

[0042] R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y; and R2 and R4 are hydrogen, or,

[0043] R1 and R2 together and R3 and R4 together, both separately and together with the atoms to which they are bound, form two aryl rings optionally substituted with 1 to 4 -SO3H groups;

[0044] R5 is an alkyl group optionally substituted with a group selected from -SO3H, -COOH and -CONH2.

[0045] Another object of the present invention relates to the corresponding conjugated dye represented by the compound of formula (II), or a pharmaceutically acceptable salt thereof,

[0046]

[0047] in

[0048] W is a group or -CH=C(R6)-CH= or -C(Cl)=, wherein

[0049] R6 is hydrogen or chlorine,

[0050] R8 is chloro or phenyl optionally substituted by a group selected from -SO3H, -COOH, -CONH-Y, -alkyl-COOH, -alkyl-CONH-Y and -R10, wherein

[0051] Y is a divalent alkyl group substituted by -SO3H or at least two hydroxyl groups,

[0052] R10 is a group -CONH-(S) m -T substituted divalent alkyl, wherein

[0053] S is a spacer group;

[0054] T is a targeting moiety; and

[0055] m is an integer equal to 0 or 1; and

[0056] * indicates bonding position;

[0057] R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y; and R2 and R4 are hydrogen, or

[0058] R1 and R2 together and R3 and R4 together, both separately and together with the atoms to which they are bound, form two aryl rings optionally substituted with 1 to 4 -SO3H groups;

[0059] R9 is optionally selected from -SO3H, -COOH, -CONH2 and -CONH-(S) m -T, wherein S, T and m are as defined above;

[0060] and wherein at least one group -CONH-(S) is present in R9 or R10 m -T.

[0061] The present invention also relates to processes for preparing compounds of formula (I) or (II) by means of synthetic transformation steps.

[0062] The present invention also comprises compounds of formula (I) or (II) as defined above for use as fluorescent probes for biomedical optical imaging applications.

[0063] In particular, the present invention comprises compounds of formula (I) or (II) as defined above, characterized by a pKa value in the range of 5.5-7.0.

[0064] definition

[0065] In this specification, and unless otherwise specified, the following terms and phrases as used herein are intended to have the following meanings.

[0066] The term "alkyl" refers to an aliphatic hydrocarbon group which may be straight or branched, having from 1 to 8 carbon atoms in the chain. For example, "C4 alkyl" includes within its meaning a straight or branched chain containing 4 carbon atoms. Similarly, "C1-C 20 "Alkyl" is an alkyl group containing 1 to 20 carbon atoms. Preferably and unless otherwise specified, the term "alkyl" refers to a C1-C6 alkyl group. Representative and preferred alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, pentyl and hexyl. Unless otherwise specified, a straight chain or branched alkyl group is a monovalent group, in other aspects it can be a multivalent group in which two or more hydrogen atoms are removed from the above hydrocarbon group and substituted, such as methylene, ethylene, isopropylene, etc.

[0067] The term "divalent alkyl group" refers to an alkyl group in which two hydrogen atoms are removed from the above hydrocarbon group and substituted.

[0068] The term "cycloalkyl" as used herein includes within its meaning a saturated (i.e., alicyclic) carbocyclic ring; for example, "C3-C7 cycloalkyl" means a saturated carbocyclic ring containing 3 to 7 carbon atoms. Suitable examples include C5-C7 carbocyclic rings, such as cyclohexyl rings.

[0069] The term "aryl" refers to an aromatic monocyclic or polycyclic ring system of 6 to about 14 carbon atoms, preferably 6 carbon atoms. Representative aryl groups include phenyl, naphthyl or benzo-fused rings. The term "benzo-fused ring" refers to an aromatic monocyclic ring of 6 carbon atoms attached to another ring to form a bicyclic aromatic system.

[0070] In this specification, term " blocking group " (Pg) represents the functional blocking group that is suitable for keeping the group to which it is combined. Specifically, blocking group is used to keep amino, hydroxyl or carboxyl functional. Suitable blocking group can include for example benzyl, carbonyl such as formyl, 9-fluoromethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), isopropoxycarbonyl or allyloxycarbonyl (Alloc), alkyl such as tert-butyl or triphenylmethyl, sulfonyl, acetyl such as trifluoroacetyl, benzyl ester, allyl, or other substituents that are generally used to protect this type of functional well-known to those skilled in the art (see for example, general reference TWGreen and PGM Wuts, Protective Groups in Organic Synthesis, Wiley, NY2007, the 4th edition, the 5th chapter).

[0071] In addition, the present invention also includes a precursor or intermediate compound suitable for preparing the desired compound of formula (I) or (II) or its salt. In such derivatives, functional groups, such as carboxylic acids or carboxamides, can be protected with appropriate protecting groups (Pg) as defined above, preferably with alkyl or ester groups. 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 appropriate protecting groups (Pg), thereby forming, for example, acetoxy, alkoxy or ester groups.

[0072] The expression "coupling reagent" refers to a reagent, for example, for forming an amide bond between a carboxyl moiety and an amino moiety. The 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 the group consisting of 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)tripyrrolidinylphosphonium hexafluorophosphate (PyBOP), 7-azabenzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate (PyAOP), [ethylcyano(hydroxyimino)acetate-O2]tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyOxim), bromotripyrrolidinylphosphonium hexafluorophosphate (PyBrOP), and 3-(dimethylamino)phosphonium hexafluorophosphate (PyBOP). ammonium / uronium-immonium 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-aza ... 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 hexafluorophosphate (TFFH) or other compounds well known to those skilled in the art.

[0073] The expression "activated carboxylic acid" refers to a derivative of a carboxyl group that is more susceptible to nucleophilic attack than the free carboxyl group; suitable derivatives may include, for example, anhydrides, thioesters, acyl halides, NHS esters, and sulfo-NHS esters.

[0074] Furthermore, the term "moiety" or "residue" is intended herein to define the remaining portion of a given molecule once suitably attached or conjugated to the rest of the molecule, either directly or via suitable linkers and / or spacers.

[0075] Targeting moiety (T)

[0076] According to the present invention, targeting moiety (T) is a molecule that binds to a biological target with specific selectivity and promotes the accumulation of contrast agents in a specific tissue or body part. Usually, it is represented by a natural or synthetic molecule for a biological system. Such specific binding can be achieved by a ligand, such as, for example, a small molecule, a protein, a peptide, a peptide mimetic, an enzyme substrate, an antibody or a fragment thereof or an aptamer, interacting with a specific biological target expressed on the surface of a target tissue or cell.

[0077] Suitable biological targets for the compounds of the 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) enzymes such as CAIX, CAII or CAXII; mucin glycoproteins such as MUC1; glucose transporters such as GLUT-1; sodium-hydrogen antiporters such as NHE1; carcinoembryonic glycoproteins such as carcinoembryonic antigen (CEA); chemokine receptors such as chemokine receptor type 4 (CXCR4); cell adhesion molecules such as ICAM, EPCAM, VCAM, E-selectin, P-selectin; hepatocyte growth factor HGFR (c-met); receptors for transferrin; ephrin receptors such as EPHA2; receptors for folate such as FR-α; ialuronic acid binding acid), such as CD44; bombesin receptors, such as BB1, BB2, BB3; N-acetyl-L-aspartyl-L-glutamate (NAAG) peptidases, such as prostate-specific membrane antigen (PSMA); and, in particular, integrin receptors, such as α v β3, α v β5, α v β6 or α5β1 integrin receptor.

[0078] For example, the integrin receptor targeting moiety is represented by a linear or cyclic peptide comprising the sequence Arg-Gly-Asp (RGD). This tripeptide has a 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.

[0079] Thus, linear and cyclic peptides and peptide mimetics containing the sequence Arg-Gly-Asp (RGD), such as, for example, cRGD, cRGDfK, cRGDyK, cRGDfC, RGD-4C, RGD-2C, AH111585, NC100692, RGD-K5 (Kapp et al., Sci Rep, 2017, 7:3905), or analogs and derivatives thereof, are well-known examples of binding motifs that target cancer tissues where cell membrane integrins are upregulated compared to healthy tissues.

[0080] In one embodiment, the compound of the present invention can be conjugated to other small molecules, peptides, proteins or antibodies, such as monoclonal antibodies for example, which have been used for treatment. Small molecules containing drug acetazolamide, such as compounds 4a, 5a, 6a, 7a and 8a (Wichert et al., Nat Chem 2015, 7: 241–249) or its analogs and derivatives, are examples of small molecules targeting enzyme CAIX. Linear and cyclic peptides and peptide mimetics, such as peptide GE11 (described in Li et al., FASEB J2005, 19: 1978-85) and / or peptide L1 (described in Williams et al., Chem Biol Drug Des 2018, 91: 605–619) or its analogs and derivatives, are examples of peptides targeting epithelial growth factor receptor (EGFR). Among these proteins, derivatives of epithelial growth factor (EGF) are examples of small proteins targeting epithelial growth factor receptor (EGFR). Among these antibodies, panitumumab and cetuximab are examples of monoclonal antibodies targeting epidermal growth factor receptor (EGFR).

[0081] Preferably, the targeting ligands of the present invention can be selectively associated with tumor cells or tissues. In particular, they can be associated with 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 can be associated with the metastatic spread of the above-mentioned cancers in tissues and organs different from the primary source. In addition, the targeting ligands can be associated with pre-neoplastic lesions and dysplasia in different tissues and organs.

[0082] Spacer S

[0083] According to the present invention, S is the optionally existing spacer, which separates the targeting moiety from the dye. The existence of the spacer is particularly relevant for some embodiments of the risk that wherein the targeting moiety and the dye interact unfavorably with each other. In addition, when the dye is relatively large and may interfere with the combination of the targeting moiety and the target site, the existence of the spacer may be needed.

[0084] The spacer can be flexible (e.g., a simple alkyl chain) or rigid (e.g., a cycloalkyl or aryl chain) to orient the dye away from the target. The spacer can also alter the pharmacokinetics and metabolism of the conjugate of formula (II) used as an imaging agent in a living organism.

[0085] Hydrophilic spacers can reduce interactions with plasma proteins, reduce blood circulation time and promote excretion. For example, if the spacer is a polyethylene glycol (PEG) moiety, the pharmacokinetics and blood clearance of the imaging agent in vivo can be changed. In such embodiments, the spacer can improve the clearance of the imaging agent from background tissues (i.e., muscle, blood), thereby producing better diagnostic images due to high target and background contrast. In addition, the introduction of a hydrophilic spacer can transfer the elimination of contrast agents from the liver to the kidneys, thereby reducing systemic retention.

[0086] Therefore, in a preferred embodiment, the spacer is a C1-C 20 Preferably, the spacer is selected from (CH2) p COO-、-(CH2CH2O) p CH2CH2COO- and -(CH2CH2O) p The group consisting of CH2CH2NH-, wherein p is an integer from 0 to 20. Preferably, p is 2, 6 or 12.

[0087] When not necessary, preferably no spacer is present, ie m is 0 and S represents a direct bond.

[0088] The spacer or alternatively the targeting moiety in the absence of the spacer can be attached to the compound of formula (I), alternatively attached at the R5 and / or R7 residues to form a compound of formula (II). The R5 / R7 linking group is a reactive functional group, such as a carboxylic acid residue suitable for conjugating the dye to the targeting moiety by forming a chemical bond.

[0089] For example, when the targeting moiety (T) containing amine is conjugated to the compound of formula (I), wherein R5 and / or R7 are alkyl substituted with carboxylic acid, the carboxylic acid can be optionally activated before conjugation by using an activating agent to convert in a more reactive form, forming, for example, N-hydroxysuccinimide (NHS) ester or mixed anhydride. Then, in order to obtain the compound of the corresponding formula (II), the targeting moiety containing amine is treated with the resulting activated acid to form an amide bond. Typically, the reaction is carried out in an aqueous buffer, in an optional co-solvent with DMSO or DMF at pH 8 to 9, or in an organic solvent with an organic base such as DIPEA, TEA or NMM.

[0090] In other aspects, direct conjugation can be performed using "non-activated" carboxylic acids.

[0091] Similarly, when the linking group of R5 is a carboxamide group, the procedure for attaching a suitable targeting moiety is similar, but generally no activation step of the linker is required, and the dye and targeting moiety are processed directly.

[0092] The compound of above formula (I) or (II) can have one or more asymmetric carbon atoms, also called chiral carbon atoms, and thus can produce diastereomers and optical isomers. Unless otherwise specified, the present invention further includes all such possible diastereomers and their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers and pharmaceutically acceptable salts thereof.

[0093] The present invention further relates to compounds of the above formula (I) or (II), wherein the functional groups of R1, R3, R5, R7, R8, R9 and Y, such as sulfonyl, carboxamide or carboxylic acid groups, may be in the form of pharmaceutically acceptable salts.

[0094] In one embodiment, the invention relates to compounds of formula (I) or (II), wherein W is a group -C(Cl)= or -CH=C(R6)-CH=, R5 and R9 are as defined above, and R1 together with R2 and R3 together with R4 and the atoms to which they are respectively bonded form two benzo-fused rings optionally substituted by 1 to 4 -SO3H groups, further represented by the following formula (Ia) or (IIa), respectively:

[0095]

[0096] wherein each of R11-R18 is independently hydrogen or -SO3H.

[0097] In another embodiment, the present invention relates to compounds of formula (I) or (II), wherein W is respectively a group R5 and R7-R9 are as defined above, and R1 together with R2 and R3 together with R4 and the atoms to which they are respectively bonded form two benzo-fused rings optionally substituted with 1 to 4 -SO3H groups, and are also represented by the following formula (Ia') or (IIa'), respectively:

[0098]

[0099] wherein each of R11-R18 is independently hydrogen or -SO3H.

[0100] In one embodiment, the invention relates to compounds of formula (I) or (II), wherein W is respectively a group R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl substituted by -SO3H or at least two hydroxyl groups, R2 and R4 are hydrogen, and R5, R7, R8 and R9 are as defined above, and are also represented by the following formula (Ib) or (IIb), respectively:

[0101]

[0102] In another embodiment, the present invention relates to compounds of formula (I) or (II), wherein W is selected from -C(Cl)=, -CH=CH-CH= and -CH=C(Cl)-CH=, R1 and R3 are independently selected from the group consisting of -H, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl substituted with -SO3H or at least two hydroxy groups, R2 and R4 are hydrogen, and R5, R7, R8 and R9 are as defined above, also represented by the following formula (Ic) or (IIc), respectively:

[0103]

[0104] In one embodiment, the invention relates to compounds of formula (I) or (II), wherein Y is a linear or branched C1-C6 alkyl substituted by two to five hydroxy groups.

[0105] In a preferred embodiment, the present invention relates to compounds of formula (I) or (II), wherein Y is selected from the group consisting of

[0106]

[0107] More preferably, the invention relates to compounds of formula (I) or (II), wherein Y is a group of formula (II) as defined above.

[0108] Another embodiment of the present invention relates to compounds 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 C1-C 20 The hydrophilic part of 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.

[0109] In further embodiments, T is a targeting moiety selected from a small molecule, a protein, a peptide, a peptidomimetic, an enzyme substrate, an antibody or any fragment thereof, and an aptamer.

[0110] Preferably, T is represented by a peptide, and in particular by a peptide that binds to an integrin receptor such as α v β3, α v β5, α v β6, α5β1, etc., preferably with α v Partial representation of β3 integrin receptor interactions.

[0111] Particularly preferred are the compounds of formula (I) listed in Table Ia and the related conjugated compounds of formula (II) listed in Table Ib.

[0112] Table Ia - Preferred compounds of formula (I)

[0113]

[0114]

[0115]

[0116] Table Ib - Preferred compounds of formula (II)

[0117]

[0118]

[0119] The present invention also relates to a process for synthesizing compounds of formula (I) or (II) prepared as shown in the following description, which are dyes with red to near infrared (650-900 nm) emission, optionally conjugated to a targeting moiety via a linker group.

[0120] Thus, the present invention provides a compound of formula (I) or (II) as defined above for use as an optical imaging agent for diagnostic biomedical applications in mammals (humans and animals). Preferably, the mammalian subject imaged is a human.

[0121] In a preferred embodiment, the compounds of the invention are used as imaging agents in the detection of normal (healthy) tissue or abnormal (pathological) tissue, in particular tumors.

[0122] Preferably, the compounds of formula (I) or (II) as defined above are used for detecting normal (healthy) tissue by means of imaging techniques including, for example, angiography, perfusion imaging, cholangiography and neuroimaging.

[0123] In a further preferred embodiment, the invention provides a compound of formula (I) or (II) as defined above for use in detecting abnormal (pathological) tissues, such as, for example, primary tumor lesions, local or distant metastases, or pre-neoplastic 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 in individual patients. A preferred use is one in which the tumor is a tumor showing overexpression of a biological epitope, for example selected from receptors, enzymes, glycoproteins, lipid rafts, transmembrane proteins located on the cell surface, and soluble factors present in serum, plasma or interstitial space. Preferably, the biological epitope is an integrin receptor for vitronectin, fibrinogen and / or transforming growth factor-β (TGF-β).

[0124] The present invention also provides compounds of formula (I) or (II) as defined above for use as fluorescent probes, wherein the detection and demarcation of tumors is performed under NIR radiation. Preferably, such detection and demarcation of tumors is performed before, during or after surgery to remove such tumor tissue. Fluorescence-guided surgery is an example of such use.

[0125] In addition, the present invention provides the use of the compound of formula (I) or (II) as defined above for detecting inflamed tissue, fibrotic tissue, ischemic tissue, or tissue with abnormal metabolic rate.

[0126] The present invention also provides a compound of formula (I) or (II) as defined above for use in a method for imaging tissues and cells, the method comprising the steps of:

[0127] i) contacting cells or tissues with a compound of formula (I) or (II);

[0128] ii) illuminating the tissue or cells at a wavelength absorbed by the imaging agent;

[0129] iii) Detection of near-infrared emission using a fluorescence camera.

[0130] Preferably, contacting the cells or tissues with the imaging agent of formula (I) or (II) is accomplished by topical (local) administration (e.g., by spraying, soaking, or application of an ointment, foam, or cream) or by systemic administration (enteral or parenteral).

[0131] The present invention further relates to a pharmaceutical diagnostic composition comprising a compound of formula (I) or a conjugate of formula (II) as defined above, and at least one pharmaceutically acceptable carrier or excipient.

[0132] 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.

[0133] Alternatively, the present invention relates to a pharmaceutical composition comprising a conjugate of formula (II) as defined above or a salt thereof, wherein R9 and / or R10 is replaced by CONH-(S) m -T substituted alkyl, and one or more pharmaceutically acceptable adjuvants, excipients or diluents.

[0134] Another aspect of the invention relates to a diagnostic kit comprising a compound of formula (I) or (II) as defined above. In addition, the kit may contain other adjuvants for implementing optical imaging. These adjuvants are, for example, suitable buffers, containers, detection reagents or instructions for use. The kit preferably contains all materials for intravenous administration of the compound of the invention.

[0135] Prior to the imaging procedure, the compounds of the invention may be administered systemically or topically to the organ or tissue to be imaged. For example, the compounds may be administered intravenously. In another embodiment, they may be administered parenterally or enterally.

[0136] The composition is administered in a dosage effective to obtain the desired optical image of the tumor, tissue or organ, which can vary widely, depending on the compound used, the tissue undergoing the imaging procedure, the imaging equipment used, and the like.

[0137] The exact concentration of the imaging agent depends on the experimental conditions and the desired results, but can generally be in the range of 1 nM to 0.1 mM. The optimal concentration is determined by systematic variation until satisfactory results with minimal background fluorescence are obtained.

[0138] Once administered, the imaging agents of the invention are exposed to light or other forms of energy that can pass through tissue layers. Preferably, the radiation wavelength or band matches the excitation wavelength or band of the photosensitizer and has low absorption by non-target cells and the rest of the subject, including blood proteins.

[0139] Typically, an optical signal is detectable by observation or instrumentation, and the response is related to fluorescence or light intensity, distribution, and lifetime.

[0140] Description of synthesis

[0141] The preparation of compounds of formula (I) or (II) as such or in the form of physiologically acceptable salts represents a further object of the invention.The cyanine dyes and dye-conjugates of the invention can be prepared, for example, according to the methods described in the following sections and in the experimental part.

[0142] General teachings on the preparation of cyanine dyes can be found in Mujumdar RB 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 that does not exist in the compounds of the art, for which it is necessary to establish appropriate synthetic methods. In fact, unlike other known cyanines, the compounds of the present invention can even be derivatized with three functional moieties (carboxylic acid or amide groups) in different ways, so that in most cases, it is necessary to use a protecting group to guide the reaction on the desired functional group.

[0143] It is well known that difficulties may arise when operating cyanines under the strong pH and temperature conditions necessary for the removal of some protecting groups, because in some cases the stability of the polymethyl backbone may be impaired and the dye may be severely degraded. In addition, when deprotecting the ester group, further obstacles may be encountered due to the possible hydrolysis and degradation of the amide group -CONH-Y (usually, amide derivatives can be hydrolyzed in concentrated alkaline media, see for example Yamana et al., Chem. Pharm. Bull., 1972, 20 (5), 881-891).

[0144] In a preferred embodiment, the protecting group of part R5 or R9 is an ester group. More preferably, an ethyl ester group can be advantageously used.

[0145] Preparation of cyanine dyes of formula (I)

[0146] According to the present invention, compounds of formula (I) can be prepared by the sequence of synthetic steps as reported in the following schemes. Depending on the substituents and the specific backbone of the cyanine dye, different synthetic routes can be chosen.

[0147] For example, in the case of cyanines of formula (Ib), wherein W is a group -cyclohexenyl-R7, wherein R7 is chlorine, R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl substituted by -SO3H or at least two hydroxyl groups, the procedure reported in the following general scheme 1 can be used.

[0148]

[0149] Solution 1

[0150] Therefore, the method of the present invention comprises the following steps:

[0151] a) reacting an appropriate amount of a 5-substituted-2,3,3-trimethylindolenine intermediate of formula (III) and (IV) with 1-formyl-3-(hydroxymethylene)-1-cyclohexene or 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene to obtain a cyanine of formula (Ib) wherein R7 is a chlorine atom.

[0152] According to step a), the reaction can be carried out using Vilsmeier's reagent in the form of dianiline or in the form of bisaldehyde (as reported in Scheme 1). The reaction can be carried out in several solvents such as, for example, ethanol, methanol, acetic anhydride or acetic acid or mixtures, with or without the addition of different bases such as trimethylamine, pyridine, sodium acetate, potassium acetate, etc., stirring the mixture for several hours (usually 2-24 hours) at different temperatures ranging from 45° C. to 120° C.

[0153] When R1 and / or R3 are independently a group -COOH, the compound of formula (Ib) may be further derivatized to obtain another compound of formula (Ib) wherein R1 and / or R3 are a group -CONHY.

[0154] According to this step, the conversion of the carboxylic acid of formula (Ib) to the corresponding carboxamide can be completed in a variety of ways and experimental conditions, which is well known in the field for the preparation of carboxamides. As an example, the carboxylic acid can first be converted in 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.

[0155] When derivatives (III) and (IV) wherein R1 and / or R3 are groups -COOH are derivatized with polyhydroxylated amines, the reaction can be carried out by activating the carboxylic acid groups with a coupling agent selected from, for example, 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 ranging from 30 minutes to several hours. This derivatization of the carboxylic acid can be carried out on an alkylated indolenine or on an indole before quaternization. In this case, it is important to protect the hydroxyl groups of the polyhydroxylated amine with a suitable protecting group such as an acetyl group before alkylation with sultone or bromohexanoic acid. The alkylation can be carried out neat or in a high boiling point solvent such as butyronitrile, sulfolane, 1,2-dichlorobenzene, dimethylacetamide, dimethylformamide or dimethylsulfoxide, stirring the solution at elevated temperature, for example 90 to 180°C for several hours, typically 12 hours to 5 days.

[0156] For embodiments relating to cyanines of formula (Ib), wherein W is a group -cyclohexenyl-R7, wherein R7 is phenyl optionally substituted by a group -SO3H, -COOH, -CONH-Y, -alkyl-COOH or -alkyl-CONH-Y, wherein Y is a divalent alkyl substituted by -SO3H or at least two hydroxy groups, and R1 and R3 are independently selected from hydrogen, -SO3H, -COOH and -CONH-Y, the procedure reported in the following general scheme 2 may be applied:

[0157]

[0158] (R7 = optionally substituted phenyl)

[0159] Solution 2

[0160] Therefore, the method of the present invention comprises the following steps:

[0161] b) reacting N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline with a suitable phenylboronic acid of formula (V) to obtain the corresponding intermediate of formula (VI); and

[0162] c) Firstly adding intermediate (IV) and then intermediate (III) to a mixture comprising intermediate (VI) to obtain a compound of formula (Ib) as defined above.

[0163] According to step b), the reaction can be carried out in several solvents such as ethanol, methanol, water or a mixture thereof, in the presence of a palladium catalyst such as palladium acetate or tetrakis(triphenylphosphine)palladium and an inorganic base such as sodium carbonate or potassium carbonate, stirring the mixture at different temperatures ranging from 70°C to 100°C for several hours (usually 2 hours).

[0164] According to step c), the reaction can be carried out stepwise in several solvents such as ethanol or methanol, first adding the modified Vilsmeier reagent and the non-alkylated indolenine (IV) prepared in b) in the presence or absence of a small amount of acetic acid and stirring the solution at 50°C-80°C for several hours (usually 4 hours). Then, the alkylated indolenine (III) is added, dissolved in an organic solvent such as ethanol or methanol, followed by the optional addition of a base such as pyridine, triethylamine, sodium acetate or potassium acetate. The deep red solution can be stirred at 50°C-80°C for several hours, generally 4 to 96 hours.

[0165] For embodiments relating to cyanines of formula (Ic), wherein W is selected from -C(Cl)=, -CH=CH-CH= and -CH=C(Cl)-CH=, and R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl substituted with -SO3H or at least two hydroxy groups, the procedure reported in the following general scheme 3 can be used.

[0166]

[0167] Solution 3

[0168] Therefore, the method of the present invention comprises the following steps:

[0169] d) reacting a suitable amount of a 5-substituted-2,3,3-trimethylindolenine intermediate of formula (III) and (IV) with the corresponding Vilsmeier reagent in the form of the dianiline hydrochloride salt (VII) to obtain the corresponding compound of formula (Ic).

[0170] According to step d), the reaction can be carried out in several solvents such as ethanol, methanol, acetic acid, acetic anhydride or mixtures thereof. The reaction can be carried out by adding indole (III) and (IV) simultaneously or by first activating the Vilsmeier reagent in a mixture of acetic acid and acetic anhydride, stirring for several hours at 50°C-70°C, and then adding one indole, typically a non-alkylated indole (IV), stirring for several hours at 50°C-70°C. The corresponding hemicyanine can be isolated, for example, by precipitation in an organic solvent such as ethyl acetate, isopropyl acetate, diethyl ether, diisopropyl ether, and then dissolved in another organic solvent such as ethanol, methanol, dimethylformamide, and reacted with a second indole, typically an alkylated indole (III), stirring for several hours at 50°C-100°C in the presence of a base such as triethylamine, pyridine, sodium acetate or potassium acetate.

[0171] For embodiments relating to cyanines of formula (Ia), wherein W is as defined in formula (I), and R1 and R2 together and R3 and R4 together with the atoms to which they are respectively bound form a benzo-fused ring optionally substituted with 1 to 4 -SO3H groups, the procedure reported in the following general scheme 4 can be applied:

[0172]

[0173] Solution 4

[0174] Therefore, the method of the present invention comprises the following steps:

[0175] e) reacting a suitable amount of 5-substituted-2,3,3-trimethylbenzindolenine intermediates of formula (VIII) and (IX) with the corresponding Vilsmeier reagent in the form of the dianiline hydrochloride salt (X) to obtain the corresponding compounds of formula (Id).

[0176] According to step e), the reaction can be carried out in several solvents such as ethanol, methanol, acetic acid, acetic anhydride or mixtures thereof, with or without the addition of a small amount of water, depending on the polarity of the R11-R18 substituents. The solution of intermediates (VIII) and (IX) is stirred at elevated temperatures ranging from 70° C. to 100° C. for several hours, usually overnight, in the presence of Vilsmeier reagent (X) and a base such as pyridine, triethylamine, sodium acetate. The crude material is then precipitated with an organic solvent and purified by precipitation or flash chromatography.

[0177] In all these embodiments, the functional groups of R5 and / or R1 / R3 may be optionally protected during the synthesis with one or more suitable protecting groups, which may need to be removed in subsequent steps. The product obtained by steps a)-d) may be deprotected according to known procedures, such as described in TW Green and PGM Wuts, Protective Groups in Organic Synthesis, Wiley, NY 2007, 4th edition, chapter 5.

[0178] Preparation of the conjugate compound of formula (II)

[0179] The cyanine derivatives of formula (I) or salts thereof can be conjugated with a suitable targeting moiety, optionally with the insertion of a spacer, to obtain a compound of the corresponding formula (II). Conjugation can be accomplished according to different procedures known in the art, such as, for example, via direct coupling of the carboxylic acid group of the compound to a nucleophilic residue of the targeting moiety, or optionally with a spacer, or by prior activation, wherein the carboxylic acid group is converted to a more reactive group, such as an ester, such as NHS, prior to coupling.

[0180] If the compounds of formula (I) or (II) prepared according to the above described methods are obtained as a mixture of isomers, it is within the scope of the present invention to separate them into the individual corresponding isomers of formula (I) or (II) using conventional techniques.

[0181] The final compounds can be isolated and purified using conventional procedures, such as chromatography and / or crystallization and salt formation.

[0182] The compound of formula (I) or (II) as defined above may be converted into a pharmaceutically acceptable salt. The compound of formula (I) or (II) as defined above, or a pharmaceutically acceptable salt thereof, may then be formulated with a pharmaceutically acceptable carrier or diluent to provide a pharmaceutical composition.

[0183] Experimental Section

[0184] The invention and specific embodiments thereof described in the following sections are exemplary only and should not be considered as limiting the invention: they show how the invention can be performed and are meant to be illustrative without limiting the scope of the invention.

[0185] Materials and Equipment

[0186] All commercially available reagents used in the synthesis were obtained from Sigma Aldrich and TCI, and they were used without further purification. c(RGDfK) was purchased from Apex Bio or Bachem. All reactions were subsequently subjected to HPLC (Agilent mod. 1100 / 1200) and HPLC-MS (Agilent mod. 1260, quadrupole LC / MSD Mod. 6120) equipped with an absorption detector set at different wavelengths (column: YMC-Triart Phenyl, 250 x 4.6 mm / S-5 μm / 12 nm). Purification was performed by flash chromatography on an automated purification system ( Rf+) using a pre-packed silica gel C18 column ( or Phenomenex) or by preparative HPLC on a preparative YMC-Triart Phenyl column. Absorbance, excitation and emission values ​​were evaluated in a plate reader to accelerate the measurement (SPARK).

[0187] List of abbreviations

[0188] DCC N,N'-Dicyclohexylcarbodiimide

[0189] DIPEA N,N-Diisopropylethylamine

[0190] DMF Dimethylformamide

[0191] DMSO Dimethyl sulfoxide

[0192] DSC N,N′-Disuccinimidyl Carbonate

[0193] 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium

[0194] HATU

[0195] 3-Oxide Hexafluorophosphate

[0196] HBTU O-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0197] HPLC High Performance Liquid Chromatography

[0198] PBS Phosphate buffered saline

[0199] NHS N-Hydroxysuccinimide

[0200] NMM N-Methylmorpholine

[0201] RT Room temperature

[0202] PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

[0203] TEA Triethylamine

[0204] TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0205] TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

[0206] μL microliter

[0207] μM micromolar

[0208] t R Retention time (HPLC)

[0209] c(RGDfK) Cyclo-(Arg-Gly-Asp-D-Phe-Lys)

[0210] 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 indicated, reference to amino acids herein is understood to be in the L-isomer configuration.

[0211] Example 1 :Synthesis of compound 1

[0212]

[0213] Preparation of intermediate (2)

[0214] Under magnetic stirring, glacial acetic acid (225mL) is added to a mixture of 4-hydrazinobenzoic acid (15g, 98.6mmol), methyl isopropyl ketone (21.1mL, 197.2mmol) and sodium acetate (16.2g, 197.2mmol) in a round-bottom flask equipped with a condenser. The orange suspension is heated under reflux and becomes a dark brown solution. After the reaction is completed for 3 hours, the solvent is concentrated under reduced pressure, and the residue is dissolved in dichloromethane (300mL) and extracted with a saturated solution of NaHCO3 (3x100 mL). The organic phase contains most of the product; the aqueous phase is re-extracted with dichloromethane (150mL), and all organic phases are combined. The whole organic phase is concentrated under reduced pressure to obtain a solid (20.43g), which is crystallized with ethyl acetate / petroleum ether 3:1 (135mL). The first solid was filtered and the mother liquor was concentrated and recrystallized again with a solution of EtOAc / PE 1:1.5 (25 mL) to obtain a second solid of high purity, which was combined with the previous solid and dried under vacuum to obtain 13.31 g, 66% yield.

[0215] Preparation of intermediate (3)

[0216] Under magnetic stirring, intermediate (2) (3.7 g, 18.2 mmol) was suspended in butyronitrile (12 mL) in a round-bottom flask equipped with a condenser. Then, 2,4-butane sultone (2.09 mL, 20 mmol) was added. The orange suspension was heated under reflux and turned into a dark brown solution. After 42 hours, the reaction was complete: the solution was cooled to room temperature and 110 mL of acetone was added under magnetic stirring. After 2 hours, the solid was filtered out and the solid was dried overnight at 35 ° C under vacuum (6.03 g, 98% yield).

[0217] HPLC purity at 270 nm: 95.1%. MS: [M+H] + 341.0.

[0218] Synthesis of compound 1

[0219] In a round-bottom flask dried under a nitrogen atmosphere, intermediate (1) (91.60 mg, 0.386 mmol, prepared as reported in U.S. Patent No. 7,408,062) and intermediate (2) (100.0 mg, 0.295 mmol) were dissolved in EtOH (40 mL) at 50°C, and then 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (50.86 mg, 0.295 mmol) was added. The mixture was then heated at 50°C and stirred under a stream of nitrogen. After 2 minutes of reaction, the red starting mixture turned purple. After 20 hours at 50°C, the reaction was stopped. The crude mixture was dried under vacuum and dissolved in a minimum amount of water. Then, 1 mL of HCl 1M was added (pH of the mixture was 1.92), and the crude product was purified by flash chromatography on a pre-packed Phenomenex AQ C18 spherical silica gel column (40 g) with a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a dark green solid (50.55 mg, 24.0% yield). HPLC purity at 510 nm: 97.3%. MS: [M+H] + 715.2.

[0220] Example 2 :Synthesis of compound 2

[0221]

[0222] Preparation of intermediate (5)

[0223] In a round-bottom flask dried under nitrogen atmosphere, intermediate (1) (1.00 g, 3.83 mmol) was dissolved in dry DMF (3 mL), and iodoethane (2.02 mL, 25.1 mmol) was added at room temperature. The orange mixture was kept at 130 ° C under magnetic stirring for 5 hours. After 1 hour at 130 ° C, the reaction mixture turned pink. After 5 hours, the crude solid was suspended in 20 mL of cold ether, and the dispersion was kept under stirring for 30 minutes. The solid was filtered and washed with cold ether, and further suspended in cold acetone. The dispersion was kept under stirring for 30 minutes and then filtered. The solid was dissolved in 1.5 mL of MeOH, and the concentrated solution was dripped into 50 mL of cold acetone. The dispersion was kept under stirring for 1 hour and then filtered, and the resulting solid was washed with cold acetone. Finally, the crude red solid was filtered on a pre-filled C18 silica gel column ( SNAP ULTRA, 60 g) with a 0.01% formic acid / acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a pink solid (297.4 mg, 29.0% yield). HPLC purity at 270 nm: 97.6%. MS: [M+H] + 268.8.

[0224] Preparation of intermediate (6)

[0225] In a round-bottom flask dried under a nitrogen atmosphere, intermediate (5) (232.4 mg, 0.869 mmol) and (2) (231.5 mg, 1.14 mmol) were dissolved in EtOH (65 mL), and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (150.1 mg, 0.869 mmol) was added at room temperature. The mixture was then heated at 50 ° C and stirred under a nitrogen stream. After reacting for 30 minutes, the yellow starting mixture turned red. After 20 hours at 50 ° C, the reaction was stopped, and the solvent was distilled under reduced pressure. Purification was performed by flash chromatography on a pre-filled C18 silica gel column ( SNAP ULTRA, 60 g) with a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a dark green solid (19.55 mg, 3.8% yield). HPLC purity at 520 nm: 98.9%, at 780 nm, 20.5%. MS: [M+H] + 607.2.

[0226] Synthesis of compound 2

[0227] In a round-bottom flask dried under nitrogen atmosphere, intermediate (6) (13.5 mg, 0.022 mmol) was dissolved in dry DMF. D-glucosamine (9.64 mg, 0.053 mmol), DIPEA (15.5 μL, 0.089 mmol) and HATU (21.1 mg, 0.056 mmol) were added. The dark red solution was kept at room temperature under magnetic stirring for 5 hours, and then cold ether (60 mL) was added. The dispersion was kept under stirring for 2 hours and then stored at -20 ° C for 48 hours. The obtained solid was filtered and washed with cold ether, dissolved in MeOH and dried under vacuum. The obtained green solid was charged on a pre-filled C18 silica gel column ( SNAP, 12 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a dark green solid (19.92 mg). HPLC purity at 520 nm: 100%. MS: [M+H] + 770.3.

[0228] Example 3 :Synthesis of compound 3

[0229]

[0230] In a round-bottom flask, compound 1 (33.16 mg, 0.046 mmol) was dissolved in 5 mL of dry DMF. Then at room temperature, D-glucosamine (16.85 mg, 0.092 mmol), HATU (35.36 mg, 0.092 mmol) and DIPEA (32.1 μL, 0.184 mmol) were added to the purple solution. The mixture was stirred at room temperature for 2 hours. 50 mL of cold ether was added to the suspension and the precipitate was filtered. The purple solid was then purified by flash chromatography on a pre-filled C18 silica gel column ( SNAP ULTRA, 30 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined and concentrated under reduced pressure. The powder was redissolved in milliQ water and the solution was freeze-dried to give a purple solid (35.0 mg, 86.6% yield). HPLC purity at 510 nm and 254 nm: 100%. MS: [M+H] + 878.

[0231] Example 4 :Synthesis of compound 4

[0232] Preparation of intermediate (7)

[0233]

[0234] In a round-bottom flask dried under a nitrogen atmosphere, N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline monohydrochloride (72.7 mg, 0.202 mmol) was dissolved in 50 mL of H2O / MeOH 1:2 degassed mixture. At room temperature, 4-carboxyphenylboronic acid (60.4 mg, 0.364 mmol), Pd tetrakis (35.0 mg, 0.0303 mmol) and Na2CO3 (38.5 mg, 0.364 mmol) were added to the yellow solution. The mixture was then heated at 80 ° C and stirred for 2 hours under a nitrogen stream. The solvent was distilled under reduced pressure, and the crude product was flash chromatographed on a pre-filled C18 silica gel column ( SNAP ULTRA, 30 g) with a milliQ water-acetonitrile gradient. The fractions containing intermediate (7) were combined and concentrated under reduced pressure. The powder was redissolved in milliQ water and the solution was freeze-dried to give a bright orange solid (50.7 mg, 75.3% yield). HPLC purity at 410 nm: 100%. MS: [M+H] + 334.1.

[0235] Preparation of intermediate (8)

[0236]

[0237] In a pressure reactor vessel, intermediate (1) (1.02 g, 4.26 mmol) was suspended in 5 mL of sulfolane; then ethyl 6-bromohexanoate (1.14 ml, 6.39 mmol) was added to the suspension at room temperature. The brown suspension was then heated at 90 ° C for 70 hours. Cold ethyl acetate (10 mL) was added to the suspension and the precipitate was filtered. The red solid was then purified by flash chromatography on a pre-filled C18 silica gel column ( SNAP, 120 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing compound 3 were combined and concentrated under reduced pressure. The powder was redissolved in milliQ water and the solution was freeze-dried to give a red solid (285 mg, 17.5% yield). HPLC purity at 270 and 254 nm: 100%. MS: [M+H] + 382.1.

[0238] Preparation of intermediate (9)

[0239]

[0240] In a round-bottom flask dried under a nitrogen atmosphere, intermediate (7) (20.1 mg, 0.0602 mmol) was dissolved in 2 mL of degassed anhydrous ethanol. Then, under a nitrogen stream, at 50 ° C, a solution of intermediate (1) (14.4 mg, 0.0602 mmol) in 2 mL of anhydrous EtOH and 20 μL of acetic acid was added dropwise to the yellow solution. After heating for a few minutes, the yellow solution turned red and then turned light brown as the temperature increased. After 4 hours, intermediate (8) (22.9 mg, 0.0602 mmol) was added to the solution. The solution was stirred at 50 ° C for 96 hours. The solvent was distilled under reduced pressure, and the crude product was purified by flash chromatography on a pre-filled C18 silica gel column ( SNAP ULTRA, 30 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing intermediate (9) were combined and concentrated under reduced pressure. The powder was redissolved in milliQ water and the solution was freeze-dried to give a purple-blue solid (8.2 mg, 16% yield). HPLC purity at 510 nm: 98.7%. MS: [M+H] + 843.2.

[0241] Preparation of intermediate (10)

[0242]

[0243] At room temperature, in a round-bottom flask, intermediate (9) (4 mg, 0.00474 mmol) was dissolved in 1.5 mL of dry DMF. Taurine, HATU and DIPEA were then added to the purple solution. The mixture was stirred for 2 hours at room temperature under a nitrogen atmosphere. Cold ether (20 mL) was added to the mixture. The precipitate was filtered, redissolved in milliQ water and purified with a milliQ water-acetonitrile gradient on a pre-filled C18 silica gel column (Redisep Gold, 5 g). The fractions containing intermediate (10) were combined, concentrated under reduced pressure and freeze-dried to give a purple-blue solid (3.5 mg, 78% yield). HPLC purity at 510 nm: 98.4%, at 780 nm: 75%. MS: [M+H] + 950.1.

[0244] Synthesis of compound 4

[0245]

[0246] In a round bottom flask, intermediate (10) (3.5 mg, 0.00368 mmol) was dissolved in 5 mL of milliQ water and NaOH 1 M was added to adjust the pH to 11. Hydrolysis was performed at pH 11 and 40°C by automatic addition of NaOH 1 M (Dosimat coupled to a pH meter). After 2 hours, HCl 1 M was added to adjust the pH to 7. The mixture was desalted on a pre-filled C18 silica gel column (Redisep Gold, 5 g) using H2O (10 CV) as eluent to remove NaCl and MeOH (5 CV) to recover the product. The eluent was distilled under reduced pressure and freeze dried to give a purple-blue solid (2.6 mg, 76% yield). HPLC purity at 510 nm: 100%. MS: [M+H] + 922.1.

[0247] Example 5 :Synthesis of compound 5

[0248] Preparation of intermediate (11)

[0249]

[0250] At 50°C, in a round-bottom flask dried under a nitrogen atmosphere, intermediate (8) (110.5 mg, 0.290 mmol) and intermediate (1) (90.0 mg, 0.377 mmol) were dissolved in EtOH (20 mL). Then, AcONa (23.8 mg, 0.290 mmol) and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (50.0 mg, 0.290 mmol) were added. The mixture was then heated at 50°C and stirred for 20 h under a nitrogen stream. The solvent was dried under vacuum, and the obtained solid was filtered on a pre-filled C18 silica gel column ( SNAP, 30 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a dark purple solid (57.65 mg, 21.9% yield). HPLC purity at 510 nm: 99.9%, at 254 nm: 93.3%. MS: [M+H] + 757.35.

[0251] Synthesis of compound 5

[0252] Intermediate (11) (57.65 mg, 0.076 mmol) was dissolved in 40 mL of H2O. The solution was adjusted to pH 11 with 0.1 M NaOH (0.548 mL) and heated at 40 ° C, and the pH was maintained constant at 11 by automatically adding 0.1 M NaOH with a Dosimat coupled to a pH meter. The reaction was stopped after 8 h, and 6.626 mL of NaOH was added. The mixture was cooled to room temperature, neutralized with 1 M HCl (1.0 mL) and distilled under vacuum. The crude product was then filtered through a pre-filled C18 silica gel column ( SNAP, 60 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a dark green solid (31.9 mg, 57.5% yield). HPLC purity at 510 nm: 99.9%, at 254 nm: 96.7%. MS: [M+H] + 729.3.

[0253] Example 6 :Synthesis of compound 6

[0254] Preparation of intermediate (13)

[0255]

[0256] At 50 ℃, in a round-bottom flask dried under a nitrogen atmosphere, intermediate (1) (90.0 mg, 0.377 mmol) is dissolved in EtOH (20 mL), and then 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde (50.0 mg, 0.290 mmol) is added. The mixture is then heated at 50 ℃ and stirred for 1.5 h under a nitrogen stream. Intermediate (12) (100.3 mg, 0.290 mmol) is added to the mixture, the mixture is heated at 50 ℃ and stirred for another 13.5 h under a nitrogen stream. Then, other intermediates (12) (30.1 mg, 0.087 mmol) are added, and the reaction is stopped after heating for 9 h. The crude mixture is dried under vacuum (182.0 mg), and is used in the following steps without further purification.

[0257] Preparation of intermediate (14)

[0258] In a round-bottom flask, the crude intermediate (13) (182.0 mg, theoretically 0.252 mmol) was dissolved in 5 mL of dry DMF. D-glucosamine (91.3 mg, 0.504 mmol), HATU (191.6 mg, 0.504 mmol) and DIPEA (158.0 μL, 0.907 mmol) were then added to the solution at room temperature. The mixture was stirred at room temperature for 4 h, and then cold ether (70 mL) was added. The dispersion was kept under stirring for 12 h, the solvent was decanted, and the obtained solid was washed with cold ether and filtered on a pre-filled C18 silica gel column ( SNAP, 60 g) with a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum and freeze-dried to give a dark green solid (114.0 mg, 44% yield from intermediate (12)). HPLC purity at 510 nm: 99.4%, at 756 nm, 40.0%. MS: [M+H] + 884.48.

[0259] Synthesis of compound 6

[0260] Intermediate (14) (114.0 mg, 0.129 mmol) was dissolved in 60 mL of H2O while adding 1 mL of EtOH. The solution was adjusted to pH 11 with 1 M NaOH (0.432 mL) and heated at 40 ° C. The pH was maintained constant at 11 by automatically adding 1 M NaOH with a Dosimat coupled to a pH meter. After 4.5 h, the hydrolysis was complete and 6.626 mL of NaOH was added. The mixture was cooled to room temperature, neutralized with 2 M HCl (1.5 mL) and distilled under vacuum. The crude product was then filtered through a pre-filled C18 silica gel column ( SNAP, 60 g) was purified using a milliQ water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under vacuum, and freeze-dried to give a dark green solid (70.62 mg, 64% yield). HPLC purity at 510 nm: 99.4%, at 254 nm, 93.9.0%. MS: [M+H] + 856.90.

[0261] Example 7 :Synthesis of compound 7

[0262] Preparation of intermediate (15)

[0263]

[0264] In a round bottom flask, intermediate (2) (1.01 g, 4.97 mmol) was dissolved in 15 mL of anhydrous DMF, followed by the addition of TBTU (2.23 g, 6.94 mmol) and DIPEA (1.73 mL, 9.95 mmol). Accordingly, D-glucosamine (1.260 mg, 6.95 mmol) was added, and the mixture was stirred at room temperature for 2.5 hours. Cold ether was added to precipitate a solid, which was filtered, dissolved in MeOH and dried under vacuum, and then purified by flash chromatography on a pre-packed C18 silica gel column ( SNAP Ultra 120g) was purified with a water-acetonitrile gradient. The fractions containing the pure product were combined and concentrated under vacuum. The batch was freeze-dried and a white solid was obtained (1.75 g, 95.9% yield). HPLC-MS purity at 270nm: 100%; MS: [M+H] + 367.0.

[0265] Preparation of intermediate (16)

[0266]

[0267] In a round-bottom flask, intermediate (15) (132.56 mg, 0.3618 mmol) was dissolved in 45 mL of anhydrous EtOH, and N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline monohydrochloride (100 mg, 0.2783 mmol) was added. The solution was heated at 50°C for 4 hours. Then, intermediate (12) (96.41 mg, 0.2783 mmol) was added, which was dissolved in 15 mL of anhydrous EtOH. The mixture was heated at 50°C for 24 hours. Intermediate (12) (48.58 mg, 0.1402 mmol) was added a second time to achieve the highest conversion to asymmetric cyanine, and the solution was stirred for another 48 hours. The mixture was cooled to room temperature and distilled under vacuum to obtain crude intermediate (16), which was used in the following reaction without further purification. HPLC purity at 510 nm: 59.9%. MS: [M+H] + 848.4.

[0268] Preparation of intermediate (17)

[0269]

[0270] In a round bottom flask, intermediate (16) (246 mg, theoretical 0.290 mmol) was dissolved in 20 mL of anhydrous DMF, followed by the addition of HATU (220.2 mg, 0.579 mmol) and DIPEA (181 μL, 1.042 mmol). Accordingly, D-glucosamine (105 mg, 0.579 mmol) was added, and the mixture was stirred at room temperature for 4 hours. Addition of cold ether resulted in the precipitation of a purple solid, which was filtered and washed twice with cold solvent, and then purified by flash chromatography on a pre-packed C18 silica gel column ( SNAP Ultra 120g) was purified with a water-acetonitrile gradient. The fractions containing the pure product were combined, distilled under reduced pressure and freeze-dried to give a purple solid (70.0 mg, 25% yield from compound 2). HPLC-MS purity at 510nm: 100%; MS: [M+H] + 1011.4.

[0271] Synthesis of compound 7

[0272]

[0273] Intermediate (17) (30.00 mg, 0.0296 mmol) was dissolved in 15 mL of a solution of H2O / EtOH 7:3. The solution was adjusted to pH 11 with NaOH 1 M and heated at 40 °C, the pH was maintained constant at 11 by automatically adding NaOH 1 M with a Dosimat coupled to a pH meter. After 2 hours the hydrolysis was complete, and 0.542 mL of NaOH 1 M was added. The mixture was cooled to room temperature, neutralized with HCl 0.1 M and distilled under vacuum to give a crude product which was subsequently purified by flash chromatography on a pre-filled C18 silica gel column ( SNAP Ultra 30g) with a water-acetonitrile gradient. The fractions containing the pure product were combined, concentrated under vacuum and freeze-dried to obtain a purple solid (15.0 mg, 51.5% yield). HPLC-MS purity at 510nm: 100%; MS: [M+H] + 983.3.

[0274] Example 8 :Synthesis of compound 8

[0275] Preparation of intermediate (18)

[0276]

[0277] The intermediate (1) (10.0 g, 0.036 mol) obtained by the procedure described in patent No. US 7,408,062 B2 was suspended in sulfolane (20.0 g). Then 6-bromo-hexanoic acid (9.0 g, 0.046 mol) was added, and the mixture was heated at 100 ° C for 72 hours. The mixture was cooled, acetone (150 mL) was then added and the resulting solid was filtered. The solid was washed three times with acetone, and then dried under vacuum at 60 ° C for 4 hours. The resulting solid was dispersed in acetonitrile (150 mL) and stirred at 65 ° C for 1 hour, and then filtered while still hot. The collected solid was washed with cold acetonitrile and then dried under vacuum at 60 ° C for 4 hours, 14.0 g was obtained at 91% (area HPLC). Yield 82%.

[0278] Synthesis of compound 8

[0279] In a 100 mL four-necked flask, pentanedialdehyde diphenylamine hydrochloride (0.24 g, 0.84 mmol), acetic acid (6.85 g) and acetic anhydride (25.2 g) were charged. The solution was heated at 60° C. for 2 hours, then intermediate (1) (0.226 g, 0.81 mmol) was added and heated for another 3 hours. The reaction mixture was then cooled to room temperature and isopropyl acetate (350 mL) was added dropwise under stirring. The solid was filtered, washed with isopropyl acetate and then dried under vacuum at 40° C. for 1 hour (0.21 g).

[0280] In another 100mL four-necked flask, intermediate (18) (0.25g, 0.53mmol), anhydrous DMF (5mL) and sodium acetate (0.065g, 0.79mmol) were loaded. The suspension was heated at 60°C to obtain a clear solution. Then, a solution of hemicyanine (0.21, 0.48mmol) in anhydrous DMF (2mL) was added dropwise, and the reaction mixture was heated at 60°C for 6 hours. Then, it was cooled and dropped into isopropyl acetate (350mL) under stirring. The solid was filtered and purified by flash chromatography on silica gel with a gradient elution of isopropyl acetate-methanol. The fractions containing the pure product were combined and concentrated under vacuum to obtain 33mg of a purple solid (7% yield).

[0281] Example 9 :Synthesis of compound 9

[0282]

[0283] Intermediate (21) and (22) are prepared as described in patent application US 2013 / 045488 A1. Under nitrogen atmosphere, intermediate (21) (9.55mg, 0.026mmol) is suspended in MeOH (2mL) in a round-bottom flask. Then, Vilsmeier reagent diphenylamine form (3.02mg, 0.0103mmol) and intermediate (22) (5mg, 0.0103mmol) are added, followed by water (200 μL) and pyridine (250 μL) required for dissolving benzindole. Then, the solution is heated at 90 ° C overnight. The mixture is concentrated under vacuum and cooled with cold EtOAc (10mL) in an ice bath to precipitate. The blue precipitate is filtered, dissolved in water and by flash chromatography on a pre-filled C18 silica gel column ( C1812g) was purified with a water-acetonitrile gradient. The fractions containing the pure product were combined and concentrated under vacuum to obtain 0.87 mg of a blue solid (9.1% yield). HPLC-MS purity at 650nm: 88%; MS: [M+H] + 922.9.

[0284] Example 10 :Synthesis of compound 10

[0285]

[0286] In a round-bottom flask, under a nitrogen atmosphere, compound 5 (10 mg, 0.0137 mmol) was dissolved in 5 mL of dry DMF. Then TBTU (4.4 mg, 0.0137 mmol) and DIPEA (6.7 μL, 0.0383 mmol) were added. The mixture was stirred at room temperature for 1 hour, and then a solution of c(RGDfK) trifluoroacetate (9.8 mg, 0.0137 mmol) in 5 mL of dry DMF was added dropwise. The mixture was stirred at room temperature overnight, and then precipitated with cold ether (150 mL) in an ice bath. The purple precipitate was filtered and washed twice with cold ether. It was then dissolved in a mixture of water / acetonitrile 2: 1, and flash chromatography was performed on a pre-filled C18 silica gel column ( SNAP Ultra 12g) was purified using a water-acetonitrile gradient. The fractions containing the pure product were combined, concentrated under reduced pressure and freeze-dried to give a blue solid (9 mg, 50% yield). HPLC purity at 510 nm: 99.3%; MS: [MH] - 1314.3.

[0287] Embodiment 11 :Synthesis of compound 11

[0288]

[0289] Preparation of intermediate (19)

[0290] In a 40mL centrifugal glass tube, compound 6 (15.60mg, 0.018mmol) was dissolved in 3mL of dry DMF. Then TSTU (10.9mg, 0.036mmol) and NMM (4 μL, 0.036mmol) were added to the purple solution at room temperature. The mixture was stirred at room temperature for 2 hours. Then, cold ether (40mL) was added to the solution. The suspension was centrifuged at 4000rpm for 55min. Then, ether was decanted to obtain a purple solid sticking to the tube wall. The crude compound was used in the following steps without further purification.

[0291] Synthesis of compound 11

[0292] In the same 40 mL centrifuge glass tube containing intermediate (19), a solution of c(RGDfK) (14 mg, 0.020 mmol) in 8 mL of borate buffer at pH 9 was added at room temperature. The purple mixture was stirred at room temperature for 2 hours. The pH of the mixture was then adjusted to 7 by adding 1 M HCl, and the crude product was purified by preparative HPLC on a YMC triart-phenyl column with a 0.1% formic acid / acetonitrile gradient. The fractions containing the pure product were combined, concentrated and desalted on a pre-filled silica gel C18 column (GE, 5 g) using 10 CV of water to remove excess formic acid and 5 CV of MeOH to recover the product. The final solution was then distilled under reduced pressure, the resulting powder was dissolved in milliQ water, and the solution was freeze-dried to give a bright purple solid (6.5 mg, 25% yield). HPLC purity: 100% at 510 nm, 89.3% at 254 nm. MS: [M+H] + 1441.4.

[0293] Example 12 :Synthesis of compound 12

[0294]

[0295] Preparation of intermediate (20)

[0296] In a 40mL glass centrifuge tube, compound 7 (10.3mg, 0.0105mmol) was dissolved in 2mL of dry DMSO. Then at room temperature TSTU (6.3mg, 0.0210mmol) and NMM (2.3 μL, 0.0210mmol) were added to the purple solution. The mixture was stirred at room temperature for 30 minutes. Then, 40mL of cold ether was added to the solution. The suspension was centrifuged at 4000rpm for 55min and the ether was decanted to obtain a purple solid sticking to the tube wall. The crude product was used in the following reactions without further purification.

[0297] Synthesis of compound 12

[0298] In the same 40 mL centrifuge tube, intermediate (20) was dissolved in 2 mL of DMSO and a solution of c(RGDfK) (8.3 mg, 0.0116 mmol) in 6 mL of pH 9 borate buffer was added at room temperature. The purple mixture was then stirred at room temperature for 1 hour. The pH of the mixture was adjusted to 6.7 by adding 0.5 M HCl. Water was distilled under reduced pressure and the crude product was purified by flash chromatography on a pre-packed C18 silica gel column ( Purify with a milliQ water-acetonitrile gradient on CLARICEP, 40 g. The fractions containing the pure product are combined and concentrated under reduced pressure. The powder is redissolved in milliQ water and the solution is freeze-dried to give a bright purple solid (4.6 mg, 27.9% yield from compound 7). HPLC purity at 510 nm: 97.8%. MS: [M+H] + 1569.5.

[0299] Example 13 : Optical properties

[0300] Analytical characterization of dyes

[0301] For each compound, analytical and optical characterizations were performed. All synthesized dyes and their conjugates were extensively characterized by HPLC-MS, obtaining high final purity values. For example, the purity calculated from the HPLC areas at 254 nm, 510 nm (maximum absorbance at neutral pH) and 780 nm for a representative compound 5 is reported in Table II below.

[0302] Table II - % purity (HPLC area) of representative compound 5 at different wavelengths.

[0303] Purity% (HPLC area) 254nm 510nm 780nm Compound 5 >95％ >98％ >95％

[0304] use Each compound was optically characterized using a multifunctional microplate reader, recording the absorbance, excitation, and emission spectra of the fluorophore buffered in phosphate buffer at several pH values ​​ranging from 3 to 8 using a specific 96-well plate.

[0305] In particular, the absorbance, excitation and emission maxima of representative compounds of formula (I) and conjugates of formula (II) are shown in Table III.

[0306] Table III - Optical characterization of pH responsive dyes and conjugates

[0307]

[0308] n / a, not applicable.

[0309] The compounds of the present invention are characterized by absorption maxima contained within the range of about 620 nm to about 790 nm, and even when conjugated to a targeting moiety, fluorescence emission is within the range of about 650 nm to 900 nm.

[0310] pK evaluation

[0311] By analyzing the changes in absorbance and emission with respect to changes in pH, the pKa of the dyes of the invention can be calculated as the inflection point of the interpolated curve.

[0312] All pH buffers used in the experiment are phosphate-based buffers, and each pH point has been accurately checked with a pH meter. First, the cyanine powder is dissolved in water at a concentration of about 1 mg / mL, and then 10 μL of the mother solution is added to 1 ml of phosphate buffer at different pH values, which is prepared in advance by mixing an appropriate amount of orthophosphoric acid and sodium hydroxide to reach the desired pH. The diluted cyanine buffer solution is then sampled and transferred to a 96-well plate (transparent for absorbance measurements and dark for emission measurements) for analysis. By plotting the absorbance value under the maximum value of the active form of each solution (e.g., 780 nm for compound 5) relative to the pH of the solution, a polynomial cubic curve is obtained, whose inflection point corresponds to the pKa value of the cyanine. An example of such a curve for representative compound 5 is shown in Figure 1 , where the inflection point provides a pKa value of 5.9.

[0313] In addition, plotting the emission values ​​of the active form at the maximum (e.g., for compound 5, at 804 nm) versus pH, a similar curve was obtained, confirming the same values ​​of pKa (results obtained from the second data source). The pKa values ​​obtained for reference compound A are also shown in Table IV.

[0314] Table IV - pK values ​​of representative compounds of the invention

[0315]

[0316] *Lee et al., Bioconjug Chem 2011; 22(4): 777–784

[0317] Residual fluorescence at different pH

[0318] For several representative compounds of the invention, the ratio of fluorescence at different pH (5, 6 and 7.5) relative to the maximum fluorescence collected at pH 3 (highly acidic) was calculated and the results are reported below in Table V. The results were also compared to the calculated values ​​for the non-alkylated reference Compound A.

[0319] Table V - Residual fluorescence at different pH

[0320]

[0321] *Lee et al., Bioconjug Chem 2011; 22(4): 777–784

[0322] These results demonstrate that the higher pKa of the compounds of the present invention, which are included in the range of 5.5-7.0, in particular 5.8-6.5, allow for improved fluorescence emission at pH 5 and 6, i.e., at pH values ​​that are more likely to characterize the extracellular environment and endosomes of tumor cells, and provide more easily detectable signals in vitro and in vivo. For example, at pH 5 and 6, they show fluorescence signals that are about 2 to 8 times higher than reference compound A. In contrast, the fluorescence at physiological pH (about 7.4) is very low, which may result in a low background signal.

[0323] References:

[0324] 1. Cherrick et al., J Clin Invest 1960;39(4):592–600

[0325] 2. Tummers Q. et al., Plos One 2015;10(6):e0129766

[0326] 3. Achilefu S. et al., J Med Chem 2002; 45, 2003-2015

[0327] 4. Fidel J. et al., Cancer Res. 2015;15;75(20):4283–4291

[0328] 5. Damaghi M. et al., Front Physiol. 2013; 4: 370

[0329] 6. WO00 / 75237

[0330] 7. Briggs M. et al., Chem. Commun. 2000, 2323-2324

[0331] 8. WO2004 / 039894

[0332] 9. Beletskii A. et al., BioTechniques 2005, 39: 894-897

[0333] 10. Lee H. et al., Bioconjug Chem. 2011, 22(4): 777–784

[0334] 11. Gilson R. et al., Mol. Pharmaceutics 2015, 12: 4237-4246

[0335] 12. TW Green and PGM Wuts, Protective Groups in Organic Synthesis, Wiley, NY 2007, 4th edition, Chapter 5

[0336] 13. Kapp et al., Sci Rep, 2017, 7: 3905

[0337] 14. Wichert et al., Nat Chem 2015, 7: 241–249

[0338] 15. Li et al., FASEB J 2005, 19: 1978-85

[0339] 16. Williams et al., Chem Biol Drug Des 2018, 91: 605–619

[0340] 17. Mujumdar RB et al., Bioconjugate Chem. 1993, 4(2): 105-111

[0341] 18. Yamana et al., Chem. Pharm. Bull., 1972, 20(5), 881-891

[0342] 19.US 7,408,062 B2

[0343] 20. US 2013 / 045488 A1

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, in W is a group or -CH=C(R6)-CH= or -C(Cl)=, wherein R6 is hydrogen or chlorine; R7 is chloro or phenyl optionally substituted by a group selected from -SO3H, -COOH, -CONH-Y, -alkyl-COOH and -alkyl-CONH-Y, wherein Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups, and * indicates bonding position; R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y; R2 and R4 are hydrogen, and R5 is an alkyl group optionally substituted with a group selected from -SO3H, -COOH and -CONH2.

2. The compound of formula (I) according to claim 1, which is represented by formula (Ib) in R5 and R7 are as defined in claim 1, and R1 and R3 are independently selected from the group consisting of -H, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl substituted with -SO3H or at least two hydroxyl groups.

3. The compound of formula (I) according to claim 1, which is represented by formula (Ic) in W is selected from -C(Cl)=, -CH=CH-CH= and -CH=C(Cl)-CH=, R1 and R3 are independently selected from the group consisting of -H, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups, and R5 is as defined in claim 1.

4. A compound of formula (II) in W is a group or -CH=C(R6)-CH= or -C(Cl)=, wherein R6 is hydrogen or chlorine; R8 is chlorine or is optionally selected from -SO3H, -COOH, -CONH-Y, -alkyl-COOH, -alkyl-CONH-Y and -CONH-(S) m -T substituted divalent alkyl group substituted phenyl, wherein Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups; S is a spacer group; T is a targeting moiety; and m is an integer equal to 0 or 1; and * indicates bonding position; R1 and R3 are independently selected from the group consisting of hydrogen, -SO3H, -COOH and -CONH-Y; R2 and R4 are hydrogen, R9 is optionally selected from -SO3H, -COOH, -CONH2 and -CONH-(S) m -T, wherein S, T and m are as defined above; and wherein at least one group -CONH-(S) is present in R8 or R9 m -T.

5. The compound of formula (II) according to claim 4, which is represented by formula (IIb) in R1 and R3 are independently selected from the group consisting of -H, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups, and R8 and R9 are as defined in claim 4.

6. The compound of formula (II) according to claim 4, which is represented by formula (IIc) in W is selected from -C(Cl)=, -CH=CH-CH= and -CH=C(Cl)-CH=, R1 and R3 are independently selected from the group consisting of -H, -SO3H, -COOH and -CONH-Y, wherein Y is a divalent alkyl group substituted with -SO3H or at least two hydroxyl groups, and R9 is as defined in claim 4.

7. A compound of formula (II) according to any one of claims 4 to 6, wherein T is a targeting moiety selected from the group consisting of small molecules, proteins, peptides, peptide mimetics, enzyme substrates, antibodies or fragments thereof, and aptamers.

8. The compound of formula (II) according to claim 7, wherein T is a moiety that interacts with an integrin receptor.

9. Use of a compound of formula (I) as defined in any one of claims 1 to 3 or a compound of formula (II) as defined in any one of claims 4 to 6 for the preparation of a fluorescent probe for biomedical optical imaging applications in mammals.

10. Use of a compound of formula (I) or a compound of formula (II) according to claim 9, wherein the imaging application is directed to the detection of abnormal tissues including primary tumor lesions, local or distant metastases, or pre-neoplastic lesions and is performed under NIR radiation.

11. A pharmaceutical composition comprising a compound of formula (I) as defined in any one of claims 1 to 3 or a compound of formula (II) as defined in any one of claims 4 to 6 and at least one pharmaceutically acceptable carrier or excipient.

12. A diagnostic kit comprising at least one compound of formula (I) as defined in any one of claims 1 to 3 or at least one compound of formula (II) as defined in any one of claims 4 to 6 and further adjuvants thereof for use in biomedical optical imaging applications.

13. Use of a compound of formula (I) as defined in any one of claims 1 to 3 or a compound of formula (II) as defined in any one of claims 4 to 6 in a method for imaging tissues and cells, the method comprising the steps of: i) contacting the cell or tissue with a compound of formula (I) as defined in any one of claims 1 to 3 or a compound of formula (II) as defined in any one of claims 4 to 6; ii) irradiating the tissue or cell at a wavelength absorbed by the imaging agent; iii) Detection of near-infrared emission using a fluorescence camera.

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