WO2012001191A1

WO2012001191A1 - Method for diagnosing farber's disease

Info

Publication number: WO2012001191A1
Application number: PCT/ES2011/070444
Authority: WO
Inventors: Gemma Fabrias Domingo; José Luis ABAD SAIZ; Josefina Casas Brugulat; Antonio Delgado Cirilo; Thierry Levade; Carmen Bedia
Original assignee: Consejo Superior De Investigaciones Científicas (Csic); Universidad De Barcelona; Institut National De La Santé Et De La Recherche Médicale, Inserm
Priority date: 2010-06-29
Filing date: 2011-06-20
Publication date: 2012-01-05
Also published as: ES2372391B1; ES2372391A1

Abstract

The invention relates to a family of compounds having a structural nucleus of amino alcohol derivatives and to the use thereof for the detection of Farber's disease, using a diagnosis method performed outside the human body, comprising the use of said compounds bound to a fluorophore, chromophore or luminophore as substrates of the enzyme acid ceramidase, the functionality of which is reduced in patients with this disease. In addition, the invention relates to a kit comprising at least one of the aforementioned compounds for use in diagnosing the disease from a biological sample isolated from a person.

Description

i

Diagnostic method of Farber's disease

The present invention relates to a family of compounds with a structural nucleus of amino alcohol derivatives and their use for the detection of Farber's disease by applying a diagnostic method outside the human body in which said compounds bound to a fluorophore are used, chromophore or luminoforo as substrates of the enzyme ceramidase acid, whose functionality is diminished in patients with said disease. Therefore the present invention is encompassed within the chemistry and more specifically of the chemistry applied to the diagnosis of diseases and the determination of enzymatic activities.

STATE OF THE PREVIOUS TECHNIQUE

Farber's disease, also called lipogranulomatosis, is a hereditary lipid storage disorder characterized by an accumulation of ceramides in lysosomes, due to a lack of enzymatic activity that degrades it, acid ceramidase. This disease has a triad of common symptoms: inflammation of the joints, subcutaneous nodules and hoarseness. Sometimes hepatosplenomegaly and malfunction of the nervous system may occur. It is a very serious disease in which patients usually die before two years of age (Levade T, Sandhoff K, Schulze H, Medin JA. Acid ceramidase deficiency: Farber lipogranulomatosis (2009) in D. Valle et al. (eds): Scriver's OMMBID (Online Metabolic and Molecular Bases of Inherited), New York, McGraw-Hill).

Currently, the diagnosis of Farber's disease is basically carried out by three types of methods: i) Determination of the acid ceramidase activity. This has been done mainly using A / -oleoyl or A / -lauroyl radiolabeled sphingosines on cell homogenates. These substrates are characterized by being very poorly soluble. and the tests must be carried out in complex mixtures of detergents. In addition, the separation and quantification of products suppose a long and tedious process: lipid extraction, migration by thin layer chromatography (TLC), reading of the plates in a radiochromatoscanner for the identification of the products and subsequent separation of the silica from the support and quantification by liquid scintillation. Another drawback of these tests is that these ceramides can be degraded by other non-lysosomal ceramidases, which are not deficient in Farber's disease.

Loading tests or load tests. These consist of the direct addition of radioactively labeled sphingolipids to the cell culture medium, for the subsequent analysis of their metabolism. The molecules used in these tests have been ceramide, cerebroside sulfate and sphingomyelin. The latter has proven to be the most suitable for this purpose since the other molecules are partially or almost completely metabolized by non-lysosomal enzymes, while sphingomyelin is primarily directed at the lysosome. Thus, in this compartment, sphingomyelin is degraded to ceramide and through the study of lipid profiles a potential accumulation of this lipid can be observed in cells of Farber patients. The analysis of these profiles requires the same separation and quantification process as the previous method, but this test has the advantage of needing less biological material. The drawbacks of these tests are that these radioactive molecules are not commercially available and that the molecules can be partially degraded by non-lysosomal enzymes.

Quantification of ceramide levels. These methods aim to measure the accumulation of ceramide that occurs in the lysosomes of the cells of Farber patients. Some authors have used ceramide derivatization, present in body fluids or tissues, to give rise to molecules that can be detected, separated and quantified by liquid chromatography (HPLC) or gas (GCL) coupled to mass detector (MS) . Another known method for measuring ceramide storage uses the enzyme diacylglycerol kinase (DAGk). This enzyme, in addition to phosphorylating diacylglycerol, has a great affinity for ceramide. Taking advantage of this, the ceramide present in the lipid extract is reacted with the DAGk in the presence of [ ³² Ρ] -γ-ΑΤΡ, to give rise to ceramide-1-phosphate. The organic phase of this reaction is migrated by TLC and the plate is exposed to a photographic film or directly displayed on a scanner that detects [ ³² P]. The ceramide-1-phosphate produced reflects the amount of ceramide accumulated in the lysosomes of the cells. The advantage of this method is that both radioactive ATP and enzyme are commercially available, but the assay is long and laborious.

Thus, existing methods for the diagnosis of Farber's disease mostly use radioactive molecules and have in common the complexity of lipid extraction and separation for analysis, either by CCF, HPLC or GLC. All these disadvantages make the diagnosis of Farber's disease reserved for a limited number of specialized laboratories.

DESCRIPTION OF THE INVENTION

The present invention describes a series of compounds and a method for determining whether an individual suffers from Farber's disease that is based on the enzymatic hydrolysis of these compounds that are a substrate for the acidic ceramidase enzyme, an enzyme involved in the dysfunctions that cause the symptoms of said disease

In a first aspect, the present invention relates to a compound of formula (I)

where

Ri is selected from C _r C ₂₄ alkyl, alkenyl , C ₂ -C ₂₄ alkynyl, C ₂ -C ₂₄ group or -R ₂ -Y ₃ wherein Y is selected from S, O, N, NH, S (O), S (0 ₂ ), C (O), NHC (O), C (0) NH, C (0) 0, OC (O),

R ₂ is selected from CrC _to alkyl, alkenyl C ₂ -C _a alkynyl , C ₂ -C _a,

R ₃ is selected from alkyl CICB, alkenyl C ₂ -Cb, alkynyl C ₂ -Cb, where a + b≤24, m is a value between 1 and 10,

X is a fluorophore, chromophore or luminophore,

with the proviso that when X is umbelliferone and m is 2, R ₁ is different from - (CH ₂ ) ₁₄ CH ₃ .

The term "alkyl" refers, in the present invention, to hydrocarbon, linear or branched chain radicals, having 1 to 24 carbon atoms, preferably 10 to 12, and which are attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, Apropyl, n-butyl, tere-butyl, sec-butyl, n-pentyl, n-hexyl, decyl, dodecyl etc. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxyl, carbonyl, cyano, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "alkenyl" refers to radicals of hydrocarbon chains, linear or branched, containing one or more double carbon-carbon bonds, for example, vinyl, 1-propyl, allyl, isoprenyl, 2-butenyl, 1, 3-butadienyl etc. Alkenyl radicals may be optionally substituted by one or more substituents such as halogen, hydroxy, azido, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

The term "alkynyl" refers to hydrocarbon chain radicals, linear or branched, which has one or more triple carbon-carbon bonds and is linked to the rest of the molecule by a single bond eg, ethynyl, 1-propyl, etc. . The alkynyl group may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxyl, cyano, carbonyl, acyl, alkoxycarbonyl, amino, nitro, mercapto and alkylthio.

"Fluorophore" refers to a small chemical group that is part of a larger molecule, which can be excited by light to emit fluorescence. In some embodiments, fluorophores produce fluorescence efficiently by excitation with light. of wavelength from about 200 nanometers to about 800 nanometers. The intensity and wavelength of the emitted radiation depend on the fluorophore and its chemical environment. Tluorophore can be selected, without limitation, from acridine, anthracene, allophycocyanin, BODIPY, harmful, coumarin, fluorescamine, tluorescein, FAM (carboxyfluorescein), HEX (hexachlorofluorescein), JOE (6-carboxy-4 ', 5'-dichloro -2 ', 7'-dimethoxy-fluorescein), Oregon Green, phycocyanin, phycoerythyrine, rhodamine, ROX (carboxy-X-rhodamine), TAMRA (carboxytetramethylrodamine), TET (tetrachloro-fluorescein), Texas red, tetramethylrodamine and xanthines.

"Luminophore" refers to an atom or chemical group that emits light that is not derived from the rise in temperature, and may be caused by chemical, biochemical or crystallographic changes. The luminophore can be selected, without limitation, from among rhodo !, rhodamine, resorufin or derivatives thereof.

"Chromophore" refers to a chemical group that absorbs excitation energy and emits it in a range of wavelengths that produces color. The chromophore can be selected, without being limited to, anthocyanins, carotenes, lycopenes, azo dyes, foichromes etc.

Preferably, m is a value between 2 and 4. More preferably m is 2.

Preferably, Ri is an alkyl - (CH ₂ ) _n CH ₃ , where n is a value between 1 and 24. More preferably n is 10.

Preferably X is umbelliferone. Umbeliferone or 7-hydroxycoumarin is an organic compound derived from coumarin by replacing its hydrogen atom in position 7 with a hydroxyl group, which has the following structure:

Another aspect of the present invention relates to a diagnostic method to determine if an individual suffers from Farber's disease comprising: a) obtaining an isolated biological sample from the individual,

b) addition of a compound of formula (I):

(I)

where

Ri is selected from C ₁ -C ₂₄ alkyl , C ₂ -C ₂₄ alkynyl, C ₂ -C _{4 February} or -R2-Y-R3 group, where Y is selected from S, O, N, NH, S ( O), S (0 ₂ ), C (O), NHC (O), C (0) NH, C (0) 0, OC (O), R ₂ , is selected from CC _a alkyl, C ₂ alkenyl - C, alkynyl , C ₂ -C _a, Re is selected from alkyl C Cb, alkenyl C ₂ -Cb, alkynyl C ₂ - Cb, where a + b <24,

m is a value between 1 and 10,

X is a fluorogenic, chromogenic or luminogenic group to the sample obtained in

(to),

c) incubation of the mixture obtained in (b)

d) oxidation of the mixture obtained in (c)

e) detection of the signal (fluorescence, color or luminescence) emitted in the mixture obtained in (d).

In a preferred embodiment, R-, is an alkyl - {CH ₂ ) _n CH ₃ , where n is a value between 1 and 24. In a more preferred embodiment, n is 10.

In another preferred embodiment, m is a value between 2 and 4. In a more preferred embodiment, m is 2.

In a preferred embodiment, X is umbelliferone.

In a preferred embodiment, step (c) of incubation can be stopped by adding methanol followed by an oxidation that can be performed by adding mixtures. known oxidants, such as, but not limited to, Nal0 ₄ dissolved in glycine / NaOH buffer solution of pH 10.6.

Preferably, the sample obtained in step (a) is a body fluid and more preferably, the fluid is blood. Leukocytes and lymphocytes can be extracted from the blood by known techniques and make the modifications required to use these extracts to perform the described method.

Preferably, the sample obtained in step (a) is epithelial tissue. Obtaining the tissue sample and the cells is performed by standard methods known to any person skilled in the art.

In a preferred embodiment, the incubation time in step (c) is between 0.5 and 5 hours.

In another aspect, the present invention relates to a method of obtaining useful data for the diagnosis of Farber's disease which comprises comparing the data obtained by the method as described above with a control sample.

The method of detection of Farber's disease described in the present invention is based on an enzymatic hydrolysis reaction on a compound of formula (I) as described above. These compounds are substrates of the enzyme ceramidase acid, which is mutated in individuals suffering from this disease, so that its functionality in cells is minimal or zero. In a first step of the method, a biological sample of the individual (blood, tissue, etc.) is obtained from which the appropriate cells have been extracted to determine the activity of ceramidase by the usual methods. This cell extract is contacted with the substrate of formula (I) and the mixture is incubated for a variable time of between 1 to 5 hours. In case the ceramidase enzyme is functional, hydrolysis of the N-acyl chain present in the compound of formula (I) will occur. This structural change causes, after an oxidation reaction, the fluorophore emits fluorescence, which can be measured by known methods, such as a suitable spectrophotometer. If the ceramidase is mutated and is not functional, the sample will not show fluorescence or it will be minimal. Based on this relationship between ceramidase activity and the fluorescence data obtained, a person skilled in the art can proceed to determine if the problem individual suffers from Farber's disease.

Preferably, it is also advisable to determine the ceramidase activity in at least one control sample which can be a cell extract of the same type of cells from a normal individual for the same experiment and on the same day. Thus, the probability that an individual suffers from Farber's disease increases when the ceramidase activity in the test sample is approximately 25% less than the ceramidase activity determined in the control sample.

The method described in the present invention has a number of advantages over the methods known at present:

■ The substrate used is not radioactive, therefore, no specific facilities or equipment or special authorizations are required.

■ The substrate used is soluble in aqueous solutions at the necessary concentrations, which does not require the use of detergents for solubilization.

■ The substrate used is stable under adequate storage conditions.

■ The substrate used is specific for acid ceramidase and, therefore, for the diagnosis of Farber's disease by not interfering with other amido hydrolases.

■ Detection is based on the measurement of fluorescence, color or luminescence through common and affordable instruments in laboratories.

• It is not necessary to separate the product from the substrate, thus avoiding tedious extraction and separation procedures (CCF, HPLC, GCL)

■ The use of halogenated organic solvents is avoided, which means that the method generates less polluting waste than the others described and implies a lower occupational risk

■ The assay is very sensitive, with a detection limit of 50 pmoles of product formed, and requires very little protein (10-20 μg) ■ The assay can be miniaturized and used for mass screening of samples and for performing multiple measurements simultaneously.

■ The assay can be applied to various types of biological samples, such as blood lymphocytes and skin fibroblasts

■ The test is rapid, requiring less than 4 hours from sample preparation to results.

In another aspect, the present invention relates to the use of a compound of formula (I):

(I)

where

Ri is selected from C1-C24 alkyl, C-2-C24 alkenyl, C-2-C24 alkynyl or a group -R2-Y- R3, where Y is selected from S, O, N, NH, S (O), S (O ₂ ), C (O), NHC (O), C (O) NH, C (O) O, OC (O), R ₂ , is selected from CrC _a alkyl, C ₂ -C alkenyl _to , C ₂ -C alkynyl _a , R ₃ is selected from CC alkyl, C-2-Cb alkenyl, C-2-Cb alkynyl, a + b <24,

m is a value between 1 and 10,

X is a fluorogenic, chromogenic or luminogenic group,

for the diagnosis of Farber's disease in an isolated biological sample of an individual.

Preferably, R is an alkyl - (CH2) _n CH ₃₎ where n is a value between 1 and 24. More preferably, n is 10.

Preferably, m is 2.

In a preferred embodiment, X is umbelliferone. In a final aspect, the present invention relates to a kit useful for determining if an individual suffers from Farber's disease from an isolated sample of said individual, which comprises the compound of formula (I) and the use of this kit for the diagnosis of Farber's disease in an isolated biological sample of an individual. Other components of the kit can be buffer solutions, preservatives, oxidants and, in general, components that contribute to the proper preservation of the reagents and that the base reaction of the test is carried out in the most efficient way possible.

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

DESCRIPTION OF THE FIGURES

Fig. 1. Shows the enzymatic activity on compounds 2-7, after 3 hours of incubation of the cells in culture (line FD1 AcCerl OX) with 40 μΜ of substrate. The results are representative of two independent experiments with triplicates. S refers to the substrate described in Bedia et al., Chembiochem. 2007, 8, 642-8.

Fig. 2. It shows the enzymatic activity on compounds 2-7, after 3 hours of incubation of the cells in culture (FD1 AcCerl OX line) with different concentrations of each substrate. The results are representative of two independent experiments with triplicates.

Flg. 3. It shows the effect of protein concentration on the hydrolysis of substrate 5, after 3 hours of incubation at 37 ° C in the presence of 20 μΜ of substrate and different amounts of cell homogenate supernatant (FD1 AcCerl OX line). The results are representative of two independent experiments Fig. 4. It shows the hydrolysis of the substrate 5 as a function of the incubation time, in the presence of 20 μΜ of substrate and 25 of protein of the cell homogenate (FD1 AcCerl Ox line). The data are representative of two independent experiments.

Fig. 5. It shows the hydrolysis of the substrate 5 as a function of the pH of the reaction mixture, in the cell homogenates of three cell lines with different degree of acid ceramidase activity: FD1 AcCerl OX (overexpress acid ceramidase), normal lymphoblasts and FD1 fibroblasts (Farber ). The tests were carried out at 37 ^e C for 3 hours in the presence of 20 μΜ of substrate and 10-25 μg of protein. For the incubation different buffer solutions without detergents were used: glycine-HCI buffer solution (pH 2.5 to 3), acetate buffer solution (pH 3.5 to 5.5), sodium phosphate buffer solution (pH 6 to 8.1) and glycine-NaOH buffer solution (pH 8.9 to 9.8). The results are representative of 4 independent experiments.

Fig. 6. Shows the kinetic analysis of substrate hydrolysis 5. Different substrate concentrations of 2.5 to 80 μΜ were incubated in the presence of 10-25 μg of cell homogenate protein (FD1 AcCerl Ox line), in the breast of a buffer solution of acetate pH 4.5 for 3 hours at 37 ^e C. The figure shows the representation of Lineweaver-Burk and the resulting kinetic parameters, which are the average of five independent experiments.

Fig. 7. Shows the measurement of the acid ceramidase activity in different lines of Farber fibroblasts and control fibroblasts. All measurements have been made with substrate 5 and a fixed amount of protein, under the standard conditions described. The results are the average of 3 independent experiments.

Fig. 8. Shows the measurement of lysosomal β-galactosidase activity in different lines of Farber fibroblasts and control fibroblasts. All measurements have been made with substrate 5 and a fixed amount of protein, under the standard conditions described. The results are the average of 3 independent experiments. Fig. 9. Shows the measurement of the acid ceramidase activity in different lines of Farber lymphoblasts and control lymphoblasts. All measurements have been made with substrate 5 and a fixed amount of protein, under the standard conditions described. The results are the average of 3 independent experiments.

Fig. 10. Shows the measurement of lysosomal β-galactosidase activity in different lines of Farber lymphoblasts and control lymphoblasts. All measurements have been made with a fixed amount of protein, under the standard conditions described. The results are the average of 3 independent experiments.

EXAMPLES

The invention will now be illustrated by examples and tests carried out by the inventors, without being limiting, which show the specificity and effectiveness of the method of the present invention.

A. Preparation of the compounds of formula (I).

General methodology: To a solution of coumarinic amine diol 1 (56 mg, 0.2 mmol), obtained as described in the literature [Bedia et al., Chembiochem. 2007, 8, 642-8.], And NEt ₃ (60 pL / 0.4 mmol), in 4 mL of CH ₂ CI ₂ under argon atmosphere at room temperature, a solution of the corresponding activated acid in 4 mL of CH is added ₂ CI ₂ . Said activated acid is prepared previously by dissolving in CH ₂ CI ₂ ECD / HOBt / acid in proportions 0.32 mmol: 0.25 mmol: 0.25 mmol). The reaction is monitored by thin layer chromatography eluting with CH ₂ Cl ₂ / MeOH 9: 1), observing after 1 h the total disappearance of the starting product. The reaction mixture is washed with 2 mL of a saturated aqueous solution of NaHC0 ₃ , the CH ₂ CI ₂ is evaporated and the resulting crude is purified by flash column chromatography using a gradient of 0 to 10% MeOH in CH ₂ CI ₂ to obtain the products in the form of white solids (yields 74-80%). An outline of this procedure is shown below:

(1'S, 2 '¾A ^ [1-hydroxymethyl-2-hydroxy-4- (2-oxo-2H-chromen-7-yioxy) butyl] buta

(compound 2, n = 2).

¹ H NMR (400 MHz; CDCI ₃ ) δ 7.62 (d, 1H, J = 9.5 Hz), 7.35 (d, 1H, J = 7.5 Hz), 6.85-6.82 (m, 2H), 6.48 (d, 1H, J = 8 Hz), 6.24 (d, 1H, J = 9.5 Hz), 4.34-4.16 (m, 2H), 4.06 (dd, 2H, J, = 3.3 and Js = 8.0 Hz), 3.92 (m, 1H) , 3.86-3.72 (m, 2H), 3.18 {brs, 1H), 2.22 (t, 2H, J = 7.0 Hz), 2.04 (m, 2H), 1.68 (sext, J = 7.5, 2H), 0.95 (t , 3H, J = 7.5 Hz)

¹³ C NMR (75 MHz; CDCI ₃ ) δ 174.2, 162.2, 161.7, 155.7, 143.9, 128.9, 113.1, 112.9, 112.6, 101.5, 70.0, 65.8, 62.2, 54.7, 38.7, 33.5, 19.3, 13.8.

(1'S, 2 '¾W- [1-hydroxymethyl-2-hydroxy-4- (2-oxo-2H-chromen-7-yioxy) butyl] octanoylamide (compound 3, n = 6).

¹ H NMR (400 MHz; CDCI ₃ ) δ 7.63 (d, 1H, J = 9.5 Hz), 7.34 (d, 1H, J = 7.5 Hz), 6.85-6.76 (m, 2H), 6.53 (d, 1H, J = 8 Hz), 6.22 (d, 1H, J = 9.5 Hz), 4.30-4.14 (m, 2H), 4.06 (brd, 2H, J = 9.6 Hz), 3.93 (m, 2H), 3.84-3.76 ( m, 1H), 3.41 (brs, 1H), 2.24 (t, 2H, J = 7.0 Hz), 2.04 (m, 2H), 1.68 (quint, J = 7.5, 2H), 1.38-1.16 (br, 8H) , 0.86 (t, 3H, J = 7.5 Hz)

¹³ C NMR (75 MHz; CDCI ₃ ) δ 174.4, 162.2, 161.7, 155.7, 143.9, 128.9, 113.1, 112.8, 112.6,

101.4, 69.9, 65.7, 62.2, 54.7, 36.9, 33.5, 31.7, 29.3, 29.1, 25.9, 22.7, 14.2.

(1'S, 2'f Af- [1-hydroxymethyl-2-hydroxy-4- (2-oxo-2H-chromen-7-yloxy) butH] decanoylamide (compound 4, n = 8).

¹ H NMR (400 MHz; CDCI ₃ ) δ 7.64 (d, 1H, J = 9.0 Hz), 7.37 (d, 1H, J = 8.0 Hz), 6.84-6.82 (m, 1H), 6.83 (s, 1H) , 6.36 (d, 1H, J = 8 Hz), 6.25 (d, 1H, J = 9.5 Hz), 4.32-4.14 (m, 2H), 4.06 (brd, 2H, J = 9.6 Hz), 3.92 (sext, 1H, J = 3.5 Hz), 3.82 (brd, 1H), 3.43 (brd, 1H, J = 5.5 Hz), 2.75 (brs, 1H), 2.25 (t, 2H, J = 8.0 Hz), 2.05 (m, 2H), 1.70-1.52 (4H), 1.38-1.16 (br, 12H), 0.87 (t, 3H, J = 7.0 Hz)

¹³ C NMR (75 MHz; CDCI ₃ ) δ 174.4, 162.2, 161.7, 155.7, 143.8, 128.9, 113.1, 112.9, 112.6,

101.5, 70.0, 65.8, 62.3, 54.7, 36.9, 33.5, 31.9, 29.6, 29.5, 29.4, 29.4, 25.9, 22.8, 14.2. (1'S, 2 '?) / \ ^ [1-hydrox »methyl-2-hydrox» -4- (2-oxo-2H-chromen-7-yloxy) butyl] dodecanoylamide (compound 5, n = 10).

H NMR (400 MHz; CDCI ₃ ) δ 7.63 (d, 1H, J = 9.0 Hz), 7.36 (d, 1H, J = 8.0 Hz), 6.84-6.82 (m, 1H), 6.82 (s, 1H), 6.41 (d, 1H, J = 8 Hz), 6.24 (d, 1H, J = 9.0 Hz), 4.32-4.14 (m, 2H), 4.07 (m, 2H), 3.92 (sext, 1H, J = 3.5 Hz ), 3.82 (dd, 1H, J, = 11.5 Hz, J¿ = 3.5 Hz), 2.24 (t, 2H, J = 8.0 Hz), 2.05 (m, 2H), 1.64 (quint, 2H, J = 7 Hz ), 1.38-1.18 (br, 16H), 0.87 (t, 3H, J = 7.0 Hz) ¹³ C NMR (75 MHz; CDCI ₃ ) δ 174.4, 162.2, 161.7, 155.7, 143.8, 128.9, 113.1, 112.9, 112.6 , 101.5, 70.0, 65.8, 62.2, 54.7, 36.9, 33.5, 32.0, 29.7, 29.7, 29.6, 29.5, 29.4, 25.9, 22.8, 14.2.

(1'S, 2 '/?) / \ Í- [1-hydrox «methyl-2-hydrox« -4- (2-oxo-2H-chromen-7-yloxy)

butyljtetradecanoylamide (compound 6, n = 12).

¹ H NMR (400 MHz; CDCI ₃ ) δ 7.63 (d, 1H, J = 9.0 Hz), 7.37 (d, 1H, J = 8.0 Hz), 6.84-6.82 (m, 1H), 6.83 (s, 1H) , 6.34 (d, 1H, J = 8 Hz), 6.26 (d, 1H, J = 9.0 Hz), 4.32-4.14 (m, 2H), 4.07 (m, 2H), 3.92 (sext, 1H, J = 3.5 Hz), 3.82 (m, 1H), 3.40 (t, 1H, 6.0 Hz), 2.70 (brs, 1H), 2.24 (t, 2H, J = 7.5 Hz), 2.06 (m, 2H), 1.65 (quint, 2H, J = 7 Hz), 1.38-1.18 (br, 20H), 0.88 (t, 3H, J = 7.0 Hz)

¹³ C NMR (75 MHz; CDCI ₃ ) δ 174.3, 162.2, 161.7, 155.7, 143.8, 128.9, 113.1, 112.9, 112.6, 101.5, 70.1, 65.8, 62.3, 54.7, 36.9, 33.5, 32.0, 29.8, 29.7, 29.6 , 29.5, 29.5, 29.4, 25.9, 22.8, 14.2.

(1'S, 2'fí) / V- [1-hydroxymethyl-2-hydroxy-4- (2-oxo-2H-chromen-7- «loxi)

butyl] tetracosanoylamide (compound 7, n = 22).

¹ H NMR (400 MHz; CDCI ₃ ) δ 7.63 (d, 1H, J = 9.6 Hz), 7.37 (d, 1H, J = 8.0 Hz), 6.84-6.82 (m, 1H), 6.83 (s, 1H) , 6.34 (d, 1H, J = 7.2 Hz), 6.26 (d, 1H, J = 9.6 Hz), 4.34-4.15 (m, 2H), 4.07 (m, 2H), 3.92 (sext, 1H, J = 3.5 Hz), 3.81 (m, 1H), 3.41 (d, 1H, J = 6 Hz), 2.81 (m, 1H), 2.73 (m, 1H), 2.25 (t, 2H, J = 7.5 Hz), 2.06 ( m, 2H), 1.65 (quint, 2H, J = 7 Hz), 1.38-1.18 (br, 40H), 0.88 (t, 3H, J = 7.0 Hz)

¹³ C NMR (75 MHz; DMSO) δ 172.2, 161.9, 160.1, 155.3, 144.1, 129.3, 112.5, 112.2, 112.1, 101.1, 67.0, 65.6, 60.6, 55.0, 35.4, 32.8, 31.1, 29.8, 29.8, 29.6, 29.5, 25.2, 21.9, 13.7. B. Biological tests.

Measurement of acid ceramidase activity

This activity can be measured in vitro with cell homogenates and on intact living cells.

For the measurement of the acidic ceramidase activity in vitro, the cells are collected and washed twice with PBS. 100 μΙ of a 0.2 M aqueous sucrose solution is added to the cell precipitates and sonic. The cell homogenates are centrifuged for 3 minutes at 15000 g and the supernatants are stored. These are used to quantify the proteins in each sample, and after calculations, the acid ceramidase activity assay is performed at equal amounts of protein. The test is carried out in 96-well plates. Each well contains 74 μΙ of sodium acetate / acetic acid buffer solution, 0.5 μΙ of the 4 mM acid ceramidase substrate solution in ethanol (final substrate concentration 20 μΜ; final ethanol concentration 0.5%) , and a fixed amount of protein (between 10 and 25 g) in a volume of 25 μΙ of 0.2 M sucrose. The plate is incubated at 37 ⁹ C for 3 hours without stirring. After this time, the reaction is stopped by adding 50 μΙ of methanol in each well and then 100 μΙ of a 2.5 mg / ml Nal0 ₄ solution in a glycine / NaOH buffer solution of pH 10.6. The plate is kept at 37 ^e C protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm).

The cell lines used to characterize the substrate are FD1 (Farber cell line with zero acidic ceramidase activity) and FD1 AcCerl OX (Farber line corrected to overexpress the acidic ceramidase). The other Farber cell lines and control lines, whether fibroblasts such as lymphoblasts (Epstein-Barr virus-transformed lymphoid cell lines), come from the Laboratoire de Biochímie Métabolíque, Instituí Fédératif de Biologíe, CHU Purpan, Toulouse, France. For the measurement of the acidic ceramidase activity in cultured cells, the cells are seeded at a density of 200,000 cells / ml (0.1 ml / well). After 24 h, the substrate dissolved in ethanol or in DMSO (0.1 μΙ, 40 mM) is added to have a final concentration of 40 μΜ. Incubate at 37 ^e C for 3 h in the CO2 incubator and then 25 μΙ of MeOH and 100 μΙ of a 2.5 mg / ml Nal0 ₄ solution in pH 10 glycine / NaOH buffer solution are added sequentially. 6. The plate is kept at 37 ^S C protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm). The white samples are prepared by adding the substrate immediately before the addition of MeOH and NalO4.

Hydrolysis of the compounds as a function of the group / V-acyl.

These experiments can be performed on cells in culture. For this, the cells are seeded at a density of 200,000 cells / ml (0.1 ml / well). After 24 h, the substrates dissolved in ethanol or in DMSO (0.1 μΙ, 40 mM) are added. The experiment can be performed at a final fixed concentration of substrate (for example, 40 μΜ) or variable, between 1 and 100 μΜ. Incubate at 37 ⁹ C for 3 h in the CO2 incubator and then 25 μΙ of MeOH and 100 μΙ of a 2.5 mg / ml NalO ₄ solution in glycine / NaOH buffer solution of pH 10.6 are added sequentially . The plate is kept at 37 ° C protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm). The white samples are prepared by adding the substrate immediately before the addition of MeOH and NalO ₄ .

Substrate hydrolysis based on protein concentration.

To determine the effect of protein concentration on substrate hydrolysis, cells in culture of the FD1 AcCerl OX line are collected and washed twice with PBS. 100 μΙ of a 0.2 M aqueous sucrose solution is added to the cell precipitates and sonic. The cell homogenates are centrifuged for 3 minutes at 15000 g and the supernatants are stored. These are used to quantify the proteins of each sample, and the acid ceramidase activity assay is performed at different amounts of cell homogenate supernatant. The test is carried out in 96-well plates. Each well contains 74 μΙ of sodium acetate / acetic acid buffer solution, 0.5 μΙ of the 4 mM acid ceramidase substrate solution in ethanol (final substrate concentration 20 μΜ; final ethanol concentration 0.5%), and different amounts of protein between 2.5 and 180 Mg in a volume of 25 μΙ of 0.2 M sucrose. The plate is maintained at 37 ^S C for 3 hours without stirring. After this time, the reaction is stopped by adding 50 μΙ of methanol and 100 μΙ of a solution of 2.5 mg / ml of Nal0 ₄ in glycine / NaOH buffer solution of pH 10.6 in each well. The plate is protected from light for two hours and subsequently read on a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm).

Substrate hydrolysis based on incubation time

To determine the hydrolysis of the substrate based on the incubation time, the cells in culture are collected and washed twice with PBS. 100 μΙ of a 0.2 M aqueous sucrose solution are added to the cell precipitates and sonicated. The cell homogenates are centrifuged for 3 minutes at 15000 g and we store the supernatants. These are used to quantify the proteins in each sample, and after calculations, the acid ceramidase activity assay is performed at equal amounts of protein. The incubation mixture of the assay is carried out in 96-well plates. Each well contains 74 μΙ of sodium acetate / acetic acid buffer solution, 0.5 μΙ of the 4 mM acid ceramidase substrate solution in ethanol (final substrate concentration 20 μΜ; final ethanol concentration 0.5%), and 25 g of protein in a volume of 25 μΙ sucrose 0.2M plate is incubated at 37 C for different ^Q timepos without agitation. After this time, the reaction is stopped by adding 50 μΙ of methanol and 100 μΙ of a solution of 2.5 mg / ml of Nal0 ₄ in glycine / NaOH buffer solution of pH 10.6 in each well. The plate is protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm). Substrate hydrolysis based on pH.

To determine the hydrolysis of the substrate based on the pH of the reaction buffer solution, three cell lines with different degrees of acid ceramidase activity are used: FD1 AcCerl OX (overexpress acid ceramidase), normal lymphoblasts and FD1 fibroblasts (Farber). Cultured cells are collected and washed twice with PBS. 100 μΙ of a 0.2 M aqueous sucrose solution is added to the cell precipitates and sonic. The cell homogenates are centrifuged for 3 minutes at 15000 g and we store the supernatants. These are used to quantify the proteins in each sample, and after calculations, the acid ceramidase activity assay is performed at equal amounts of protein. The incubation mixture of the assay is carried out in 96-well plates. Each well contains 74 μΙ of different buffer solutions without detergents: glycine-HCI buffer solution (pH 2.5 to 3), acetate buffer solution (pH 3.5 to 5.5), sodium phosphate buffer solution (pH 6 at 8.1) and glycine-NaOH buffer solution {pH 8.9 to 9.8), 0.5 μΙ of the 4 mM acid ceramidase substrate solution in ethanol (final substrate concentration 20 μΜ; final concentration of 0.5% ethanol), and a fixed amount of protein (between 10 and 25 μg) in a volume of 25 μΙ of 0.2 M sucrose. The plate is incubated at 37 ^S C for 3 hours without stirring. After this time, the reaction is stopped by adding 50 μΙ of methanol and 100 μΙ of a solution of 2.5 mg / ml of Nal0 ₄ in glycine / NaOH buffer solution of pH 10.6 in each well. The plate is protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm).

Kinetic analysis of substrate hydrolysis.

To perform the kinetic analysis of the substrate hydrolysis, the cells in culture (FD1 AcCeM Ox line) are collected and washed twice with PBS. 100 μΙ of a 0.2 M aqueous sucrose solution is added to the cell precipitates and sonic. The cell homogenates are centrifuged for 3 minutes at 15000 g and the supernatants are stored. These are used to quantify the proteins in each sample, and after calculations, the acid ceramidase activity test is performed at equal amounts of protein. The test is carried out in 96-well plates. Each well contains 74 μΙ of sodium acetate / acetic acid buffer solution, 0.5 μΙ of the acid ceramidase substrate solution at different concentrations in ethanol (final substrate concentration of 2.5 to 80 μΜ); final concentration of ethanol 0.5%), and a fixed amount of protein (between 10 and 25 μς) in a volume of 25 μΙ of 0.2 M sucrose. The plate is incubated at 37 ^e C for 3 hours without stirring. After this time, the reaction is stopped by adding 50 μΙ of methanol and 100 μΙ of a solution of 2.5 mg / ml of Nal0 ₄ in glycine / NaOH buffer solution of pH 10.6 in each well. The plate is protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm).

Measurement of acid ceramidase activity and lysosomal 3-qalactosidase activity in different Farber cell lines and control cells.

Cultured cells are collected and washed twice with PBS. 100 μΙ of a 0.2 M aqueous sucrose solution is added to the cell precipitates and sonic. The cell homogenates are centrifuged for 3 minutes at 5000 g and the supernatants are stored. These are used to quantify the proteins in each sample, and after calculations, the acid ceramidase activity assay is performed at equal amounts of protein. The test is carried out in 96-well plates. Each well contains 74 μΙ of sodium acetate / acetic acid buffer solution, 0.5 μΙ of the 4 mM acid ceramidase substrate solution in ethanol (final substrate concentration 20 μΜ; final ethanol concentration 0.5%), and a fixed amount of protein (between 10 and 25 μg) in a volume of 25 μΙ of 0.2 M sucrose. The plate is incubated at 37 ^S C for 3 hours without stirring. After this time, the reaction is stopped by adding 50 μΙ of methanol and 100 μΙ of a solution of 2.5 mg / ml of Nal0 ₄ in glycine / NaOH buffer solution of pH 10.6 in each well. The plate is protected from light for two hours and the fluorescence is then measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm).

The activity, lysosomal β-galactosidase is measured with the same protein extracts used for the measurement of the acid ceramidase activity. The essay is based on the Fluorogenic method already published in the literature ² with some modifications in order to adapt it to the 96-well plate format. Thus, a fixed amount of protein (between 5 and 10 pg) in a volume of 10 μΙ, is mixed in a well together with 20 μΙ of 0.5 M acetic acid / sodium acetate buffer solution pH 4.5, 20 μΙ of 20 mM EDTA, 40 μΙ of the 3 mM 4-methylumbelliferil-pD-galactopyranoside substrate (final concentration 400 μΜ) and 10 μΙ of ultrapure water. This mixture is incubated at 37 ^Q C for 30 minutes and then add 100 μΙ of glycine buffer / NaOH at pH 10.6. Subsequently the fluorescence is measured in a plate fluorimeter (excitation wavelength 340 nm, emission 460 nm).

Claims

1. Compound of formula

(I)

where

Ri is selected from C1-C24 alkyl, C2-C24 alkenyl, C2-C24 alkynyl or a group -F½-Y-

R ₃ , where Y is selected from S, O, N, NH, S (O), S (O ₂ ), C (O), NHC (O), C (0) NH,

C (O) O, OC (O), R ₂ is selected from CrC _to alkyl, alkenyl C ₂ -C _a alkenyl, C2-C, 3 is selected from CC _b alkyl, C2-C _b, C ₂ -Cb alkynyl, where a + b <24, m is a value between 1 and 10,

X is a fluorophore, chromophore or luminophore,

with the proviso that when X is umbelliferone and m is 2, Ri is different from -

2. Compound according to claim 1 wherein m is a value between 2 and 4.

3. Compound according to claim 2 wherein m is 2.

4. A compound according to any of claims 1 to 3 wherein R1 is an alkyl - (CH ₂₎ n CH _3, where n is a value between 1 and 24.

5. Compound according to claim 4 wherein n is 10.

6. Compound according to the preceding claims wherein X is umbelliferone.

7. Diagnostic method to determine if an individual suffers from Farber's disease that includes: a) Obtaining an isolated biological sample from an individual

b) addition of a compound of formula (I):

(

where

R is selected from CRC2 ₄ alkyl, C2-C24 alkenyl, C2-C2 ₄ or a group

-R2-Y-R3, where Y is selected from S, O, N, NH, S (O), S (O ₂ ), C (O), NHC (O),

C (O) NH, C (O) O, OC {O), R ₂ is selected from C _r C alkyl, alkenyl C ₂ -C _a alkenyl, C2-C, R3 is selected from alkyl d-Cb , C2-Gb alkenyl, C2- alkynyl

Cb, being a + b <24,

m is a value between 1 and 10,

X is a fluorophore, chromophore or luminophore

to an isolated biological sample of an individual, c) incubation of the mixture obtained in (b)

d) oxidation of the mixture obtained in (c)

e) detection of the fluorescence produced in the mixture obtained in (d).

8. Method according to claim 7 wherein m is a value between 2 and 4.

9. Method according to claim 8 wherein m is 2.

10. Method according to any of claims 7 to 9 wherein R1 is an alkyl - (CH ₂ ) nCH ₃ , where n is a value between 1 and 24.

11. Method according to claim 10 wherein n is 10.

12. Method according to any of claims 7 to 11 wherein X is umbelliferone.

13. Method according to any of claims 7 to 12 wherein the isolated biological sample is a body fluid.

14. Method according to claim 13 wherein the body fluid is blood.

15. Method according to any of claims 7 to 12 wherein the isolated biological sample is epithelial tissue.

16. Method according to any of claims 7 to 15 wherein the incubation time in step (b) is between 0.5 and 5 hours.

17. Use of a compound of formula (I):

(I)

where

R is selected from CrC2 ₄ alkyl, C2-C24 alkenyl, C2-C24 alkynyl or a group -Ffe-Y-

R ₃ , where Y is selected from S, O, N, NH, S (O), S (0 ₂ ), C (O), NHC (O), C <0) NH,

C (0) 0, OC (O), R2, is selected from CrC alkyl, alkenyl C2-C, C2-C, R3 is selected from C _r C _b alkyl, alkenyl C ₂ -C _b alkynyl C ₂ -C _b , being a + b <24, m is a value between 1 and 10,

X is fluorophore, chromophore or luminophore,

18. Use according to claim 17 wherein m is a value between 2 and 4.

19. Use according to claim 18 wherein m is 2.

20. Use according to any of claims 17 to 19 wherein Ri is an alkyl - (CH ₂ ) nCH ₃ , where n is a value between 1 and 24.

21. Use according to claim 20 wherein n is 10.

22. Use according to any of claims 17 to 21 wherein X is umbelliferone.

23. A useful kit to determine if an individual suffers from Farber's disease from an isolated sample of said individual, which comprises the compound of formula (I).

24. Use of a kit according to claim 23 for the diagnosis of Farber's disease in an isolated biological sample of an individual.