ES2847240B2 - HALOACETAMIDES DERIVED FROM ANTENNAS OF LANTANIDES AND THEIR APPLICATION AS LABELING REAGENTS OF THE LUMINESCENCE OF LANTANIDES - Google Patents

Description

DESCRIPTION

HALOACETAMIDES DERIVED FROM LANTANIDE ANTENNAS AND THEIR

APPLICATION AS LABELING REAGENTS OF THE LUMINESCENCE OF

LANTANIDES

The present invention refers to haloacetamides derived from quinolin-2(1H)-ones of formula (I), which sensitize the luminescence of lanthanides, and their application as reagents for marking the luminescence of lanthanides in various biomolecules, among others, in peptides and proteins.

BACKGROUND OF THE INVENTION

Understanding the structure and functions of living cells, organs and organisms is a constant challenge in modern biology and medicine, requiring the development of ingenious and innovative visualization and analytical techniques and appropriate biosensors. Among these, optical biosensors occupy a prominent place due to their speed of response, their sensitivity and the possibility of simultaneous detection of several analytes. Within this group, in recent years, luminescent biosensors have been among the most widely used, due to their high selectivity, sensitivity and versatility, and can even be used in in vivo applications with high spatial and temporal resolution (Morris, MC ACS Med Chem Lett 2014, 5, 99). This technique involves the excitation of biomolecules, by irradiation with UV/visible light, followed by relaxation to their ground state, by emitting much of the absorbed energy as light.

In recent years, sensors or probes based on lanthanide luminescence are emerging as powerful tools in the fields of Molecular Biology, Biochemistry and Biomedicine, due to the advantages that their use presents compared to organic fluorophores and fluorescent proteins. , among which the following stand out: a) Long luminescence lifetimes (ps-ms), which enable time-resolved fluorimetry, where a delay is applied between the excitation pulse and the detection window, eliminating interference by autofluorescence biological media and significantly increasing the signal-to-noise ratio and, therefore, the sensitivity; b) Very fine emission bands that do not overlap, allowing the use simultaneous use of different lanthanide probes to quantitatively detect different analytes without cross-interference (multichannel detection). c) Large Stokes shifts between excitation and emission wavelengths, avoiding concentration-dependent self-absorption problems (Heffern, MC et al. Chem. Rev. 2014, 114, 4496). The absorption and emission bands of the lanthanide cations correspond to electronic transitions between the 4f orbitals that are prohibited. Therefore, they have low absorbance coefficients, which makes their excitation difficult. This problem is avoided by taking advantage of the possibility of indirect excitation by energy transfer from organic chromophores with a high molar absorption coefficient. This process is known as sensitization or antenna effect and the chromophore is called "antenna". Heffern, MC et al. Chem. Rev. 2014, 114, 4496).

Despite the exceptional photophysical properties of lanthanide antennae, a very limited number of compounds of this type are currently available for the study of biological systems and their application is limited due to the lack of tools that allow the labeling of biomolecules. (eg peptides and proteins) or nanoparticles in an efficient and simple way (Sy, M. Chem. Commun. 2016, 52, 5080). Luminescent labeling of biological material with lanthanide antennas requires the introduction of a reactive functionality in the antenna for its covalent binding specifically to the material or target of interest, generally through nucleophilic amino or thiol groups (Sy, M. Chem Commun. 2016, 52, 5080; Hagan, AK Anal. Bioanal. Chem. 2011, 400, 2847). Isocyanates, N-hydroxysuccinimide esters or sulfonic acid chlorides have been used for labeling in amino groups, while maleimide, haloacetamide, dithiopyridine or methanesulfonate groups have been used for labeling in thiol groups (Sy, M. Chem. Commun. 2016, 52, 5080). These groups are attached through a spacer to the antenna. This functionalization requires the appropriate chemical design and manipulation of the antenna, which should not significantly affect the photophysical properties of the antenna. The use of these reagents generally requires the washing of the labeled sample to eliminate the excess of labeling reagent and avoid its interference in the photophysical analysis of the sample.

The present invention arose within a broad research project for the design and synthesis of new fluorophores with application in the development of fluorescence biosensors, in which a new family of lanthanide antennas was discovered.

Given the high interest of these antennas as tools for the development of various luminescent biosensors, haloacetamides derived from these antennas were prepared, which are the objective of the present invention, as well as their application for the luminescent labeling of biomolecules, such as peptides and proteins. The haloacetamide grouping is one of the most widely used reactive groups for the attachment of fluorescent probes to biomolecules, which is known as fluorescent labeling (Corrie, JET et al. J. Chem. Soc. Perkin Trans. 11994, 2975; Loving, G. et al Bioconjug Chem.

2009, 20, 2133; Ge, P. et al. Bioconjug. Chem. 2003, 14, 870).

DESCRIPTION OF THE INVENTION

The inventors have designed and synthesized haloacetamides derived from quinolin-2(1H)-ones of general formula (I) capable of sensitizing the luminescence of lanthanide cations and with application in the luminescent labeling of various biomolecules and, therefore, in the development of luminescent biosensors, as shown in the following scheme where R1, R2, R3 and X are defined below:

Among the advantages of the present invention, it should be noted that:

a) The biomolecules labeled with the compounds of formula (I) efficiently sensitize the luminescence emission of lanthanide cations, preferably that of Eu3+, which emits in the red, and that of Tb3+, which emits in the green.

b) Large Stokes shifts between the absorption wavelength of the antenna ( ~ 300 nm) and the emission wavelengths of the lanthanides, which avoid self-absorption problems.

c) Long luminescence lifetimes of lanthanides (ps-ms), which enable time-resolved fluorimetry, where a delay is applied between the excitation pulse and the detection window, eliminating interferences by media autofluorescence biological and significantly increasing the signal-to-noise ratio and, therefore, the sensitivity.

d) Very fine emission bands of the lanthanides that do not overlap, allowing the simultaneous use of probes of different lanthanides to quantitatively detect different analytes without cross interference (multichannel detection).

e) The compounds of formula (I) constitute luminescent marking tools for both in vitro and in cell studies.

Therefore, in a first aspect, the present invention relates to compounds of formula (I):

or any of its pharmaceutically acceptable salts or solvates, where

X is a halogen selected from Cl, Bryl;

R2 and R3 are independently selected from H, optionally substituted (Ci-C6)alkyl, or R2 and R3 form an alkanediyl bridge of the type -(CH2)n-, where n is selected from 2 to 3; and

R1 is selected from -C02H, -P03H2, -S03H, -S02NH2, -CONHR5, -PO(OH)NHR5, -PO(NHR5)2 and -S02NHR5, R5 being selected from alkyl(Ci-C6), structure (A) and structure (A'):

(A) (A')

where m is selected from 2, 3 and 4; and Ln3+ is a lanthanide, which is selected from La3+, Ce3+, Pr3+, Nd3+, Pm3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, and Lu3+.

In a preferred embodiment X is Br.

In another preferred embodiment R2 is H.

In another preferred embodiment R3 is methyl.

In another preferred embodiment R2 and R3 form a -CH2-CH2- bridge.

In another preferred embodiment R1 is -C02H.

In another preferred embodiment, R1 is -CONHR5, with R5 being a group of structure (A), where m is selected from between 2, 3 and 4; and more preferably m is 2.

In another preferred embodiment, R1 is -CONHR5, R5 being a group of structure (A'), where m is selected from 2, 3 and 4 and Ln3+ is selected from La3+, Ce3+, Eu3+, and Tb3+; more preferably m is 2 and Ln3+ is La3+, Eu3+, or Tb3+.

In another preferred embodiment, the compound of the invention has a structure with the general formula (la):

or any of its pharmaceutically acceptable salts or solvates, where

X is a halogen selected from Cl, Bryl;

R2 and R3 are independently selected from H, optionally substituted (Ci-C6)alkyl; and

R1 is selected from -C02H, -P03H2, -S03H, -S02NH2, -CONHR5, -PO(OH)NHR5, -PO(NHR5)2 and -S02NHR5, R5 being selected from alkyl(Ci-C6), structure (A) and structure (A'):

(A) (A')

where m is selected from 2, 3 and 4; and Ln3+ is a lanthanide, which is selected from La3+, Ce3+, Pr3+, Nd3+, Pm3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, and Lu3+.

In another preferred embodiment, the compound of the invention has a structure of general formula (Ib):

or any of its pharmaceutically acceptable salts or solvates, where

X is a halogen selected from Cl, Br and l; and

R1 is selected from -C02H, -P03H2, -S03H, -S02NH2, -CONHR5, -PO(OH)NHR5, -PO(NHR5)2 and -S02NHR5, R5 being selected from alkyl(Ci-C6), structure (A) and structure (A'):

(A) (A')

where m is selected from 2, 3 and 4; and Ln3+ is a lanthanide, which is selected from La3+, Ce3+, Pr3+, Nd3+, Pm3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, and Lu3+.

Likewise, it is to be understood that the present invention encompasses all isomers of the compounds of formula (I), that is, all geometric, tautomeric and optical forms, and their mixtures (eg racemic mixtures).

The term "tautomer" or "tautomeric form" as used herein refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) that include interconversions via migration of a proton, such as keto-enol or imine-enamine isomerizations. Valence tautomers include interconversions by rearrangement of some bonding electrons.

The present invention also includes isotope-labeled compounds, which are identical to those cited in formula (I) except that one or more atoms have been replaced by an atom having an atomic mass or mass number different from the atomic mass or number mass commonly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 18F, 123I, and 125I.

In this sense, pharmaceutically acceptable salts of said compounds containing the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labeled compounds of the present invention, eg, those into which radioactive isotopes such as 3 H or 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritium, ie, 3H, and carbon-14, ie, 14C, isotopes are particularly preferred for their ease of preparation and detectability. The 11C and 18F isotopes are particularly useful in PET (positron emission tomography), and the 125l isotopes are particularly useful in SPECT (single photon emission computed tomography), all useful in brain imaging. In addition, substitution with heavier isotopes such as deuterium, i.e. 2H, may provide some therapeutic advantages resulting from increased metabolic stability, for example longer in vivo half-life or lower dosage requirements, and thus in some cases may be preferred. Isotopically labeled compounds of formula (I) can generally be prepared by carrying out the procedures described in the following examples, substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

On the other hand, the salts mentioned above will be physiologically and pharmaceutically acceptable salts. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19. Thus, the term "pharmaceutically acceptable salts" refers to salts prepared from non-toxic pharmaceutically acceptable bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium salts, manganic, manganous, potassium, sodium, zinc salts and the like. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary, secondary, and tertiary amines, substituted amines including natural substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N '-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, resins polyamine, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When the compound of the present invention is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic acids. , sulfuric, tartaric, p-toluenesulfonic and the like.

Preferred examples of pharmaceutically acceptable salts include ammonium, calcium, magnesium, potassium and sodium salts, and those formed from maleic, fumaric, benzoic, ascorbic, pamoic, succinic, hydrochloric, sulfuric, bismethylene salicylic, methanesulfonic, ethanedisulfonic, propionic acids. , tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric.

Likewise, the compounds of formula (I) may be in crystalline form as free compounds or as solvates and both forms are within the scope of the present invention. Solvation methods are generally known in the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvates are hydrates or else the solvation molecules are solvent molecules, such as alcohols.

Another aspect of the invention refers to the use of the compounds of formula (I) as defined above as reagents for the luminescent labeling of biological samples containing peptides, proteins, and/or nanoparticles, useful in the development of luminescent biosensors.

In a preferred embodiment, the labeling of peptides is selected from peptide sequences of kinase substrates, specifically, from peptides of 15 amino acids derived from tau protein.

In a more preferred embodiment, the peptide sequence to be marked is selected from among those that have a single cysteine.

In a more preferred embodiment, the peptide luminescent labeling is selected from between labeling in solution or labeling in solid phase.

Another aspect of the invention relates to luminescent biosensors comprising at least one compound of formula (I) as defined above.

Another aspect of the invention relates to a method for luminescent labeling of isolated biological samples that comprises contacting the biological sample with at least one compound of formula (I) as defined above. When the compound of formula (I) does not incorporate the lanthanide in its structure, it must be added in situ through its corresponding salt.

In the present invention, the term "alkyl" refers to radicals of hydrocarbon chains, linear or branched, that have from 1 to 6 carbon atoms, preferably from 1 to 4, and that are joined to the rest of the molecule by means of a simple bond. , for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert-butyl, sec-butyl, n -pentyl, n-hexyl etc. Optionally, it can be substituted by at least one substituent, where the substituents are selected from OR', =0, SR', SOR', SO2R', NO2, NHR', N(R')2, =N-R', NHCOR', N(COR')2, NHSO2R', NR'C(=NR')NHR', CN, halogen, C(O)R', COOR', OC(O)R', CONHR', CON( R')2, substituted or unsubstituted (C1-C10) alkyl, substituted or unsubstituted (C2-C10) alkenyl, substituted or unsubstituted (C2-C10) alkynyl, substituted or unsubstituted (C6-Cis) aryl and heterocycle (Cs-Cis) substituted or unsubstituted, where each R' group is independently selected from H, OH, NO2, NH2, SH, CN, halogen, O, C(O)H, C(O)alkyl, COOH, alkyl (C1-C5) substituted or unsubstituted, (C2-C5) alkenyl substituted or unsubstituted, (C2-C5) alkynyl substituted or unsubstituted.

In the present invention the term "halogen", as understood in the present invention, includes fluorine, chlorine, bromine and iodine.

In the present invention, the term "lanthanide" refers to any of the chemical elements of period 6 of the periodic table between atomic numbers 57 and 71 (lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium , Dysprosium, Holmium, Erbium, Thulium, Ytterbium and Lutetium), preferably Europium or Terbium.

In the present invention the term "biological sample" refers to materials of both natural and synthetic origin that present biological activity, fundamentally comprising biomolecules, such as, for example, peptides and proteins, nanoparticles and cells.

In the present invention, the term "nanoparticle" refers to particles of various materials with a size of less than 100 nm.

In the present invention "marking in solution" is understood when the material to be marked is dissolved in a solvent, such as various aqueous buffers; and "solid phase marking" when the material to be marked is anchored to a solid polymeric resin.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. Other objects, advantages and features of the invention will be apparent to those skilled in the art in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Diagram of the luminescent labeling of peptides in solid phase with reagent (8).

FIG. 2: Graph of luminescence of labeled and unlabeled peptides with the fluorophore (8), in the presence and absence of Ln3+ cations.

FIG. 3. Diagram of the luminescent labeling of peptides in solid phase with reagent (13).

FIG. 4. Graph of luminescence of labeled and unlabeled peptides with the fluorophore (13), in the presence and absence of Ln3+ cations.

EXAMPLES

The invention is illustrated below by means of tests carried out by the inventors, which shows the effectiveness of the product of the invention.

Example 1: Synthesis of 7-(2-(2-bromoacetamido)ethoxy)-4-methyl-2-oxo-1.2-dihyvdroquinoline-3-carboxylic acid (8)

The title compound was synthesized from 2-amino-4-methoxy-acetophenone (1), as indicated in the following reaction scheme:

Synthesis of 2-amino-4-hydroxy-acetophenone (2)

A solution of 2-amino-4-methoxy-acetophenone (1) (320 mg, 1.93 mmol) and AICI3 in CH2CI2 (20 mL) was stirred at 50 °C for 2 h. It was then poured onto ice generating a precipitate which was filtered to obtain the title product (233 mg, 80%) as a white solid. Mp: 236 °C). HPLC-MS (gradient 15-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic acid solution in H2O, 10 min) ^ir = 5.21 min. 1H-NMR (400 MHz, CD3OD) 5: 2.46 (s, 3H), 6.18 (s, 1H), 6.19 (d, 1H, J = 8.5 Hz), 7.66 (d, 1H, J = 8.5 Hz).13C -NMR (100 MHz, CD3OD) 5: 27.4, 103.1, 107.4, 114.0, 135.8, 153.0, 164.5, 200.7. HRMS (ESI) m/z Caled, for C8H9N02 ([M+H]+): 152.0706, found: 152.0707.

Synthesis of methyl 3-((2-acetyl-5-hydroxyphenyl)amino)-3-oxopropanoate (3)

To a solution of acetophenone (2) (150 mg, 0.99 mmol) in CH2Cl2 (4 mL)/ACN (1 mL) was added 3-chloro-3-oxypropionate (106 mL, 0.99 mmol) and stirred for 6 h at room temperature. The solvent was then evaporated and the residue was recrystallized from MeOH to give the title product (220 mg, 90%) as a clear needle-like crystalline solid. Mp: 170.3 0C. HPLC-MS (30-95% gradient of 0.1% formic acid solution in acetonitrile in 0.1% formic solution in H20, 10 min) tR = 1.96 min. 1H-NMR (400 MHz, CI3CD) 5: 2.52 (s, 3H), 3.45 (s, 2H), 3.73 (s, 3H), 6.54 (dd, 1H, J = 9 and 2.5 Hz), 7.74 (d, 1H, J = 9 Hz), 8.05 (d, 1H, J = 2.5 Hz), 12.42 (s, 1H). 13C-NMR (100 MHz, CI3CD) 5: 28.2, 45.0, 52.7, 106.6, 111.2, 115.0, 134.5, 142.5, 163.5, 165.2, 167.8, 201.3. HRMS (ESI) m/z\ Caled, for C12H13NO5 ([M+H]+): 252.0867, found: 252.0874.

Synthesis of methyl 7-hydroxy-4-methyl-2-oxo-1.2-dihydroquinoline-3-carboxylate (4) To a solution of (3) (230 mg, 0.91 mmol) in MeOH (8 mL) was added K2CO3 ( 139 mg, 1.07 mmol) and heated at 700C by MW irradiation for 30 min. The reaction mixture was cooled and evaporated under reduced pressure. The reaction crude was then redissolved in CH2Cl2 (50 mL) and the resulting solution was washed with H2O (20 mL), brine (20 mL), dried over Na2SO4 and evaporated to dryness to obtain quinolin-2- One (4) (190 mg, 90%) as a yellow solid. Mp: 187°C. HPLC-MS (5-95% gradient of 0.1% formic acid solution in acetonitrile in 0.1% formic acid solution in H20, 10 min) tR = 5.87 min. 1H-NMR (400 MHz, DMSO-de) 5: 2.16 (s, 3H), 3.69 (s, 3H), 5.79 (s, 1H), 6.04 (dd, 1H, J=9 and 2.5 Hz), 7.13 ( d, 1H, J=9 Hz), 10.53 (s, 1H). 13C-NMR (100 MHz, DMSO-de) or: 15.6, 51.3, 100.4, 104.6, 119.7, 125.5, 142.5, 145.2, 159.8, 166.4, 168.5, 170.3. HRMS (ESI) m/z Caled, for C12H11NO4 ([M+H]+): 234.0761, found: 234.0775.

Synthesis of methyl 7-(2-((tert-butoxycarbonyl)amino)ethoxy)-4-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (5)

To a solution of the quinolin-2-one (4) (115 mg, 0.49 mmol) in dry DMF (5 mL) was added Na2C03 (76 mg, 0.73 mmol) and W-Boc-2-bromoethylamine (164 mg , 0.735mmol). The reaction mixture was stirred at 1200C in a sealed tube for 3 hours. It was then cooled and evaporated under reduced pressure. The reaction crude was then dissolved in CH2Cl2 (50 mL) and washed with H2O (20 mL), brine (20 mL), dried over Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography on silica gel, using a 0-1.5% gradient of MeOH in CH2CI2 as eluent to obtain the title compound (50 mg, 30%) as a brown solid. Mp: 1700C. HPLC-MS (30-95% gradient of 0.1% formic acid solution in acetonitrile in 0.1% formic solution in H20, 10 min) tR = 3.89 min. 1H-NMR (300 MHz, CD3OD) 5: 1.37 (s, 9H), 2.38 (s, 3H), 3.46 (q, 2H, J= 5.5 Hz), 3.87 (s, 3H), 4.01 (t, 2H, J= 5.5 Hz), 5.67 (t, 1H, J = 5.5 Hz), 6.70 (d, 1H, J = 2.5 Hz), 6.79 (dd, 1H, J = 9 and 2.5 Hz), 7.57 (d, 1H, J =9Hz).

13C-NMR (75 MHz, CD3OD) 5: 16.5, 28.4, 40.0, 52.7, 67.4, 80.0, 99.1, 112.9, 114.1, 123.1, 127.0, 139.9, 147.4, 156.8, 160.9, 167.7, 167.7. HRMS (ESI) m/z\ Caled, for C19H25N2O6 ([M+H]+): 377.1707, found: 377.1717.

Synthesis of methyl 7-(2-aminoethoxy)-4-methyl-2-oxo-1.2-dihydroquinoline-3-carboxylate (61

To a solution of the quinolin-2-one (5) (50 mg, 0.132 mmol) in CH2Cl2 (4 mL) with 0.5% MeOH and TFA (508 pl, 6.6 mmol) was added. The solution was stirred at room temperature for 2 hours. Subsequently, it was evaporated, dissolved in H2O (4 ml) and lyophilized, obtaining the compound of title (6) (52 mg, 100%) as a colorless syrup. 1H-NMR (500 MHz, DMSO-de) 5: 2.36 (s, 3H), 3.28 (m, 2H), 3.81 (s, 3H), 4.22 (t, 2H, J= 5.5), 6.85 (dd, 1H , J = 2.5 and 1 Hz), 6.92 (dd, 1H, J = 9 and 2.5 Hz), 7.77 (dd, 1H, J= 9 and 1 Hz), 8.04 (m, 2H), 11.95 (s, 1H). 13C-NMR (125 MHz, DMSO-de) or: 16.0, 38.2, 52.2, 65.7, 99.3, 110.9, 113.0, 123.8, 127.4, 139.8, 144.9, 159.0, 160.2, 166.8. HRMS (ESI) m/z\ Caled, for C14H17N2O4 ([M+H]+): 277.1183, found: 277.1181.

Synthesis of 7-(2-aminoethoxy)-4-methyl-2-oxo-1.2-dihydroquinoline-3-carboxylic acid (7) The methyl ester (6) (25 mg, 0.09 mmol) was dissolved in 1N NaOH solution HCl (3 mL) and the solution heated at reflux for 4 hours. The reaction mixture was then cooled and lyophilized. The residue was purified by semi-preparative reverse phase HPLC (Cis) to obtain the desired derivative (7) (7 mg, 30 %) as a white solid. Mp: 167 0C. HPLC-MS (gradient 5-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic acid solution in H2O, 10 min) tR = 7.64 min. 1H-NMR (500 MHz, D20) 5: 2.44 (s, 3H), 3.45 (t, 2H, J= 5.5 Hz), 4.11 (t, 2H, J= 5.5 Hz), 6.73 (d, 1H, J= 2.5 Hz), 6.92 (dd, 1H, J = 2.5 and 9 Hz), 7.69 (d, 1H, J = 9 Hz). 13C-NMR (125 MHz, D20) 5: 15.78, 40.57, 68.0, 99.1, 112.70, 114.8, 126.6, 128.6, 137.7, 143.3, 160.1, 161.4, 174.3.

Synthesis of 7-(2-(2-bromoacetamido)ethoxy)-4-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (8)

Dihydroquinoline (7) (7 mg, 0.025 mmol) was dissolved in dry DMF (3 mL) under Ar2 atmosphere. The solution stirred for 1 min and DIPEA (5 pl, 0.03 mmol) and bromoacetic bromide (2.6 pl, 0.03 mmol) were added and allowed to react for 30 min. The solvent was then evaporated and the residue was purified by reverse phase flash chromatography, using a gradient of 0-10% ACN in H20 as eluant, to obtain the desired compound (8) (5 mg, 53%) as syrup. colorless. HPLC-MS (gradient 5-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic solution in H20, 10 min) ir = 7.30 min. 1H-NMR (500 MHz, DMSO-cfe) 5: 2.63 (s, 3H), 3.51 (q, 2H, J= 5 Hz), 3.89 (s, 2H), 4.09 (m, 2H), 6.86 (d, 1H, J= 2.5 Hz), 6.95 (dd, 1H, J= 9 and 2.5 Hz), 7.87 (d, 1H, J= 9 Hz), 11.50 (s, 1H). 13 C-NMR (125 MHz, DMSO-de) 5: 16.3, 29.4, 38.7,66.5,99.0, 112.1, 113.9, 120.0, 128.0, 139.6, 149.7, 161.2, 161.5, 166.4, 166.9. HRMS(ESI): Caled, for Ci5Hi6BrN205 ([M+H]+): 383.0198, found: 383.0204.

Example 2: Synthesis of 8-(2-(2-bromoacetamido)ethoxy)-2-oxo-1.2.4.5-tetrahydrocyclopentarctelquinoline-3-carboxylic acid (13)

The title compound was synthesized from methyl 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-carboxylate (González-Vera, JA eí al. Chem. Commun. 2016 , 52, 9652), as indicated in the following reaction scheme:

Synthesis of 8-hydroxy-2-oxo-1,2,4,5-tetrahydrocyclopentafdelquinoline-3-carboxylate methyl (9)

To a solution of methyl 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[cfe]quinoline-3-carboxylate (50 mg, 0.19 mmol) in CH2Cl2 (5 mL) was added AICl3(50 mg, 0.38 mmol) and heated at 500C for 12 h. At the end of this time, the solvent was evaporated and H20 was added to the residue, generating a solid precipitate, which was filtered and dried under vacuum, to obtain the title compound (9) as a dark pink solid (32 mg, 82%). Mp: 2470C. HPLC-MS (gradient 5-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic acid solution in H2O, 10 min) ir = 6.94 min. 1H-NMR (DMSO-de, 400 MHz) 5: 3.10 (t, 2H, J= 5.5 Hz), 3.23-3.33 (m, 2H), 3.76 (s, 3H), 6.89 (d, 1H, J = 8 Hz), 7.00 (d, 1H, J= 8 Hz), 9.74 (s, 1H), 10.89 (s, 1H). 13 C-NMR (DMSO-de, 126 MHz) 5: 29.5, 31.8, 51.5, 116.4, 117.2, 119.5, 125.5, 125.8, 137.6, 140.7, 160.3, 164.1, 165.3. HRMS (ESI) m/z\ Caled, for C i3HnN04([M+H]+): 246.076, found: 246.077.

Synthesis of methyl 8-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-oxo- 1,2.4.5 -tetrahydrocyclopenta- del-quinoline-3-carboxylate (10)

To a solution of the carboxylate (9) (450 mg, 1.83 mmol) in anhydrous DMF (15 mL) was added 2-(Boc-amino)ethyl bromide (491 mg, 2.19 mmol) and K2CO3 (304 mg, 2.19 mmol) and the mixture was heated at 900C, in an Ar2 atmosphere for 24 h. The next day, the solvent was evaporated and the residue was purified by flash chromatography, using a gradient 0-4% MeOH in CH2Cl2 as eluent, obtaining the title compound (10) as a light brown solid (90 mg, 13% ). Mp: 169.5 0C HPLC-MS (30-95% gradient of 0.1 % formic acid solution in acetonitrile in 0.1% formic acid solution in H20, 10 min) ir = 4.31 min. 1H-NMR (DMSO-de, 400 MHz) 5: 1.40 (s, 9H), 3.17 (t, 2H, J=5.5 Hz), 3.32 - 3.37 (m, 2H), 3.38 - 3.43 (m, 2H), 3.79 (s, 3H), 3.98 (t, 2H, J=5 Hz), 7.01 (d, 1H, J=8 Hz), 7.15 (d, 1H, J=8Hz), 7.42 (t, 1H, J= 6Hz), 11.28 (s, 1H). 13C-NMR (DMSO-de, 100 MHz) 5: 28.2, 29.5, 31.9, 51.5, 68.3, 77.9, 115.7, 116.9, 117.0, 125.6, 126.6, 138.7, 142.1, 155.5, 160.16, 163.9, 163.9, 163.9 HRMS (ESI) m/z: Caled, for C2oH24N20e([M+H]+): 389.1707, found: 389.1729.

Synthesis of methyl 8-(2-aminoethoxy)-2-oxo-1,2,4,5-tetrahydrocyclopentafdelquinoline-3-carboxylate (11)

To a solution of compound (10) (90 mg, 0.31 mmol) in CH2Cl2 (4 mL) was added TFA (1.2 mL, 15.5 mmol) and the reaction stirred at room temperature for 12 h. After the reaction mixture was evaporated to dryness, the residue was dissolved in H20 HCl (3 mL) and the solution lyophilized to obtain the product of title (11) (90 mg, 100%) as a brown syrup. HPLC-MS (gradient 5-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic solution in H2O, 10 min) fe = 5.01 min. 1H-NMR (500 MHz, D20) 52.44 (s, 3H), 3.45 (t, J = 5.4 Hz, 2H), 4.11 (t, J = 5.4 Hz, 2H), 6.73 (d, J = 2.5 Hz, 1H ), 6.92 (d, 1H), 7.69 (d, 1H). 13C NMR (125 MHz, D20) 5 15.8, 40.6, 68.0, 99.1, 112.7, 114.7, 126.6, 128.6, 137.7, 143.3, 160.1, 164.2, 174.2.

Synthesis of 8-(2-aminoethoxy)-2-oxo-1.2.4.5-tetrahydrocyclopentaidelauinoline-3-carboxylic acid (12)

The methyl ester (11) was dissolved in 1N NaOH solution (3 mL) and the reaction mixture was refluxed for 3 h. The reaction mixture was then allowed to cool to room temperature and lyophilized. The residue was purified by reverse phase flash chromatography, using a gradient of 0-10% acetonitrile in H20 as eluent, obtaining the desired acid (12) as a brown syrup (37 mg, 60%). HPLC-MS (gradient 5-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic solution in H20, 10 min) fe = 4.55 min. 1H-NMR (400 MHz, DMSO-d6) 3.15 (t, 2H, J = 5.5 Hz), 3.51 (t, 2H, J = 5.5 Hz), 4.03 (t, 2H, J = 4.5 Hz), 6.92 (d , 1H, J = 7.5 Hz), 7.01 (d, 1H, J = 7.5 Hz). HRMS (ESI) m/z Caled, for Ci4Hi4N204 ([M+H]+): 275.1026, found: 275.1021.

Synthesis of 8-(2-(2-bromoacetamido)ethoxy)-2-oxo-1,2.4.5-tetrahydrocyclopentaidel-quinoline-3-carboxylic acid (13)

A solution of 2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline (12) (27 mg, 0.098 mmol) in dry DMF (3 ml), and under Ar2 atmosphere, was stirred for 1 minute and then DIPEA (17.17 pl, 0.098 mmol) and bromoacetic bromide (9.46 pl, 0.1078 mmol) were added and allowed to react for 1 hour at room temperature. At the end of this time, the solvent was evaporated and the residue was dissolved in CH2Cl2 (25 ml). The solution was washed successively with H20 (5 ml) and saturated NaCl solution (5 ml), dried over Na2S 04 and evaporated to dryness. The residue was purified by flash chromatography using a gradient of 0-4% MeOH in CH2Cl2 as eluant to give the title compound (13) (20 mg, 53%) as a colorless syrup. HPLC-MS (gradient 5-95% of 0.1% formic acid solution in acetonitrile in 0.1% formic solution in H20, 10 min) fe = 7.74 min.

Example 3: Luminescent labeling in peptide solution with the bromoacetamide (8)

The peptides used in this study were derived from the Tau protein (Wada, Y. et al. J. Biochem. 1998, 124, 738), which incorporate a single cysteine residue in position -2 with respect to a threonine, were synthesized by solid phase synthesis and after isolation and purification, they were dissolved in Hepes buffer at pH 7.4 and labeled on the cysteine residue by reaction with compound (8) in excess (5-10 times), for 16h at 4 0C . Subsequently, the excess antenna was removed by purification on NAP-5 columns (GE Healthcare; 100% yield by analytical HPLC-MS) and the resulting labeled peptides were coordinated with the corresponding lanthanide after in situ addition of the respective lanthanide salt ( TbCh, EuCh, Sc(OTf)3, Y(OTf)3 or La(OTf)3) in excess (5-100 times) at 250C in Hepes buffer at pH 7.4.

Example 4: Solid phase luminescent labeling of peptides with bromoacetamide (8)

As in example 3, the peptides used in this study were derived from the Tau protein (Wada, Y. et al. J. Biochem. 1998, 124, 738), incorporate a single cysteine residue and were synthesized in phase solid. As shown in simplified form in Figure 1, these resin-bound peptides (PAL-PEG, 50 mg, 0.0095 mmol, 1 equiv.), which incorporated the protected cysteine residue as Cys(Mmt), labile in medium acid, swollen with CH2Cl2 (5 min) and DMF (5 min). The Mmt protecting group was removed by treatment with 1% TFA and 5% triisopropylsilane (TIS) in CH2Cl2 under N2 atmosphere (4 x 20 min or until most of the yellow color due to the Mmt cation disappeared). The resin was then subjected to a stringent wash with CH2Cl2 (5x) and DMF (5x), DIEA (0.0475 mmol, 5 equiv.) in anhydrous DMF (200 pL) was added, and the resulting mixture was stirred for 2-3 min at 25°C. Compound (8) (17 mg, 0.0285 mmol, 3 equiv.) was then added to the resin in anhydrous DMF (150 pL) and, after 12 hours stirring at room temperature, the resin was filtered to remove the reagents. excess and washed with DMF, CH2Cl2, MeOH and CH2Cl2 (5 x each). Detachment of the peptide from the resin and removal of the protecting groups was carried out by treating the peptides bound to the resin with TFA/EDT/H2O/TIS (94:2.5:2.5:1% v/v). The resulting solution was concentrated under a stream of N2 and precipitated by addition of cold Et20. The pellet was triturated with cold Et20 (3x), redissolved in water, filtered and lyophilized. Peptides were purified by HPLC-MS semi-preparative reverse phase. The resulting labeled peptides were coordinated with the corresponding lanthanide after in situ addition of the respective salt (TbCh, EuCh, Sc(OTf)3, Y(OTf)3 or La(OTf)3) in excess (5-100 times). at 250C in Hepes buffer at pH 7.4. Coordination of the labeled peptide with (8) and TbCh resulted in a 2,069-fold increase in luminescence, while coordination with EuCh resulted in a 490-fold increase in luminescence (Figure 2).

Example 5: Luminescent labeling in peptide solution with bromoacetamide (13)

As in example 3, the peptides were dissolved in Hepes buffer at pH 7.4 and labeled at the cysteine residue by reaction with compound (13) in excess (5-10 times), for 16h at 40C. Subsequently, the excess antenna was removed by purification on NAP-5 columns (GE Healthcare; 100% yield by analytical HPLC-MS) and the resulting labeled peptides were coordinated with the corresponding lanthanide after in situ addition of the respective lanthanide salt ( TbCh, EuCh, Sc(OTf)3, Y(OTf)3 or La(OTf)3) in excess (5-100 times) at 250C in Hepes buffer at pH 7.4.

Example 6: Solid phase luminescent labeling of peptides with bromoacetamide (13)

As in example 4, the peptides used in this study were derived from the Tau protein (Wada, Y. et al. J. Biochem. 1998, 124, 738), incorporate a single cysteine residue and were synthesized in phase solid. As shown in simplified form in Figure 3, these resin-bound peptides (PAL-PEG, 50 mg, 0.0095 mmol, 1 equiv.), which incorporated the protected cysteine residue as Cys(Mmt), labile in medium acid, swollen with CH2Cl2 (5 min) and DMF (5 min). The Mmt protecting group was removed by treatment with 1% TFA, 5% triisopropylsilane (TIS) in CH2Cl2 under N2 atmosphere (4 x 20 min or until most of the yellow color due to the Mmt cation disappeared). The resin was then subjected to a stringent wash with CH2Cl2 (5x) and DMF (5x), DIEA (0.0475 mmol, 5 equiv.) in anhydrous DMF (200 pL) was added, and the resulting mixture was stirred for 2-3 min at 25°C. Compound (13) (17 mg, 0.0285 mmol, 3 equiv.) was then added to the resin in anhydrous DMF (150 pL) and after 12 hours stirring at room temperature, the resin was filtered to remove excess reagents. and washed with DMF, CH2Cl2, MeOH, CH2Cl2 (5 x each). The Detachment of the peptide from the resin and removal of the protecting groups was carried out by treating the peptides bound to the resin with TFA/EDT/H20/TIS (94:2.5:2.5:1% v/v). The resulting solution was concentrated under a stream of N2 and precipitated by addition of cold Et20. The pellet was triturated with cold Et20 (3x), redissolved in water, filtered and lyophilized. Peptides were purified by semi-preparative reverse phase HPLC-MS. The resulting labeled peptides were coordinated with the corresponding lanthanide after in situ addition of the respective salt (TbCh, EuCh, Sc(OTf)3, Y(OTf)3 or La(OTf)3) in excess (5-100 times). at 250C in Hepes buffer at pH 7.4. Coordination with TbCh resulted in a 1,097-fold increase in luminescence, while coordination with EuCh resulted in a 7,600-fold increase in luminescence (Figure 4).

Claims (19)

1. Compound of formula (I):

or any of its pharmaceutically acceptable salts or solvates, where: X is a halogen selected from Cl, Br and I; R2 and R3 are independently selected from H, optionally substituted (Ci-C6)alkyl, or R2 and R3 form an alkanediyl bridge of the type -(CH2)n-, where n is selected from 2 to 3; and R1 is selected from -C02H, -P03H2, -S03H, -S02NH2, -CONHR5, -PO(OH)NHR5, -PO(NHR5)2 and -S02NHR5, R5 being selected from alkyl(Ci-C6), structure (A) and structure (A'):

(A) (A') where m is selected from 2, 3 and 4; and Ln3+ is a lanthanide, which is selected from La3+, Ce3+, Pr3+, Nd3+, Pm3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, Yb3+, and Lu3+. 2. Compound according to claim 1, where X is Br. 3. Compound according to any of the preceding claims, wherein R2 is H. 4. Compound according to any of the preceding claims, wherein R3 is methyl. 5. Compound according to any of claims 1 to 2, wherein R2 and R3 form a -CH2-CH2- bridge. 6. Compound according to any of the preceding claims, wherein R1 is -C02H. 7. Compound according to any of claims 1 to 5, where R1 is -CONHR5, R5 being a group of structure (A), where m is selected from 2, 3 and 4. 8. Compound according to any of claims 1 to 5, where R1 is -CONHR5, R5 being a group of structure (A'), where m is selected from between 2,3 and 4 and Ln3+ is selected from La3+, Ce3+, Eu3+, and Tb3+. 9. Compound according to any of claims 7 to 8, where m is 2. 10. Compound according to any of claims 7 to 8, where Ln3+ is La3+, Eu3+, or Tb3+. 11. Compound according to claim 1, wherein the compound of formula (I) is: - 7-(2-(2-bromoacetamido)ethoxy)-4-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid; or - 8-(2-(2-bromoacetamido)ethoxy)-2-oxo-1,2,4,5-tetrahydrocyclopenta-[de]quinoline-3-carboxylic acid. 12. Use of the compounds of formula (I) according to claims 1 to 11 as reagents for luminescent labeling of biological samples. 13. Use according to the preceding claim where the biological samples contain peptides, proteins, and/or nanoparticles. 14. Use according to the preceding claim, where the peptide is selected from peptide sequences of kinase substrates and/or peptides of 15 amino acids derived from tau protein. 15. Use according to the preceding claim, where the peptide sequence is selected from among those that have a single cysteine. 16. Use according to any of claims 12 to 15, where the labeling is carried out in solution or in solid phase. 17. Luminescent biosensors comprising at least one compound of formula (I) according to any of claims 1 to 11. 18. Procedure for luminescent labeling of isolated biological samples comprising contacting said biological sample with at least one compound of formula (I) according to any of claims 1 to 11. 19. Labeling procedure according to the previous claim where the lanthanide is added in situ through its corresponding salt.