Activatable fluorescent probes for optical urinalysis of kidney disease

Abstract

The current invention relates to compounds suitable for detecting the presence of N-Acetyl-β- D-glucosaminidase (NAG). The current invention further relates to methods of detecting the NAG in a sample obtained from a subject suspected of having increased levels of NAG comprising a step of providing a formulation comprising the compounds.

Description

[0001] ACTIVATABLE FLUORESCENT PROBES FOR OPTICAL URINALYSIS OF KIDNEY DISEASE

[0002] Field of Invention

[0003] The current invention relates to compounds suitable for detecting the presence of M-Acetyl-p- o-glucosaminidase (NAG) and methods of detecting the NAG in a sample obtained from a subject suspected of having increased levels of NAG comprising a step of providing a formulation comprising the compounds.

[0004] Background

[0005] The listing or discussion of a prior-pubiished document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] A / -Acetyl-[3-D-glucosaminidase (NAG) is an enzyme mainly distributed in the cytoplasm of the proximal tubular cell. In normal conditions, the NAG from the plasma cannot be filtered through the glomerulus due to its high molecular weight which is 130-140 kDa. However, if renal tubule injury happens during kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), NAG will be extensively secreted by the proximal tubular cells into the urine. Therefore, NAG has received widespread research attention for its clinical significance in sensitive detection and accurate prediction of kidney disease.

[0007] The common method for detecting NAG in urine is by its activity using substrate conversion assay that is based on ultraviolet-visible (UV-vis) spectroscopy. However, the results from this method are not always reliable due to low sensitivity and significant background interference.

[0008] It is apparent that breakthrough is still needed to overcome the current hurdles so that more reliable results can be obtained. The present invention aims to address the current limitation with a method based on fluorescent spectroscopy as it will be more feasible for its stronger robustness towards background interferences. Therefore, it is critical to design fluorophores, especially, activatable fluorescent probes that turn-on signals in response to the biomarkers of interest. The high signal-to-background ratio from the probes grand them with high sensitivity and excellent ability to detect diseases in advance.

[0009] Summary of Invention Aspects and embodiments of the invention will now be described by reference to the following numbered clauses.

[0010] 1. A compound of formula I: where:

[0011] X is a bond or , where the dotted line represents the point of attachment to Y and the wavy line represents the point of attachment to the rest of the molecule;

[0012] Y represents:

[0013] where the wavy line represents the point of attachment to the rest of the molecule; where: the wavy line represents the point of attachment to the rest of the molecule;

[0014] Z represents O, S or Se;

[0015] Ri represents H, Me, CF3, CH2OH;

[0016] where the wiggly line represents the point of attachment to the rest of the molecule;

[0017] R3represents O or is selected from:

[0018] where the wavy line represents the point of attachment to the rest of the molecule;

[0019] FU represents:

[0020] where the wavy line represents the point of attachment to the rest of the molecule;

[0021] Rs represents H, Me, OH, or halo;

[0022] where the wavy line represents the point of attachment to the rest of the molecule;

[0023] R7represents H, NH2 or N(Me)2;

[0024] where the wavy line represents the point of attachment to the rest of the molecule, or a pharmaceutically acceptable salt or solvate thereof.

[0025] 2. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 1 , wherein when Y represents:

[0026] where the wavy line represents the point of attachment to the rest of the molecule X is a bond;

[0027] Ri represents CF3, CH2OH;

[0028] R2represents H or is selected from: where the wiggly line represents the point of attachment to the rest of the molecule; and where the wavy line represents the point of attachment to the rest of the molecule.

[0029] 3. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 2, wherein

[0030] Ri represents CF3; where the wiggly line represents the point of attachment to the rest of the molecule; and

[0031] R3represents O or is selected from: where the wavy line represents the point of attachment to the rest of the molecule. 4. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 1 , wherein when Y represents: where the wavy line represents the point of attachment to the rest of the molecule, X is a bond;

[0032] FU represents: where the wavy line represents the point of attachment to the rest of the molecule; and

[0033] Rs represents Me, or OH.

[0034] 5. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 4, wherein

[0035] FU represents: where the wavy line represents the point of attachment to the rest of the molecule; and

[0036] R5represents Me, or OH.

[0037] 6. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 1 , wherein when Y represents:

[0038] where the wavy line represents the point of attachment to the rest of the molecule;

[0039] Re represents: where the wavy line represents the point of attachment to the rest of the molecule;

[0040] R? represents H, NH2or N(Me)2; and where the wavy line represents the point of attachment to the rest of the molecule.

[0041] 7. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 6, wherein

[0042] X is , where the dotted line represents the point of attachment to Y and the wavy line represents the point of attachment to the rest of the molecule;

[0043] Rs represents: where the wavy line represents the point of attachment to the rest of the molecule;

[0044] R? represents NH2or N(Me)2; and

[0045] Rs represents: where the wavy line represents the point of attachment to the rest of the molecule.

[0046] 8. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Clause 1 , wherein the compound of formula I is selected from one or more of the group consisting of:

[0047]

[0048] 9. A method of detecting the presence of / V-Acetyl-p-D-glucosaminidase (NAG) in a sample obtained from a subject suspected of having increased levels of NAG, the method comprising the steps of:

[0049] (a) providing a formulation comprising a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, and a sample obtained from a subject;

[0050] (b) contacting the sample with the formulation for a period of time and detecting the presence or absence of a signal associated with NAG. 10. The method according to Clause 9, wherein the method is in vitro.

[0051] 11. The method according to Clause 9 or Clause 10, wherein the signal detected is an fluorescence signal.

[0052] 12. The method according to any one of Clauses 9 to 1 1 , wherein the sample is selected from one or more of tissues fluids, urine, a microorganism culture, and a plant culture, optionally wherein the sample is a urine sample.

[0053] 13. The method according to any one of Clauses 9 to 12, wherein the method further comprises use of a control sample, where the control sample’s NAG signal is compared to the sample obtained from a subject and an increase in the detection signal indicates the presence of an increased amount of NAG.

[0054] 14. The method according to any one of Clauses 9 to 13, wherein the method provides a quantitative determination of the amount of NAG in the sample.

[0055] Drawings

[0056] FIG. 1 depicts the high-performance liquid chromatography (HPLC) analysis of compounds P1 , P2, and P3 in the absence or presence of NAG in PBS.

[0057] FIG. 2 depicts (a) the ultraviolet-visible (UV-vis) spectra and (b) the fluorescence spectra of compound P1 in the absence or presence of / V-Acetyl-p-o-glucosaminidase (NAG) in phosphate-buffered saline (PBS).

[0058] FIG. 3 depicts (a) the UV-vis spectra and (b) the fluorescence spectra of compound P2 in the absence or presence of NAG in PBS.

[0059] FIG. 4 depicts (a) the UV-vis spectra and (b) the fluorescence spectra of compound P2 in the absence or presence of NAG in PBS.

[0060] FIG. 5 depicts the detection mechanisms of (a) compound P1 and (b) compound P3 towards NAG. FIG. 6 depicts the UV-vis spectra (a, c, e) and the fluorescence spectra (b, d, f) of compounds P1 , P2, and P3, respectively, in the absence or presence of NAG in PBS with bovine serum albumin (BSA).

[0061] Description

[0062] It has been surprisingly found that specific fluorescent molecular probes can be used to detect NAG conditions in urine. In the present invention, bright activatable fluorescent probes for the detection of NAG have been designed and synthesized.

[0063] Thus, in a first aspect of the invention, there is provided a compound of formula I: where:

[0064] X is a bond or , where the dotted line represents the point of attachment to Y and the wavy line represents the point of attachment to the rest of the molecule;

[0065] Y represents:

[0066] where the wavy line represents the point of attachment to the rest of the molecule; where: the wavy line represents the point of attachment to the rest of the molecule;

[0067] Z represents O, S or Se;

[0068] Ri represents H, Me, CF3, CH2OH;

[0069] R2 represents H or is selected from: where the wiggly line represents the point of attachment to the rest of the molecule;

[0070] R3represents O or is selected from:

[0071] where the wavy line represents the point of attachment to the rest of the molecule;

[0072] FU represents:

[0073] where the wavy line represents the point of attachment to the rest of the molecule;

[0074] Rs represents H, Me, OH, or halo;

[0075] Rs represents:

[0076] where the wavy line represents the point of attachment to the rest of the molecule;

[0077] R7represents H, NH2 or N(Me)2;

[0078] or a pharmaceutically acceptable salt or solvate thereof. In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0079] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0080] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an oxygen carrier” includes mixtures of two or more such oxygen carriers, reference to “the catalyst” includes mixtures of two or more such catalysts, and the like.

[0081] In some embodiments that may be mentioned herein, Y may represent: where the wavy line represents the point of attachment to the rest of the molecule X is a bond;

[0082] Ri represents CF3, CH2OH;

[0083] R2represents H or is selected from: where the wiggly line represents the point of attachment to the rest of the molecule; and where the wavy line represents the point of attachment to the rest of the molecule.

[0084] In such embodiments that may be mentioned herein,

[0085] Ri may represent CF3; where the wiggly line represents the point of attachment to the rest of the molecule; and

[0086] R3may represent O or is selected from: where the wavy line represents the point of attachment to the rest of the molecule.

[0087] In some embodiments that may be mentioned herein, wherein when Y may represent: where the wavy line represents the point of attachment to the rest of the molecule, X is a bond;

[0088] R4 represents: where the wavy line represents the point of attachment to the rest of the molecule; and

[0089] R5represents Me, or OH.

[0090] In such embodiments that may be mentioned herein, R4 may represent: where the wavy line represents the point of attachment to the rest of the molecule; and

[0091] Rs represents Me, or OH.

[0092] In some embodiments that may be mentioned herein, when Y may represent: where the wavy line represents the point of attachment to the rest of the molecule;

[0093] Rs represents:

[0094] where the wavy line represents the point of attachment to the rest of the molecule;

[0095] R? represents H, NH2or N(Me)2; and where the wavy line represents the point of attachment to the rest of the molecule.

[0096] In such embodiments that may be mentioned herein,

[0097] X may be , where the dotted line represents the point of attachment to Y and the wavy line represents the point of attachment to the rest of the molecule;

[0098] R6may represent: where the wavy line represents the point of attachment to the rest of the molecule;

[0099] R7may represent NH2or N(Me)2; and where the wavy line represents the point of attachment to the rest of the molecule.

[0100] In some embodiments that may be mentioned herein, the compound of formula I may be selected from one or more of the group consisting of:

[0101]

[0102] References herein (in any aspect or embodiment of the invention) to compounds of formula I includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.

[0103] Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

[0104] Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0105] Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic) , lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1 - hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

[0106] Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.

[0107] As mentioned above, also encompassed by formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

[0108] The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.

[0109] For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn etal., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3. “Pharmaceutically functional derivatives” of compounds of formula I as defined herein includes ester derivatives and / or derivatives that have, or provide for, the same biological function and / or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I.

[0110] The term “prodrug” of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).

[0111] Prodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

[0112] Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. 1-92, Elsevier, New York-Oxford (1985).

[0113] Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula I”.

[0114] Compounds of formula I may contain double bonds and may thus exist as E entgegen) and Z (zusammeri) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.

[0115] Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.

[0116] Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and / or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a 'chiral pool’ method), by reaction of the appropriate starting material with a 'chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.

[0117] Further embodiments of the invention that may be mentioned include those in which the compound of formula I is isotopically labelled. However, other, particular embodiments of the invention that may be mentioned include those in which the compound of formula I is not isotopically labelled.

[0118] The term "isotopically labelled", when used herein includes references to compounds of formula I in which there is a non-natural isotope (or a non-natural distribution of isotopes) at one or more positions in the compound. References herein to "one or more positions in the compound" will be understood by those skilled in the art to refer to one or more of the atoms of the compound of formula I. Thus, the term "isotopically labelled" includes references to compounds of formula I that are isotopically enriched at one or more positions in the compound.

[0119] The isotopic labelling or enrichment of the compound of formula I may be with a radioactive or non-radioactive isotope of any of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine and / or iodine. Particular isotopes that may be mentioned in this respect include2H,3H,11C,13C,14C,13N,15N,15O,170,180,35S,18F,37CI,77Br,82Br and125l).

[0120] When the compound of formula I is labelled or enriched with a radioactive or nonradioactive isotope, compounds of formula I that may be mentioned include those in which at least one atom in the compound displays an isotopic distribution in which a radioactive or nonradioactive isotope of the atom in question is present in levels at least 10% (e.g. from 10% to 5000%, particularly from 50% to 1000% and more particularly from 100% to 500%) above the natural level of that radioactive or non-radioactive isotope. In a second aspect of the invention, there is provided a method of detecting the presence of A / -Acetyl-|3-D-glucosaminidase (NAG) in a sample obtained from a subject suspected of having increased levels of NAG, the method comprising the steps of:

[0121] (a) providing a formulation comprising a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, and a sample obtained from a subject;

[0122] (b) contacting the sample with the formulation for a period of time and detecting the presence or absence of a signal associated with NAG.

[0123] In some embodiments that may be mentioned herein, the method may be in vitro.

[0124] Visualising methods that may be mentioned include spectroscopic detection methods (e.g. fluorescence detection, magnetic resonance imaging, etc.) or, when the compound of formula I is isotopically labelled or enriched with a radioisotope (such as3H,11C,35S,18F, or125l), radioactivity detection methods (e.g. alpha-, beta- or gamma-detection by standard autoradiography, phosphor or scintillation methods known to those skilled in the art, or positron emission tomography (which latter method may be employed, for example, when the compound of formula I is isotopically labelled or enriched with11C, or, particularly,18F)).

[0125] In some embodiments that may be mentioned herein, the signal detected may be an fluorescence signal. Details of fluorescence measurement technique are provided in the examples below.

[0126] In some embodiments that may be mentioned herein, the sample may be selected from one or more of tissues fluids, urine, a microorganism culture, and a plant culture. For example, the sample may be a urine sample.

[0127] In some embodiments that may be mentioned herein, the method may further comprise use of a control sample, where the control sample’s NAG signal is compared to the sample obtained from a subject and an increase in the detection signal indicates the presence of an increased amount of NAG.

[0128] In some embodiments that may be mentioned herein, the method may provide a quantitative determination of the amount of NAG in the sample.

[0129] The terms “patient and “patients" include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0130] A typical method for detecting NAG in urine is by detecting its activity using substrate conversion assay that is based on ultraviolet-visible (UV-vis) spectroscopy. However, the results from this method are not always reliable due to low sensitivity and significant background interference. As will be appreciated, the present invention provides great improvement as it is based on fluorescent spectroscopy, which will be more feasible for its stronger robustness towards background interferences. The high signal-to-background ratio from the probes grand them with high sensitivity and excellent ability to detect diseases in advance.

[0131] Further embodiments of the invention may relate to the following embodiments.

[0132] In some embodiments of the present invention, there may be provided a compound of formula

[0133] I:

[0134] Wherein:

[0135] A represents NAG responsive moiety, with the following chemical structure:

[0136] B is a self-immolative linker group, n represents 0 or 1 , with the following chemical structure:

[0137] C represents a fluorophore, such as the activatable fluorescent substrates:

[0138]

[0139] X represents O, S or Se atoms; where the wavy line represents the point of attachment to the rest of the caging group.

[0140] Ri represents the H atom, methyl group (-CH3), trifluoromethyl group (-CF3) and methyl hydroxyl group (-CH2OH). When Ri represents the H atom, methyl group (-CH3), trifluoromethyl group (-CF3) and methyl hydroxyl group (-CH2OH), the fluorescent probes are shown as below:

[0141] R2 represents the electron-withdrawing moiety to redshift the fluorescence emission, e.g. those specifically shown in Table 1.

[0142] Table 1

[0143]

[0144] R3 represents the acceptor moiety to redshift the fluorescence emission, e.g. those specifically shown in Table 2.

[0145] Table 2 where the wavy line represents the point of attachment to the rest of the molecule.

[0146] The mentioned fluorescent substrates are also included in embodiments of the invention. FU represents an acceptor moiety to redshift the fluorescence emission to red region, e.g., those specifically shown in Table 3.

[0147] Table 3 where the wavy line represents the point of attachment to the rest of the molecule.

[0148] Rs represents the H atom, hydroxyl group (-OH), and halogen substituents (F / CI / Br / l). When

[0149] Rs represents H and methyl group (-OH), the fluorescent probes are shown as below:

[0150] When R5represents the halogen substituents (e.g. F, Cl, Br and I), the fluorescent probes are shown as below:

[0151]

[0152] The mentioned fluorescent substrates are also included in embodiments of the invention.

[0153] R6represents dimethylamine and modified amine groups, e.g., and those specifically shown in Table 4.

[0154] Table 4 where the wavy line represents the point of attachment to the rest of the molecule.

[0155] R? represents H atom, amine, and dimethylamine groups, separately. When R7 represents H atom, amine, and dimethylamine groups, the fluorescent probes are shown as below:

[0156] RB represents other moieties, and those specifically shown in Table 5.

[0157] Table 5

[0158]

[0159] The mentioned fluorescent substrates may also be included in embodiments of the present invention.

[0160] It will be appreciated that embodiments of the present invention may be combined in any technically sensible manner.

[0161] In another aspect of the invention, there is a method to detect the presence of M-Acetyl-p-D- glucosaminidase (NAG) in a subject, the method comprising the steps of providing a formula I, as defined herein and technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof to a subject, suspected of suffering from the presence of NAG to the irradiation with light and detecting fluorescence from the irradiated subject, wherein an increase in fluorescence signal compared to a control group indicates the presence of NAG.

[0162] In another aspect of the invention, there is provided a compound of formula I, as defined herein and any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof for use in the detection of NAG in a sample.

[0163] Compounds of formula I represents the activatable fluorescent probes (such as compounds P1 , P2, and P3 disclosed in the Example section), which can be specially hydrolyzed by the W-Acetyl-p-o-glucosaminidase (NAG), and demonstrate enhanced fluorescence signal. Advantageously, the fold enhancement or reduction generated by the activatable fluorescent probes could reflect directly on the activity and of NAG in various of biological systems, such as human or animal tissue fluids, urine, microorganisms, plants and other biological systems of liquid form.

[0164] In some embodiments that may be mentioned herein, the specific reaction conditions may be as follows:

[0165] The working concentrations of the activatable fluorescent probes are 50 pM. The system is PBS-based and the pH value may be in between of 5.5-10.5. The best working pH value is 7.4. The incubation temperature is 37 °C; a lower or a higher working temperature may have effects on the time needed for a complete hydrolysis thus the incubation temperature may be adjusted accordingly. The incubation time is 60 minutes. Activatable fluorescent probes (e.g. P1 -3) could be completely hydrolyzed within 60 minutes.

[0166] As demonstrated in the examples below, the detecting performance of the presently claimed activatable fluorescent probes against NAG in PBS have been tested with an interference, such as bovine serum albumin (BSA), which is a protein found predominantly in the circulatory system of cows and is widely used as a testing model to study the interactions between exogenous molecules and albumin due to its structural homology with human serum albumin. After the co-incubation of NAG and BSA, there are still 2-fold, 7-fold, and remarkable 28-fold signal differences from P1-3, respectively.

[0167] Further aspects and embodiments of the invention are described in the following numbered statements. 1 . In one aspect of the present invention, there is provided a compound of formula I: wherein A represents a NAG responsive moiety, with the following chemical structure:

[0168] Wherein B is optionally present and if present, is a self-immolative linker group, with the following chemical structure: wherein C represents a fluorophore; and n is independently 1 or 2.

[0169] In some embodiments of the present invention, C may represent a fluorophore, such as the activatable fluorescent substrates:

[0170] wherein

[0171] X represents O, S or Se atoms; the wavy line represents the point of attachment to the rest of the molecules;

[0172] R2 represents a carboxylic group or methyl acetate group.

[0173] In some embodiments of the present invention, when R3 may represent carboxylic group, or methyl acetate group, the fluorophore are shown as below:

[0174] In some embodiments of the present invention, R3 may represent an acceptor moiety to redshift the fluorescence emission, e.g. those specifically shown in Table 6.

[0175] Table 6 where the wavy line represents the point of attachment to the rest of the molecule.

[0176] The mentioned fluorescent substrates are also included in embodiments of the invention.

[0177] In some embodiments of the present invention, Ri may represent H atom, methyl group (- CH3), trifluoromethyl group (-CF3) or methyl hydroxyl group (-CH2OH).

[0178] In some embodiments of the present invention, when R1 may represent H atom, methyl group (-CH3), trifluoromethyl group (-CF3), or methyl hydroxyl group (-CH2OH), the fluorescent probes are shown as below:

[0179] In some embodiments of the present invention, R4may represent an acceptor moiety to redshift the fluorescence emission to red region, e.g., those specifically shown in Table 7.

[0180] Table 7

[0181] where the wavy line represents the point of attachment to the rest of the molecule.

[0182] In some embodiments of the present invention, when Rs may represent H atom, hydroxyl group (-OH), or halogen substituent (e.g. F, Cl, Br and I), the fluorescent probes are shown as below:

[0183] In some embodiments of the present invention, when R5may represent halogen substituent

[0184] (e.g. F, Cl, Br and I), the fluorescent probes are shown as below:

[0185] The mentioned fluorescent substrates are also included in embodiments of the invention.

[0186] In some embodiments of the present invention, R7may represent H atom, amine, and dimethylamine groups, separately. In some embodiments of the present invention, when R7may represent H atom, amine, or dimethylamine group, the fluorescent probes are shown as below:

[0187] In some embodiments of the present invention, Re may represent dimethylamine, or modified amine group, e.g., those specifically shown in Table 8.

[0188] Table 8

[0189] where the wavy line represents the point of attachment to the rest of the molecule.

[0190] The mentioned fluorescent substrates are also included in embodiments of the invention.

[0191] In some embodiments of the present invention, Rs may represent other moieties, and those specifically shown in Table 9:

[0192] Table 9

[0193]

[0194] 2. In another aspect of the invention, there is provided a method to detect NAG in a subject, the method comprising the steps of providing a compound of formula I, as defined above to a sample, or a pharmaceutically acceptable method like transdermal drug delivery to a subject, and detecting fluorescence signal from the samples or from the subject.

[0195] Some other advantages of the current invention over existing methods may include one or more of the following.

[0196] (i) The dual-modality allows for flexible application scenarios, where colorimetric changes support rapid, naked-eye detection without specialized equipment, ideal for point-of-care diagnostics or resource-limited settings.

[0197] (ii) Fluorescence imaging from the designed probes (P3) can provide high sensitivity and spatial resolution even in the presence of BSA.

[0198] Further aspects and embodiments of the invention will now be described by reference to the following non-limiting examples. Examples

[0199] Materials

[0200] All chemicals used in the experiments were purchased from Sigma-Aldrich unless otherwise described. Fetal bovine serum (FBS) was purchased from Gibco.

[0201] Statistics Analysis

[0202] All in vitro and in vivo data were expressed as the mean ± standard deviation unless otherwise stated. Statistical comparisons between the two groups were determined by Student's t-test (2-tailed, unpaired) and P** values less than 0.01 were considered as statistically significant meaning.

[0203] Characterisation Methods

[0204] Nuclear Magnetic Resonance (NMR) Measurements

[0205] 1H NMR spectra were determined with a Broker BBFO 400 MHz NMR.

[0206] Ultraviolet-Visible (UV-Vis) Absorption Measurements

[0207] UV-Vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer.

[0208] Fluorescence Spectra Measurements

[0209] Fluorescence measurements were carried out on a Fluorolog 3-TCSPC spectrofluorometer (Horiba Jobin Yvon).

[0210] High Performance Liquid Chromatography (HPLC) Analysis

[0211] High performance liquid chromatography (HPLC) analyses were performed on an Agilent 1260 system equipped with a G131 1 B pump, a UV detector and an Agilent Zorbax SB-C18 RP (9.4 x 250 mm) column, with methanol (0.1% of trifluoroacetic acid (TFA)) and H2O (0.1 % of TFA) as the eluent.

[0212] Firstly, the biological sample with prove (e.g. prove P3) was incubated at 37°C for 30-60 min. Upon completion of the reaction, a stop solution (e.g. glycine-NaOH buffer, pH 10.0) was added into the reaction. The solution was then filtered through a 0.22 pm membrane and 20- 50 pL of the resulting filtrate was injected to the HPLC for analysis. Detection: For UV-vis substrate: Absorbance at 650 nm for P3. To determine the activity, the peak area was compared against a standard curve. Fluorescence Images

[0213] ALSM800 confocal laser scanning microscope (Carl Zeiss, Germany) was used to capture the fluorescence images.

[0214] Fluorescence Probe Imaging

[0215] Fluorescence in vivo imaging of probes were measured by an IV IS spectrum imaging system.

[0216] Fluorescence Probe Imaging of NAG in Cells

[0217] Cell seeding was firstly performed by plating cells on C in 12- or 24-well plates and culture to ~70% confluence. Then, probes (e.g. probe P3, typically 1-10 pM in culture medium) were added and allowed to incubate for 30 minutes to 2 hours at 37°C. Upon completion, the plates were washed 2-3 times with PBS to remove excess probes. Fixation with 4% paraformaldehyde for 15 minutes were optionally performed when imaging fixed cells was desired. For the imaging, the fluorescence channel settings were adjusted depending on the probe (e.g. excitation and emission (Ex / Em) = 640 / 680 nm for red fluorophores). For image analysis, Imaged or other suitable software were used to quantify the fluorescence intensity.

[0218] Fluorescence Probe Imaging for NAG Detection (In-Vitro)

[0219] First step of fluorescence probe imaging for NAG detection in vitro involved incubating the sample or introducing the probe into urine or cells. This was followed by a circulation and activation period of 1 to 3 hours, depending on the probe’s kinetics. After sufficient activation, fluorescence was measured using either a plate reader with excitation and emission settings matched to the probe, or confocal microscopy for cellular or tissue-level visualization.

[0220] The activatable fluorescent probes (compounds P1 , P2, P3) were developed herein for optical urinalysis of NAG to detect kidney diseases at an early stage.

[0221] Example 1. Synthesis of Compound P1

[0222] Compound P1 is designed by caging the optically tunable atoms (oxygen or nitrogen) on the coumarin derivative and was afforded by following the protocol below.

[0223]

[0224] First Step: Synthesis of Compound 3 (3S,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-chlorotetrahydro-2H-pyran-3,4-diyl diacetate (0.15 mmol, compound 1) and coumarin derivative (0.1 mmol, compound 2) was dissolved in 2 mL dichloromethane (DCM) and 2 mL of 1 N NaOH (sodium hydroxide) was vigorously stirred at room temperature. After 4 h stirring, the reaction mixture was diluted with dichloromethane (DCM) and the organic layer was washed with 1 N NaOH. The organic layer was then dried with anhydrous MgSO4, filtered and concentrated under the reduce pressure. The crude product was purified by flash column chromatography to give compound 3 as white solid (80%).

[0225] Second Step: Synthesis of Compound P1 from Compound 3

[0226] Compound 3 (23 mg) was dissolved in 1 mL MeOH (methanol) and stirred in an ice-water bath. Then, MeONa (0.1 mmol) was added and the reaction was stirred at 0 °C for 30 min. 1 N HCI was added to neutralize the reaction mixture and the crude product was purified by HPLC using ACN (acetonitrile) / water or MeOH / water as an eluent to give the compound P1 as white solid (16 mg, 90%).

[0227] Results and Discussion

[0228] NMR analysis was performed to confirm that compound P1 was successfully afforded.

[0229] 1H NMR (400 MHz, DMSO-d6) of Compound P1 : 5 8.80 (s, 1 H), 7.91 - 7.81 (m, 2H), 7.04 (s, 1 H), 6.99 (d, J = 8.7 Hz, 1 H), 5.18 (d, J = 8.4 Hz, 1 H), 3.82 (s, 3H), 3.72 (t, J = 9.1 Hz, 4H), 3.21 (t, J = 8.9 Hz, 2H), 1 .81 (s, 3H). (DMSO: dimethyl sulfoxide)

[0230] Example 2. Synthesis of Compound P2 Compound P2 is designed by caging the optically tunable atoms (oxygen or nitrogen) on dicyanomethylene-4H-pyran (DCM)-based molecule and was afforded by following the protocol below.

[0231] First Step: Synthesis of Compound 6

[0232] The mixture of acceptor dicyanomethylene-4H-derivative (0.2 mmol, compound 4), piperidine (0.02 mmol), and (2R,3S,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-(4-formylphenoxy)- tetrahydro-2H-pyran-3,4-diyl diacetate (0.05 mmol, compound 5) was dissolved in anhydrous acetonitrile (ACN, 2.5 ml) and refluxed for 2-8 h under nitrogen (N2) atmosphere. After completing the reaction, the mixture was extracted with brine (2 x 20 ml) and dichloromethane (2 x 20 ml), and then the organic solution was collected and dried over anhydrous Na2SO4. The organic solvent was removed in vacuo to give the crude product. The crude product was further purified by HPLC using methanol and water as an eluent to afford corresponding products.

[0233] Second Step: Synthesis of Compound P2 from Compound 6

[0234] To a solution of compound 6 (0.05 mmol) in MeOH was added MeONa (6 eq.) and the mixture was stirred for 1-2 h at room temperature. The crude product was directly purified by HPLC using methanol and water as an eluent to afford P2 (50 %).

[0235] Results and Discussion

[0236] NMR analysis was performed to confirm that compound P2 was successfully afforded.1H NMR (400 MHz, DMSO) of Compound P2: 5 8.93 (d, J = 8.4 Hz, 1 H), 8.09 (d, J = 8.8 Hz, 1 H), 7.95 - 7.91 (m, 1 H), 7.86 (d, J = 8.8 Hz, 1 H), 7.78 (d, J = 8.4 Hz, 2H), 7.61 (t, J= 7.6 Hz, 1 H), 7.41 (s, 2H), 7.04 (d, J = 8.4 Hz, 2H), 7.02 (s, 1 H), 5.14 (dd, J = 13.2, 4.4 Hz, 2H), 5.06 (d, J= 8.0 Hz, 1 H), 4.66 (t, J = 5.2 Hz, 1 H), 4.56 (d, J = 6.8 Hz, 2H), 3.75 - 3.66 (m, 2H), 3.23 - 3.16 (m, 4H), 1 .82 (s, 3H), 1 .40 (t, J = 6.8 Hz, 3H).

[0237] Example 3. Synthesis of Compound P3

[0238] Compound P3 is designed by caging the optically tunable atoms (oxygen or nitrogen) on the methylene blue derivative (MB) and was afforded by following the protocol below.

[0239] First Step: Synthesis of Compound 8

[0240] (2R,3S,5R,6S)-5-acetamido-2-(acetoxymethyl)-6-(4-formylphenoxy)tetrahydro-2H-pyran-3,4- diyl diacetate (compound 4) (0.1 mmol), 4-dimethylaminopyridine (DMAP) (0.25 mmol), and 3 ml anhydrous DCM were added into round bottom flask (RBF), together with a magnetic stirring bar. The system was then transferred into an ice bath and stirred for 10 min. Leuco methylene blue (LMB, compound 7) (0.12 mmol) dissolved in 2 mL anhydrous DCM was then added into the system slowly and stirred for 5 min. After 5 min, the system was taken out of the ice bath, and stirred at room temperature for 2 h. After the reaction was completed, the system was extracted with brine and ethyl acetate for 3 times. The organic phases were collected and dried over anhydrous Na2SO4, and then concentrated for column chromatography. The crude product was directly used in next step without further purification.

[0241] Second Step: Synthesis of Compound P3 from Compound 3

[0242] To a solution of compound 8 (0.05 mmol) in MeOH was added MeONa (6 eq) and the mixture was stirred for 1-2 h at room temperature. The crude product was directly purified by HPLC using methanol and water as an eluent to afford corresponding products.

[0243] Results and Discussion

[0244] NMR analysis was performed to confirm that compound P3 was successfully afforded.

[0245] 1H NMR (400 MHz,MeOD) of Compound P3: 67.30 (d, J = 8.4 Hz, 1 H), 6 7.29 (d, J= 8.8 Hz, 1H), 6 7.28 (d, J = 8.8 Hz, 1 H), 6 7.26 (d, J = 8.4 Hz, 1 H), 6 7.03 (d, J = 8.8 Hz, 1 H), 5 7.01 (d, J= 8.8 Hz, 1H), 6 6.72 (d, J = 8.8 Hz. 1 H), 6 6.69 (d, J = 8.8 Hz, 1 H), 6 6.66 (s, 1 H) 66.64 (s, 1H), 6 5.13 (s, 2H), 6 5.06 (d, J = 8.8 Hz, 1H), 6 3.95-3.90 (m, 1H), 6 3.94 (t, J = 3.6 Hz, 1H), 63.72-3.70 (m, 1 H), 6 3.90-3.58 (m, 1H), 6 3.45 (d, J = 8.8 Hz, 1 H), 62.94 (s, 6H), 6 2.94 (s, 6H), 6 2.00 (s, 3H). (MeOD: deuterated methanol)

[0246] Example 4. High-Performance Liquid Chromatography (HPLC) Analyses of Activated NAG

[0247] To determine if the NAG enzymes were successfully activated upon addition of compounds P1 , P2, P3, HPLC analysis of the samples after mixing the compounds with the NAG enzyme were carried out.

[0248] Results and Discussions

[0249] After incubation with NAG enzyme for 1 hour, HPLC profile of compounds P1 , P2, P3 showed new elution peaks at 3.8, 16.4 and 26.8 min, respectively, indicating that the corresponding activated forms have been successfully generated (FIG. 1 ).

[0250] Example 5. Detection Sensitivity Investigation

[0251] To investigate the detection sensitivity of compounds P1 , P2, P3, the UV-vis absorption and fluorescence spectra were measured in the absence or presence of NAG. Detection of N-Acetyl-fi-D-glucosaminidase (NAG) Activity

[0252] Step 1: Preparation of Substrate Solution

[0253] The designed NAG probes (P1 , P2 or P3) was dissolved in 1 x PBS buffer (pH 7.4) to a final concentration of 100 pM. Note that the solution needed to be shielded from light exposure.

[0254] Step 2: Reaction Setup before Fluorescence Measurement

[0255] In each well of a black 96-well plate, 50 pL of enzyme or sample and 50 pL of designed NAG substrate solution were added accordingly. The samples were then mixed gently and allowed to incubate at 37°C for 60 minutes.

[0256] Step 3: Fluorescence Measurement

[0257] The fluorescence of the samples were measured using a microplate reader according to the corresponding excitation and emission of selected NAG probe.

[0258] Step 4: Quantification

[0259] A standard curve was prepared using serial dilutions of activated NAG probe in the final solution (e.g., 0-10 pM). Then, the enzyme activity was calculated based on fluorescence intensity relative to the standard curve.

[0260] Results and Discussions

[0261] After incubation of compound P1 with NAG, the absorption maxima shifted from 340 to 405 nm, and the fluorescent signals at 440 nm were enhanced by 40-fold (FIG. 2). Upon treatment of NAG with compound P2 (FIG. 3), its absorption maxima at 455 nm were reduced by 30 % and slightly blue-shifted to 450 nm. As a result, the fluorescent signal at 565 nm shrunken by 10-fold. As for compound P3 (FIG. 4), the characteristic absorption peak of MB at 665 nm disappeared due to the formation of leucomethylene blue (LMB) which has no n -system. After the incubation of NAG with compound P3, LMB was released after the cleavage of / V-Acetyl- P-D-glucosaminide and the leaving of the self-immolative linker. LMB can be easily oxidized back to MB by oxygen and the corresponding fluorescent singles at 685 nm show a remarkable 86-fold enhancement.

[0262] FIG. 5 shows the detection mechanisms of (a) compound P1 and (b) compound P3 towards W-Acetyl-p-o-glucosaminidase (NAG). After incubation of compound P1 with NAG, the absorption maxima shifted from 340 to 405 nm, and the fluorescent signals at 440 nm were enhanced by 40-fold. Example 6. Incubation with / V-Acethyl-p-o-Glucosaminidase (NAG) and Bovine Serum Albumin (BSA)

[0263] To demonstrate the feasibility of compounds P1 , P2, and P3 to detect NAG with interferences, compounds P1 , P2, and P3 were co-incubated with both NAG and BSA (FIG. 6).

[0264] Incubation and Detection of Activatable Fluorescent Probes with NAG and BSA

[0265] Step 1: Preparation of Substrate Solution

[0266] The designed NAG probes (P1 , P2 or P3) in 1 x PBS buffer (pH 7.4) to a final concentration of 100 pM. Note that the solution needed to be shielded from light exposure.

[0267] Step 2: Reaction Setup before Fluorescence Measurement

[0268] In each well of a black 96-well plate, 50 pL of enzyme or sample with BSA (20 mg / ml) and 50 pL of designed NAG substrate solution were added accordingly. The samples were then mixed gently and allowed to incubate at 37°C for 60 minutes.

[0269] Step 3: Fluorescence Measurement

[0270] The fluorescence of the samples were measured using a microplate reader according to the corresponding excitation and emission of selected NAG probe.

[0271] Step 4: Quantification

[0272] A standard curve was prepared using serial dilutions of activated NAG probe in the final solution (e.g., 0-10 pM) with BSA (20 mg / ml). Then the enzyme activity was calculated based on fluorescence intensity relative to the standard curve.

[0273] Results and Discussions

[0274] Bovine serum albumin (BSA), which is a protein found predominantly in the circulatory system of cows, is widely used as a testing model to study the interactions between exogenous molecules and albumin due to its structural homology with human serum albumin. More importantly, serum albumin in urine is a sign of kidney disease and a significant risk factor for complications.

[0275] For compound P1 , after the co-incubation, the absorption maxima shifted from 340 to 405 nm, and the fluorescent signals at 440nm were enhanced 2 times. There was a 60% reduction for the absorption maxima of compound P2 at 455 nm, and the fluorescent signals at 565 nm were reduced by 7-fold. As for compound P3, after the co-incubation of NAG and BSA, the absorbance at 665 nm recovered and the fluorescent signals at 685 nm enhanced by 28-fold.

[0276] Example 7. Detection of NAG in Urine Samples of Patients with chronic kidney disease (CKD)

[0277] A total of 38 patients with chronic kidney disease (CKD) were enrolled in the study, with a mean age of 58 years and 63% of the participants being male. Additionally, 12 healthy individuals were recruited as controls. Informed consent was obtained from all participants at the time of urine sample collection.

[0278] Detection of NAG in urine samples were performed by following the protocol disclosed in Example 6.

[0279] Results and Discussions

[0280] After the co-incubation of NAG and serum albumin, a widespread biological interference found in urine from patients with kidney disease, there are still 2-fold, 7-fold, and remarkable 28-fold signal differences from compounds P1-P3, respectively (FIG. 6b, 6d, and 6f). All the probes, especially compound P3, demonstrate promising potential for clinical translation.

Claims

Claims1. A compound of formula I:where:X is a bond or , where the dotted line represents the point of attachment to Y and the wavy line represents the point of attachment to the rest of the molecule;Y represents:where the wavy line represents the point of attachment to the rest of the molecule; where: the wavy line represents the point of attachment to the rest of the molecule;Z represents O, S or Se;Ri represents H, Me, CF3, CH2OH;where the wiggly line represents the point of attachment to the rest of the molecule;R3represents O or is selected from:where the wavy line represents the point of attachment to the rest of the molecule;FU represents:where the wavy line represents the point of attachment to the rest of the molecule;Rs represents H, Me, OH, or halo;Rs represents:where the wavy line represents the point of attachment to the rest of the molecule;R? represents H, NH2 or N(Me)2;where the wavy line represents the point of attachment to the rest of the molecule, or a pharmaceutically acceptable salt or solvate thereof.

2. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 1 , wherein when Y represents:where the wavy line represents the point of attachment to the rest of the molecule X is a bond;Ri represents CF3, CH2OH;R2represents H or is selected from:where the wiggly line represents the point of attachment to the rest of the molecule; andwhere the wavy line represents the point of attachment to the rest of the molecule.

3. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 2, whereinRi represents CF3;where the wiggly line represents the point of attachment to the rest of the molecule; andR3represents O or is selected from:where the wavy line represents the point of attachment to the rest of the molecule.

4. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 1 , wherein when Y represents:where the wavy line represents the point of attachment to the rest of the molecule, X is a bond;F represents:where the wavy line represents the point of attachment to the rest of the molecule; andRs represents Me, or OH.

5. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 4, whereinF represents:where the wavy line represents the point of attachment to the rest of the molecule; andR5represents Me, or OH.

6. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 1 , wherein when Y represents:where the wavy line represents the point of attachment to the rest of the molecule;Re represents:where the wavy line represents the point of attachment to the rest of the molecule;R? represents H, NH2or N(Me)2; andwhere the wavy line represents the point of attachment to the rest of the molecule.

7. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 6, whereinX is , where the dotted line represents the point of attachment to Y and the wavy line represents the point of attachment to the rest of the molecule;Rs represents:where the wavy line represents the point of attachment to the rest of the molecule;R? represents NH2or N(Me)2; andRs represents:where the wavy line represents the point of attachment to the rest of the molecule.

8. The compound of formula I or a pharmaceutically acceptable salt or solvate thereof, according to Claim 1 , wherein the compound of formula I is selected from one or more of the group consisting of:

9. A method of detecting the presence of M-Acetyl-|3-D-glucosaminidase (NAG) in a sample obtained from a subject suspected of having increased levels of NAG, the method comprising the steps of:(a) providing a formulation comprising a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof, and a sample obtained from a subject;(b) contacting the sample with the formulation for a period of time and detecting the presence or absence of a signal associated with NAG.

10. The method according to Claim 9, wherein the method is in vitro.

11. The method according to Claim 9 or Claim 10, wherein the signal detected is an fluorescence signal.

12. The method according to any one of Claims 9 to 11 , wherein the sample is selected from one or more of tissues fluids, urine, a microorganism culture, and a plant culture, optionally wherein the sample is a urine sample.

13. The method according to any one of Claims 9 to 12, wherein the method further comprises use of a control sample, where the control sample’s NAG signal is compared to the sample obtained from a subject and an increase in the detection signal indicates the presence of an increased amount of NAG.

14. The method according to any one of Claims 9 to 13, wherein the method provides a quantitative determination of the amount of NAG in the sample.

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