Novel urea derivatives of amphoteric macrolides, and solubilisation of amphoteric antibiotics by carbohydrate urea derivatives

Abstract

The present invention relates to obtaining novel derivatives of amphoteric macrolides (such as amphotericin B) by modifying an insoluble amine in their structures (for example the amine group of the mycosamine of AmB) so as to introduce a urea function linked to a hydroxylated alkyl chain, making it possible to improve the solubility thereof, maintain or improve the activity thereof, and substantially reduce the toxicity thereof. In one embodiment, the derivatives are obtained by grafting carbohydrate urea derivatives. The invention also relates to the use of said carbohydrate urea derivatives as a function for solubilising amphoteric antibiotics and as a solubilisation adjuvant.

Description

[0001] Description

[0002] Title: New urea derivatives of amphoteric macrolides and solubilization of amphoteric antibiotics by urea derivatives of carbohydrates

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The invention relates to urea derivatives of amphoteric macrolides, and the uses of these molecules in particular as medicaments. The invention also relates to urea derivatives of carbohydrates and their uses in the solubilization of amphoteric medicaments, either by grafting with said medicaments or as a solubilization adjuvant.

[0005] STATE OF THE ART

[0006] There are many molecules with an amine function that are insoluble or poorly soluble. The insoluble nature of these molecules generally makes their use difficult.

[0007] This is particularly the case for amphoteric molecules such as polyene macrolides.

[0008] Polyene macrolides are a class of antifungal antibiotics with a broad spectrum of action against fungi and yeasts pathogenic to humans. This is particularly true for Amphotericin B (AmB), an antifungal with broad-spectrum fungistatic and fungicidal action: Condida albicans, Coccidioides immitis, Sporotcrichum, Cryptococcus neoformans, Histoplasma, Blastomyces, Rhizopus orizae, Aspergillus niger, etc. It is still, despite the introduction of imidazoles, the most effective drug for many conditions. AmB is also useful as an alternative therapy for the treatment of various forms of leishmaniasis. AmB is also used to treat primary meningoencephalitis caused by amoebae. It also has immunomodulatory properties in mice and to some extent in humans. It also enhances the action of a number of anti-cancer drugs.

[0009] However, due to their macrocyclic nature and amphoteric character, polyene macrolides such as AmB are poorly soluble in water and tend to form micelles in aqueous solution.

[0010] Furthermore, the toxicity of AmB to animal cells, particularly renal cells, lymphocytes and erythrocytes, requires significant precautions for clinical use. This is particularly the case for the treatment of deep and systemic mycoses, which must be treated by intravenous administration.

[0011] These considerations have led many researchers to search for non-toxic and more water-soluble derivatives. A large number of AmB derivatives, modified either at the acid function or at the primary amine function, have been proposed. Attempts have also been made to increase the solubility of AmB by adding surfactants or by forming salts. Unfortunately, all these attempts have resulted in derivatives that are unstable in solution or have lost their antibiotic properties. The use of expensive liposomal forms partially reduces the nephrotoxic risk but can cause acute IV poisoning. There is therefore a need for a solution to improve the solubility of amphotericin B in particular and of insoluble molecules with an amine function in general, while making them stable in an aqueous medium, maintaining or improving their efficacy and reducing their toxicity.

[0012] International patent application WO2022 / 003181 proposes urea derivatives of amphotericin B with improved solubility, said compounds being obtained by grafting the compounds to the primary amine of the mycosamine of amphotericin B with the aim of preserving or increasing the basic character of the molecule. International patent application WO 2013 / 186384 proposes glucamine thiourea derivatives with increased solubility. Patent application US 2017 / 0430029 also proposes derivatives of amphotericin B. However, there is still a need for new solutions to increase the efficacy of amphotericin B and other amphoteric antibiotics, and to reduce its toxicity.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention relates to novel amphoteric macrolide compounds comprising a structure having a macrocycle substituted by at least one amino sugar, said amino sugar being grafted with a molecule of formula (I) below, a pharmaceutically acceptable salt or a stereoisomer, a racemic mixture, a geometric isomer of said molecule of formula (I) or a mixture thereof:

[0015] The invention also relates to the use of said compound of formula (I), a pharmaceutically acceptable salt or a stereoisomer, a racemic mixture, a geometric isomer of said molecule of formula (I') or a mixture of those as a solubilizing function of an amphoteric antibiotic medicament comprising an amine group and a carboxyl group, and in which said compound of formula (I) is grafted to replace said amine group. According to one embodiment, said amino sugar corresponds to a mycosamine group, and said compound of formula (I) is grafted to replace the C3 amino group of mycosamine.

[0016] The invention also relates to the use as a solubilizing function of an amphoteric antibiotic medicament comprising an amine group and a carboxyl group, and in which said compound of formula (I) is grafted to replace said amine group.

[0017] The invention also relates to compounds of formula (I') below, a pharmaceutically acceptable salt, a stereoisomer, a racemic mixture, a geometric isomer of said molecule of formula (I') or a mixture thereof, and its use as a solubilization adjuvant for an amphoteric macrolide drug.

[0018] In particular, in said formulas I, and the:

[0019] R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkylamino, C1-C6 alkoxy, C1-C6 alkoxy substituted with C1-C3 alkyl-hydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom;

[0020] R3 represents a hydrogen atom, or one of the groups selected from an amine group, a C 1 -C 6 alkylamino, a hydroxyl, and a C 1 -C 6 alkoxy;

[0021] R4 represents a hydrogen atom, or one of the groups selected from a -NH2 and a -OH; n = 0 to 16, in particular n = 0 to 12, and more particularly n = 1 to 12 m = 0 to 16, in particular m = 0 to 12, and more particularly m = 1 to 12.

[0022] In one embodiment, R 1 and R 2 independently represent a hydrogen atom, or one of the groups selected from C 1 -C 3 alkyl, C 1 -C 3 hydroxyalkyl, C 1 -C 3 alkylamino, C 1 -C 3 alkoxy substituted with C 1 -C 3 alkyl-hydroxyl, and wherein at least one of R 1 or R 2 is a hydrogen atom; R 3 represents a hydrogen atom, or one of the groups selected from — NH 2 , — OH and C 1 -C 3 alkoxy; R 4 represents a hydrogen atom, or one of the groups selected from — NH 2 and — OH, preferably — OH; n = 0 to 6, in particular 1 to 6; preferably 1 to 3; and m = 0 to 6, in particular 1 to 6, preferably 1 to 3.

[0023] In another embodiment, R 1 and R 2 independently represent a hydrogen atom, or one of the groups selected from C 1 -C 3 alkyl, C 1 -C 3 hydroxyalkyl, C 1 -C 3 alkylamino, C 1 -C 3 alkoxy substituted with C 1 -C 3 alkyl-hydroxyl, and wherein at least one of R 1 or R 2 is a hydrogen atom; R s represents a hydrogen atom, or one of the groups selected from a group — NH 2 , — OH, and C 1 -C 3 alkoxy; R 4 represents — OH; n = 1 to 2; and m = 1 to 2.

[0024] According to a preferred embodiment, R1, R2 independently represent a hydrogen atom, or one of the groups selected from — CH3, — CH2-CH3, — CH2-OH, — CH2-CH2-OH, — CH2- NH2, — CH2-CH2-NH2, — CH2-O-CH2-OH, — CH2-CH2-O-CH2-CH2-OH, and wherein at least one of R1 or R2 is a hydrogen atom; R3 represents a hydrogen atom or an — OH group; R4 represents — OH; n = 1; and m = 2.

[0025] According to a second preferred embodiment, - R1, R2 respectively represent a hydrogen atom; R3 represents a hydrogen atom or a C1 to C3, preferably C1 to C2, alkoxy; R4 represents — OH; n= 1; and m= 2.

[0026] When said compound of formula I is grafted to a macrolide compound, said macrolide compound is preferably amphotericin B or nystatin, or one of their pharmaceutically acceptable salts, and said amino sugar corresponds to mycosamine, and said compound of formula (I) is grafted to replace the C3 amino group of mycosamine. New urea derivatives of amphotericin B and nystatin are thus obtained.

[0027] Molecules referred to as Amb-urea 2, Amb-urea 3, Amb-urea 7, Amb-urea 9, Amb-urea 10, Amb-urea 12 and Amb-urea 14 are preferred novel macrolide compounds according to the invention. Table I shows the structure of said molecules.

[0028] Likewise, the use of a compound of formula (I') as a solubilization adjuvant of an amphoteric macrolide compound is preferably intended for the solubilization (in aqueous medium) of amphotericin B and nystatin, and said use and said use comprises the preparation of a mixture of said compound of formula I' with amphotericin B or nystatin. The invention also relates to said amphoteric macrolide compounds grafted with a compound of formula (I) as described above for its use as an anti-infectious, antibiotic or antifungal agent, or even as an anti-cancer agent. Naturally, this use involves the administration to a patient or subject of a therapeutic (therapeutically effective dose) or prophylactic (effective dose for prevention) dose of said compound, by any type of general or systemic route (oral, intravenous, nasal, etc.), by local or in situ route (cutaneous, ocular, etc.).By subject or patient we mean any animal, including humans and any animal that may be undergoing anti-infectious veterinary treatment.

[0029] The invention also relates to a pharmaceutical composition comprising an amphoteric macrolide compound grafted with a compound of formula (I) as described above, and further comprising a pharmaceutically acceptable carrier and / or at least one pharmaceutically acceptable excipient and / or at least one pharmaceutically acceptable diluent.

[0030] Salts of the compounds of the invention are prepared according to techniques well known to those skilled in the art and include those with mineral or organic acids which allow suitable separation or crystallization of the compounds, as well as pharmaceutically acceptable salts. By "pharmaceutically acceptable" is meant that which is useful in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for veterinary as well as human pharmaceutical use. Such salts include:

[0031] (1) acid addition salts formed with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like; or

[0032] (2) salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or coordinates with an organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0033] Finally, the invention also relates to a method for inhibiting the growth of a fungus ex vivo, comprising contacting a fungus with an effective amount of one of said amphoteric macrolide compounds.

[0034] Other features and advantages will emerge from the detailed description of the invention, the examples and the test results which follow.

[0035] LIST OF FIGURES Fig. 1 illustrates the modification of the solubility pH of amphotericin B after grafting of a molecule of formula I) according to the invention, the urea derivative of amphotericin B obtained being referenced Amb Urea 2.

[0036] Fig. 2 illustrates the production of the AmB urea derivative 2 by grafting N-formyl-1-amino-deoxy-glucitol.

[0037] Fig. 3 illustrates the production of the AmB-urea derivative 2 by grafting a D-glucamine fragment according to the invention.

[0038] Fig. 3 illustrates the production of a urea derivative of amphotericin B Amb-urea 3 by grafting a methyl-glucamine fragment according to the invention.

[0039] Fig. 4 illustrates the production of a urea derivative of nystatin according to the invention by grafting a D-glucamine fragment.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to novel urea derivatives of amphoteric macrolides. In particular, the invention aims to increase the solubility, efficacy and / or reduce the toxicity of amphoteric macrolides whose structure carries an amine making them unstable in solution, such as amphotericin B (AmB) and nystatin.

[0042] AmB is used in the treatment of fungal infections, particularly systemic fungal infections, but dosage is often limited by systemic toxicity. The toxicity of AmB is partly related to its low solubility, specifically a self-associated form called micelles. In addition, AmB in aqueous media gives rise to time-dependent concentration, ionic strength, and polydisperse systems.

[0043] The present invention proposes to obtain new derivatives of amphoteric macrolides by modifying their insoluble amine to introduce a urea substituted by a hydroxylated alkyl chain. The urea function makes it possible to reduce the formation of micelles while the hydroxylated chain increases the solubility, and this without loss of biological activity.

[0044] In particular, the invention proposes to use a compound of formula (I) to substitute an amphoteric macrolide, in particular at the level of an amino sugar, such as that of the mycosamine of amphotericin B.

[0045] In the compound of formula (I) below:

[0046] R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkylamino, C1-C6 alkoxy optionally substituted with C1-C3 alkylhydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom; Rs represents a hydrogen atom, or one of the groups selected from an amine group, C1-C6 alkylamino, hydroxyl, and C1-C6 alkoxy;

[0047] R4 represents a hydrogen atom, or one of the groups selected from a -NH2 and a -OH; n = 0 to 16, in particular n = 0 to 12, and more particularly n = 1 to 12; and m = 0 to 16, in particular m = 0 to 12, and more particularly m = 1 to 12.

[0048] These compounds have been found to be able to improve the solubility and decrease the toxicity of amphotericin B.

[0049] Fig. 1 shows an example of the modification of solubility of amphotericin B after grafting of a compound of formula I as represented in formula (Ia) below, this derivative according to the invention is referenced AmB urea 2 in the present invention.

[0050] The solubility of the derivative has been modified to dissolve in a pH range compatible with the body environment, and with a significantly increased solubility between pH 4 to 10 compared to amphotericin B. This compound also exhibits an MIC50% equivalent to that of amphotericin B against Cryptococcus neoformans and Candida Albicans. Advantageously, the toxicity of the amphotericin B derivative of formula (Ia) shows a notable reduction in toxicity (see experimental part, Tables 3-7) and therefore represents an excellent candidate to replace amphotericin B in therapeutic applications.

[0051] It appears that this decrease in toxicity and increase in solubility are related to the effects of the urea bridge of the compound of formula (I), which prevents the formation of micelles and promotes the protonation of the carboxyl function of amphotericin B. The hydroxylated alkyl chain linked to the urea bridge also allows amphotericin B to be better solubilized, without loss of biological activity.

[0052] In one embodiment of the compounds of formula (I), this hydroxylated alkyl chain (bearing the substituents R3 and R4) comprises at least 3 carbons bearing — OH groups (i.e. R3 and R4 are — OH groups, and m=1 and n=0), preferably at least 5 carbons, and at least 3 — OH substituents, preferably at least 4 or 5 — OH substituents.

[0053] It has also been demonstrated that biological activity is retained with reduced toxicity when at least R1 and R2 in formula I is a hydrogen atom because a physiologically protonatable amine is then retained. Thus, one of R1 and R2 can be selected from the groups mentioned for formula (I) above.

[0054] In a preferred embodiment, the compounds of formula I are obtained by grafting substituted or unsubstituted amino carbohydrates, such as 1-amino-1-deoxy-sorbitol also called D-glucamine (see Table I, Amb Urea 2), N-methyl-D-glucamine (see Table I, AmB Urea 3), N-ethyl-D-glucamine (see Table I, AmB Urea 7), 2-amino-2-deoxy-D-glucose also called D-glucosamine (see Amb Urea 9), N-hydroxyethyl-D-glucamine (AmB Urea 10), and N-(2-aminoethyl)-glucamine (see Amb Urea 14). The urea derivatives of amphotericin B are illustrated in Table 1 of the experimental part. The invention also relates to the use of isomers, and stereoisomers of said carbohydrates, as well as the use of amino carbohydrates modified at the amine level to introduce substituents as defined for introducing R1 and R2.

[0055] Compounds of formula I can also be grafted into the structure of other macrolide antibiotics such as nystatin.

[0056] In one embodiment, this nystatin derivative comprises, for example, the following formula.

[0057] The invention also relates to the use of a compound of formula I as a solubilizing function of an amphoteric antibiotic comprising an amine substituent and an acid function, in particular a carboxyl substituent, said compound of formula I being grafted onto said antibiotic so as to substitute said amine.

[0058] In one embodiment, the compound of formula I is used as a solubilizing function of cephalexin. For example, cephalexin-glucamine urea of ​​formula IV is obtained below: In another embodiment, the compound of formula I is used as a solubilizing function of daptomycin. For example, daptomycin-glucamine urea of ​​formula IV is obtained below:

[0059] Finally, the invention also relates to compounds of formula (Ia) and their use as a solubilization adjuvant for amphoteric antibiotics, such as amphotericin B, nyastatin, cephalexin and daptomycin.

[0060] In particular, the urea derivatives of the alkyl hydroxylated aminos of the invention have the structure of the compounds of formula I. However, they are not grafted onto another molecule, the proximal nitrogen atom on formula I is therefore substituted by a hydrogen atom, as illustrated in formula I' below.

[0061] In particular, the invention relates to urea derivatives of amino carbohydrates such as

[0062] 1-amino-1-deoxy-sorbitol also called D-glucamine, N-methyl-D-glucamine, N-ethyl-D-glucamine,

[0063] 2-amino-2-deoxy-D-glucose also known as D-glucosamine, N-hydroxyethyl-D-glucamine, and N (2 aminoethyl) glucamine and their use as solubilization aids, including polyene macrolides such as amphotericin B, and nystatin, as well as amphoteric antibiotics such as daptomycin, and cephalexin.

[0064] For example, an amino carbohydrate urea derivative according to the invention is glucamine urea of ​​formula (V) illustrated below:

[0065] (2,3,4,5,6-pentahydroxyhexyl)urea.

[0066] Experimental Part

[0067] I. Synthesis methods

[0068] Example I. Synthesis of a molecule of formula (Ia)

[0069]

[0070] In a first embodiment (Fig. 2), formula Ia is obtained by modification of the mycosamine of amphotericin B, in particular by grafting N-formyl-1-amino-deoxy-glucitol at its C3 amine. To a solution of amphotericin B in a solvent (THF, DMF, or DMSO) is added at room temperature, N-formyl-1-amino-deoxy-glucitol. The solution is left stirring for between 30 minutes and two hours at room temperature, then precipitated in an acetone / diethyl ether mixture or in an ethyl acetate / hexane mixture. The final product was isolated by centrifugation at 6000 rpm, and dried overnight under vacuum. The resulting solid was verified by LC-MS and C-NMR. In another embodiment (Fig. 3), glucamine (1-Amino-1-deoxy-D-glucitol) is directly grafted following the same process.

[0071] Other methods for obtaining the urea derivatives of the invention are also conceivable. For example, an intermediate urea at the C3 carbon of mycosamine (such as AmB-NHCONHMe) can be obtained in a first step using a base (DI PEA, pyridine) in the reaction, and in a second step the desired carbohydrate fragment is grafted.

[0072] Example II. Synthesis of a molecule of formula (Ia) Dissolve 1 mmol of D-Glucamine in DMSO with magnetic stirring. Slowly add Carbonyldiimidazole GDI in stoichiometric quantity (1 mmol) still with magnetic stirring at a temperature of 30-50 °C. Reaction for 2 hours. Pour the solution into an ice-cold acetone / ether (1:1) volume for precipitation and filtration by centrifugation. Dry the solid obtained under vacuum for 1 night at 40 °C under reduced pressure.

[0073] II. Microbiological and toxicological studies of urea derivatives of Amphotericin B

[0074] The toxicity and MIC of the different amphotericin B derivatives according to the invention were compared with amphotericin B. Table I below shows the structure of the derivatives tested.

[0075] Table 1. Structure of amphotericin B derivatives

[0076] a) Determination of the MIC against Candida Albicans and Cryptococcus Neoformans

[0077] The minimum inhibitory concentration (MIC) of each antifungal was determined according to the broth microdilution method recommended by the American Committee for Clinical Laboratory Standards (NCCLS), and the culture medium for yeasts was RPMI-1640 medium in MOPS buffer. The method includes the following specific steps:

[0078] (1) Preparation of stock solutions: Solutions of amphotericin B (AmB) and urea derivatives according to the invention are adjusted to a final concentration of between 0.007 and 8 pg / mL in a MOPS buffer (Sigma) pH 7.0 at 0.165 M.

[0079] (2) Preparation of a culture medium: The culture medium for the tested yeasts is Sabouraud agar in a tube (Bio Mérieux, France). The fungal growth culture medium for MIC determination is RPM1 1640 medium (Sigma-Aldrich) buffered with MOPS according to NCCLS M27-A,

[0080] (3) MIC Determination: Broth microdilution tests were performed according to NCCLS M27-A. An inoculum suspension adjusted to a turbidity of 0.5 McFarland, is diluted to a concentration of 1.0 x 10 3 at 5.0 x 10 3cells per ml, normalized by spectrophotometry, and a 0.1 ml aliquot is added to each well of a microdilution plate (final inoculum, 0.5 x 10 3 at 2.5 x io 3 cells / mL). Inoculation size is verified by colony counting. Microdilution plates are incubated at 35 °C. MIC parameters were read visually after 72 h of incubation. The MICs of amphotericin B and urea derivatives were defined as the lowest concentrations that produced complete growth inhibition (first clear well).

[0081] (4) Quality Control: QC testing was performed according to NCCLS M27-A using Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019. QC determinations performed each day were within the control limits for amphotericin B established by Barry et al.

[0082] The results obtained on the antifungal activities of the molecules tested against 12 isolates of C. Neoformans and 20 isolates of Candida albicans determined by the microdilution method are presented in Table 2 below.

[0083] Table 2- MIC where 50 and 90% of the tested isolates are inhibited The results show that all the amphotericin B derivatives according to the invention exhibit antifungal activity. However, the antifungal activity of the derivatives varies slightly depending on the strain tested. When looking at the MIC 50%, the best agents for treating Cryptococcus neoformans are the Amb Urea 3 and 9 derivatives, which provide superior effects to amphotericin B, while AmB Urea 2 and 7 have similar effects to amphotericin B. To treat Candida albicans, the best agents are the Amb Urea 2, 9, 10 derivatives, which have an efficacy equivalent to that of amphotericin B. b) Toxicity studies on red blood cells and nucleated cells

[0084] The toxicological properties of the derivatives were studied on human and murine red blood cells as well as on lymphoid cells of peripheral blood, bone marrow, thymus in humans, on spherical T and B lymphocytes of mice and on two of the 10 tumor lines: XG3 (mouse), Daudi (human). All the solutions of the AmB derivatives in accordance with the invention; and of AmB are prepared externally by diluting 4 mg of the diluted molecules in 1 ml of 5% glucose.

[0085] The cells are prepared by depositing 1 ml of peripheral blood collected and diluted half on Ficoll-hypaque to remove lymphocytes, then centrifuging for 20 min at 2000 rpm. The red blood cell pellet is recovered and washed. A 1.25% suspension is then made in a 150 mM KCI, 0.5 mM Tris HCl, pH 7.4 solution and 150 μl is distributed per well as well as 50 μl of each of the derivative dilutions, plus a control well (5% glucose solution). The cells are then incubated for 1 hour 30 minutes at 37 ° C in a humid incubator. To evaluate cell lysis, hemoglobin is measured by taking 100 μl of supernatant from each well and diluting it in 1 ml of distilled water. Hemoglobin concentration is calculated by measuring the optical density at 540nm and taking into account the absorption of Amphotericin B derivatives.

[0086] The LD 50 is the dose that results in 50% lysis of red blood cells (a 100% lysis control is obtained with distilled water, a 0% lysis control is obtained with the 5% glucose solution). The results are shown below.

[0087] Table 3: In vitro toxicity of AmB derivatives on different mouse cells Table 3: In vitro toxicity of AmB derivatives on different mouse cells

[0088] Table 4: In vitro toxicity of AmB derivatives on mouse cells

[0089] Table 5: In vitro toxicity of AmB derivatives on mouse cells The results show that all the tested derivatives have a lower toxicity than amphotericin B. In particular, Amb Urea 2, 7, 14 show systematically very high LD50s compared to Fungizone (amphotericin B) on all the cells tested. c) Study of toxicity on nucleated cells

[0090] Amphotericin B derivative solutions are prepared in a similar way to those mentioned in the previous paragraph. The cells are prepared by depositing 20 ml of blood diluted 1 / 2 with 0.9% NaCl, on 10 ml of Ficoll-hypaque and centrifuging for 30 min at 2000 rpm. The cell ring is recovered and washed twice in PBS buffer containing 5% fetal calf serum. The cells are prepared by suspending 1, 14.106 cells / ml in RPMI 1640 containing 2.5% fetal calf serum and distributing 150 μl per well. 50 μl of each dilution of the derivatives are added as well as a control (5% glucose solution). The cells are then incubated for 1 h 30 in a humid incubator at 5% CO2, at 37°C or at room temperature 25°C.To assess the toxicity of the derivatives, a cell count is performed in the presence of trypan blue and the LD 50 (concentration of derivative causing 50% mortality) is calculated compared to the control (5% glucose solution) for each concentration of each derivative.

[0091] The tables below show that the urea derivatives of AmB prepared according to the invention are much less toxic than Amphotericin B for human and murine cells, this being able to vary from one derivative to another.

[0092] Table 6 In vitro toxicity of urea derivatives of AmB on different human cells Table 7: In vitro toxicity of urea derivatives of AmB on different human cells

[0093] III. Use of compounds of formula I as solubilizing adjuvants for amphotericin B

[0094] Methodology Dissolve 10 mg of AmB in 1 mL of methanol, then filter the solution through a 0.45 cm membrane to remove impurities. In parallel, dissolve 10 to 20 mg of glucamine urea (formula a previously synthesized by coupling) in a phosphate buffer at pH 7. Slowly add the AmB solution to the glucamine urea solution, with continuous stirring at room temperature, for 30 minutes, then adjust the final solution with the same phosphate buffer. Results

[0095] The observed solubility of AmB increases from 0.08 mg / ml for AmB alone to 30 mg / ml for AmB with glucamine urea (formula (Ia)) as an adjuvant.

Claims

CLAIMS 1. Macrolide antibiotic compound comprising a structure having a macrocycle substituted by at least one amino sugar, said amino sugar being grafted with a molecule of formula (I) below, a salt, a stereoisomer, a racemic mixture, a geometric isomer of said compound of said formula (I) or a mixture thereof: in which R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkylamino, C1-C6 alkoxy, C1-C6 alkoxy substituted with C1-C3 alkyl-hydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom; R3 represents a hydrogen atom, or one of the groups selected from an amine group, a C1-C6 alkylamino, a hydroxyl, and a C1-C6 alkoxy; R4 represents a hydrogen atom, or one of the groups selected from -NH2 and -OH; n = 0 to 16, in particular n = 0 to 12, and more particularly n = 1 to 12; and m = 0 to 16, in particular m = 0 to 12, and more particularly m = 1 to 12.

2. Compound according to claim 1, wherein in formula (I): R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 alkylamino, C1-C3 alkoxy substituted with C1-C3 alkyl-hydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom; R3 represents a hydrogen atom, or one of the groups selected from a group — NH2, — OH and a C1 to C3 alkoxy R4 represents a hydrogen atom, or one of the groups selected from — NH2 and — OH, preferably — OH; n= 0 to 6, in particular 1 to 6; preferably 1 to 3; and m=0 to 6, in particular 1 to 6, preferably 1 to 3.

3. Compound according to claim 1 or 2, wherein in formula (I): R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C3 alkyl, C1-C3 hydroxyalkyl, C1-C3 alkylamino, C1-C3 alkoxy substituted with C1-C3 alkylhydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom; R3 represents a hydrogen atom, or one of the groups selected from a group — NH2, — OH, and a C1 to C3 alkoxy ; R4 represents — OH; n= 1 to 2; and - m=1 to 2.

4. Compound according to one of claims 1 to 3, in which in formula (I): Ri, R2 independently represent a hydrogen atom, or one of the groups selected from — CH3, — CH2-CH3, — CH2-OH, — CH2-CH2-OH, — CH2-NH2, — CH2-CH2-NH2, — CH2-O-CH2-OH, — CH2-CH2-O-CH2-CH2-OH, and wherein at least one of Ri or R2 is a hydrogen atom; R3 represents a hydrogen atom or an —OH group; R4 represents — OH; - n= 1 ; and - m= 2.

5. Compound according to one of claims 1 to 4, chosen from the following molecules or one of its pharmaceutically acceptable salts: AmB urea -7, formula (Ic); AmB urea 14, formula (Ig).

6. Compound according to one of claims 1 to 3, in which in formula (I): - Ri, R2 respectively represent a hydrogen atom; - R3 represents a hydrogen atom or a C1 to C3 alkoxy, preferably C1 to C2; - R4 represents — OH; n= 1; and m= 2.

7. Compound according to claim 6, chosen from the following molecules: AmB urea -9, formula (Id) 8. A compound according to any one of claims 1 to 4 or 6, wherein the grafted macrolide antibiotic is amphotericin B or nystatin, or one of their pharmaceutically acceptable salts, and the amino sugar corresponds to mycosamine, and said compound of formula (I) is grafted to replace the C3 amino group of mycosamine.

9. A compound according to claim 8, wherein the grafted macrolide antibiotic compound is nystatin, and has the following structure:

10. Use of a compound of formula (I) below or a salt, a stereoisomer, a racemic mixture, a geometric isomer of said compound of said formula (I) or a mixture thereof as a solubilizing function of an amphoteric antibiotic drug comprising an amine group and a carboxyl group, and in which said compound of formula (I) is grafted to replace said amine group: and in which R 1 and R 2 independently represent a hydrogen atom, or one of the groups selected from C 1 -C 6 alkyl, C 1 -C 6 hydroxyalkyl, C 1 -C 6 alkylamino, C 1 -C 6 alkoxy, C 1 -C 6 alkoxy substituted with C 1 -C 3 alkyl-hydroxyl, and wherein at least one of R 1 or R 2 is a hydrogen atom; R3 represents a hydrogen atom, or one of the groups selected from an amine group, a C 1 -C 6 alkylamino, a hydroxyl, and a C 1 -C 6 alkoxy; R4 represents a hydrogen atom, or one of the groups selected from a -NH2 and a -OH; n = 0 to 16, in particular n = 0 to 12, and more particularly n = 1 to 12 m = 0 to 16, in particular m = 0 to 12, and more particularly m = 1 to 12 11. Use of a compound of formula I' below or a salt, a stereoisomer, a racemic mixture, a geometric isomer of said compound of said formula (I) or a mixture thereof as a solubilizing adjuvant for an amphoteric macrolide drug, and wherein; R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkylamino, C1-C6 alkoxy, C1-C6 alkoxy substituted with C1-C3 alkyl-hydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom; R3 represents a hydrogen atom, or one of the groups selected from an amine group, a C 1 -C 6 alkylamino, a hydroxyl, and a C 1 -C 6 alkoxy; R4 represents a hydrogen atom, or one of the groups selected from a -NH2 and a -OH; n = 0 to 16, in particular n = 0 to 12, and more particularly n = 1 to 12 m = 0 to 16, in particular m = 0 to 12, and more particularly m = 1 to 12.

12. Use according to claim 11, wherein the amphoteric macrolide drug is amphotericin B or nystatin, and said use comprises preparing a mixture of said compound of formula Ib with amphotericin B or nystatin.

13. Use according to one of claims 10 to 12, in which the substituents R1, R2, R3 and R4, as well as the value of m and n of said compound of formula (I) or of formula (Ib) are selected as defined in one of claims 2 to 4 or 6.

14. Use according to claim 13, in which R1, R2 and R3 respectively represent a hydrogen atom, R4 represents -OH, n=1 and m=2.

15. Compound according to one of claims 1 to 9 for its use as an antiinfectious, antibiotic or antifungal agent, preferably in the prevention and / or treatment of infections by at least one of the following fungi: Candida albicans, Coccidioïdes immitis, Sporotcrichum, Cryptococcus neoformans, Histoplasma, Blastomyces, Rhizopus orizae, Aspergillus niger; or for its use in the prevention or treatment of leishmaniasis and / or primary meningoencephalitis due to amoebae and / or as an immunomodulatory drug.

16. Pharmaceutical composition comprising a compound according to one of claims 1 to 9, further comprising a pharmaceutically acceptable carrier and / or at least one pharmaceutically acceptable excipient and / or at least one pharmaceutically acceptable diluent.

17. A method of inhibiting the growth of a fungus ex vivo, comprising contacting a fungus with an effective amount of a compound according to one of claims 1 to 9.

18. A compound having the following formula I', a salt, or a stereoisomer, a racemic mixture, a geometric isomer of said compound of said formula (I') or a mixture thereof: in which R1 and R2 independently represent a hydrogen atom, or one of the groups selected from C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkylamino, C1-C6 alkoxy, C1-C6 alkoxy substituted with C1-C3 alkyl-hydroxyl, and wherein at least one of R1 or R2 is a hydrogen atom; R3 represents a hydrogen atom, or one of the groups selected from an amine group, a C1-C6 alkylamino, a hydroxyl, and a C1-C6 alkoxy; R4 represents a hydrogen atom, or one of the groups selected from -NH2 and -OH; n = 0 to 16, in particular n = 0 to 12, and more particularly n = 1 to 12; and m = 0 to 16, in particular m = 0 to 12, and more particularly m = 1 to 12.

19. A compound according to claim 18, wherein the substituents R1, R2, R3 and R4, as well as the value of m and n of said compound of formula (I) or formula (Ib) are selected as defined in one of claims 2 to 4 or 6.

20. Compound according to one of claims 18 to 19, having the following formula:

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