MXPA01008781A - Echinocandin/carbohydrate complexes - Google Patents

Abstract

A complex of an echinocandin compound with a carbohydrate is described having improved thermal stability and water solubility. A process for making the echinocandin/carbohydrate complex is also described as well as the use of the complex in pharmaceutical formulations and treatments of fungal infections.

Description

COMPOUNDS OF EOUINOCANDIN / CARBOHYDRATE FIELD OF THE INVENTION The present invention relates to pharmaceutically active echinocandin materials, in particular a crystalline complex between an echinocandin compound and a carbohydrate to improve the stability and solubility in water.

BACKGROUND OF THE INVENTION Echinocandin compounds containing helaminal functionality are generally susceptible to ring opening in the aminal bond, especially at high temperatures. In addition, the amorphous forms of the compounds are sensitive both in humidity and in temperatures above -10 ° C, the amorphous material is not stable above freezing temperatures. This not only accepts the shelf life of the drug in bulk form, but also makes it more difficult to handle the compounds in an industrial process. A solution to eliminate the opening of the ring in the aminal bond is to remove or functionalize the hydroxy group of the hemiaminal function; however, this requires an additional synthesis stage. Although this is an effective way to increase the stability of the modified compound, any additional step in the manufacturing process reduces productivity, increases waste potential and increases costs. US 4,876,241 describes the use of a sugar as a stabilizer in biological and pharmaceutical products; however, the process is aimed at the stabilization of products during the thermal inactivation of viral and bacterial contaminants in solution. The sugar is removed after the thermal inactivation process. Consequently, this process does not solve the long-term stability of the product. The effects of stabilization of sugars in a thermal process have been demonstrated. For example, the effects of sugars, pH and calcium on the thermal denaturation of whey proteins are discussed in Ibrahim et al. Egyptian J. Dairy Sci. , 23: 177-188 (1995). As with the previous reference, stabilizing effects are obtained in a liquid form. No reference suggests that stability can be improved by incorporating a carbohydrate into the crystalline form of a compound. In addition to thermal instability, lipopeptide compounds, such as echinocandins, are also known to have very poor water solubility (<0.1 mg / ml) which makes them particularly difficult for formulation for parenteral (ip) applications and the purification of materials. Generally, amorphous materials are harder to purify than crystalline materials. Therefore, there is a need for improved thermal stability and water solubility of echinocandin compounds without altering the bioavailability or without making structural changes to the compound as well as providing a means to further purify the echinocandin.

BRIEF DESCRIPTION OF THE INVENTION It has now been found that crystallization of an echinocandin compound in the presence of a carbohydrate (or simple sugar) produces a crystalline product having improved thermal stability and water solubility without compromising the bioavailability of the active compound. In one embodiment of the present invention, a crystalline complex is provided between an echinocandin compound and a carbohydrate. The complex is characterized in that the echinocandin / carbohydrate complex has a more crystalline form (i.e., a more ordered matrix) as compared to the echinocandin compound without the carbohydrate. In another embodiment of the present invention, there is provided a method for making an echinocandin / carbohydrate complex described above, comprising the steps of: (a) providing an echinocandin compound; (b) mixing the echinocandin compound and a carbohydrate in a solvent to form a mixture; (c) heat the mixture for solubility the echinocandin compound and to solubilize or disperse the carbohydrate; (d) allowing the mixture to cool to produce the echinocandin / carbohydrate complex; and (e) isolating the echinocandin / carbohydrate complex. In still another embodiment of the present invention, there is provided a process for forming a parenteral formulation comprising the step of mixing the echinocandin / carbohydrate complex described above, in an aqueous solvent. In another embodiment of the present invention, a pharmaceutical formulation is provided which includes the echinocandin / carbohydrate complex described above and a pharmaceutically acceptable carrier. In another embodiment of the present invention, there is provided a method of treating a fungal infection in a mammal in need thereof, which comprises administering to the mammal the echinocandin / carbohydrate complex described above. In another embodiment, a method is provided for treating a fungal infection in a mammal in need thereof, which comprises contacting the echinocandin / carbohydrate complex described above with body fluids of the mammal, wherein the complex collapses to an amorphous form. when it comes into contact with bodily fluids.

Definitions The term "complex" refers to an association between the echinocandin compound and the carbohydrate, such that the complex has a more crystalline form (e.g., a more ordered unitary matrix) than the corresponding echinocandin compound without the carbohydrate. The term "carbohydrate" refers to an aldehyde or ketone derivative of polyhydric alcohols represented by the formulas Cn (H20) n (e.g. glucose, C6 (H20) 6; sucrose (C12 C20)?: L). Carbohydrates include compounds with relatively small molecules, such as simple sugars (for example monosaccharides, disaccharides, etc.), as well as macromolecular (polymeric) substances such as starch, glycogen and cellulose polysaccharides. Sugars are carbohydrates (saccharides) that have the general composition (CH20) n and the simple derivatives thereof. Although simple monomeric sugars (glycoses) are described as polyhydroxyaldehydes or ketones, for example HOCH2- (CHOH) 4-CHO for aldohexoses (eg glucose) or HOCH2- (CHOH) 3-CO-CH2OH for 2-ketoses (e.g. fructose), sugars are commonly written as cyclic ethers with a ring of five (furanose) or six (pyranose) members, for example The D and L enantiomers, as well as the alpha and beta anomers of the compounds are also included within the definition of carbohydrates. The term "echinocandin" refers to a compound having the following general structure: wherein: R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or combinations thereof, - R1 (R2, R3, R6, R7 and Rx0 are independently hydroxy or hydrogen, - R4 is hydrogen, methyl or -CH2C (0) NH2; R5 and RX1 are independently methyl or hydrogen; R8 is -OH, -OS03H2, -OP03H2, -0P03HRa, or -OP02HRa, where Ra is hydroxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, phenyl, phenoxy, p-halophenyl, p-halofenoxy, p-nitrophenyl, p-nitrophenoxy, benzyl, benzyloxy, p-halobenzyl, p-halobenzyloxy, p-nitrobenzyl or p -nitrobenzyloxy, R9 is -H, -OH or -OSO3H, and pharmaceutically acceptable salts or hydrates thereof Although a specific chiral form is shown above, other chiral forms are within the spirit of the present invention. "or" ECB "refers to an echinocandin compound as described above, wherein R1 # R2, R3, R6, R7, R8 and R10 are hydroxyl groups i; R4, R5 and R1X are methyl groups, - R9 is a hydrogen. In the natural product, R is a linoleoyl group. In a particularly useful semi-synthetic compound, R has a rigid and a flexible component, for example a compound wherein R is represented by the following formula: The term "alkyl" refers to a hydrocarbon radical of the general formula C n H 2n + 1 containing 1 to 30 carbon atoms, unless otherwise indicated. The alkane radical can be linear, branched, cyclic or multicyclic. The alkane radical can be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy or alkanoate group has the same definition as in the above. The term "alkenyl" refers to an acyclic hydrocarbon containing at least one carbon-carbon double bond. The radical alkene can be linear, branched, cyclic or multicyclic. The alkene radical can be substituted or unsubstituted. The term "alkynyl" refers to an acyclic hydrocarbon containing at least one carbon-carbon triple bond. The alkyne radical can be linear or branched. The alkyne radical can be substituted or unsubstituted. The term "aryl" refers to aromatic portions that have simple ring systems (for example phenyl) or fused (for example naphthalene, anthracene, phenanthrene, etc.). The aryl groups can be substituted or unsubstituted. The substituted aryl groups include a chain of aromatic portions (for example biphenyl), terphenyl, phenylnaphthalyl). The term "heteroaryl" refers to aromatic portions containing at least one heteroatom within the aromatic ring system (eg, pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.) . The aromatic portion may consist of a single or fused ring system. Heteroaryl groups can be substituted or unsubstituted. Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows substituents which are classical alkyl groups such as methyl, ethyl, isopropyl, isobutyl, tertiary butyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term specifically embraces and allows for substitutions in alkyl which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamate, etc., as well as those which include the unsubstituted alkyl portion. However, it is generally understood by those skilled in the art that substituents can be selected so as not to adversely affect the pharmacological characteristics of the compound or to adversely interfere with the use of the medicament. Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkylamino, quaternary ammonium salts, aminoalkyl, hydroxyalkylamino, aminoalkylthio , carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl and combinations thereof.

DETAILED DESCRIPTION Attempts to crystallize echinocandin B from a solvent such as methanol provide a crystalline product containing the solvent in sufficient purity; however, this material degrades as the solvent evaporates. Applicants have discovered that when the crystallization process is carried out in the presence of a carbohydrate (or sugar), a crystalline complex is formed between the echinocandin compound and the carbohydrate. Although it is not desired to join any particular theory, it is considered that the carbohydrate is incorporated into the open spaces within the crystal unit cell of the echinocandin. As a result, the carbohydrate acts as a non-volatile solvate. An analogous complex has been reported by Etter et al. Using triphenylphosphine oxide (J. Am. Chem. Soc., 110: 639-640 (1988)). An advantage of an inclusion complex is the extraction of the carbohydrate (or sugar) from the matrix, thereby causing the remaining crystalline structure to collapse to an amorphous solid. Amorphous solids are generally considered more bioavailable. As a result, the echinocandin / carbohydrate complex can be reverted to an amorphous form in vivo (e.g., when contacted with bodily fluids of the mammal in question, and therefore bioavailability is optimized during treatment.

It is considered that the aminal group is stabilized by hydrogen bonding between the carbohydrate and the aminal functionality. This theory is based on the observation that the carbohydrate is released immediately before the dispersion of the crystalline complex in water. Complexes are formed using standard or conventional crystallization procedures such as those typically performed to purify compounds by recrystallization. Echinocandin material and carbohydrate dissolve at elevated temperature (about 40 to 60 ° C, preferably less than 55 ° C) in a solvent. The solution is then cooled slowly until crystallization begins. A seed crystal (such as a pre-crystallized complex or an insoluble sugar) can be added to initiate crystallization. Suitable solvents include any solvent, or solvent mixtures, inert to the reaction that is carried out and that sufficiently solubilize the reagents to provide a medium within which to carry out the complex formation that is desired between the carbohydrate and the echinocandin compound, such as protic solvents or ketone including methanol, ethanol, benzyl alcohol as well as mixtures of benzyl alcohol with solvents such as methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-butanol, t-butanol, 2-pentanol, 2-methyl-1-propanol, MEK, acetone, ethyl acetate, toluene, acetonitrile, fluorobenzene, methylene chloride, nitromethane or cyclic ketones such as cyclopentanone and cyclohexanone. Preferred solvents include methanol, ethanol, benzyl alcohol and mixtures of benzyl alcohol with methyl ethyl ketone, ethyl acetate and acetonitrile. Suitable carbohydrates include adonitol, arabinose, arabitol, ascorbic acid, chitin, D-cellobiose, 2-deoxy-D-ribose, dulcitol (S) - (+) - erythrulose, fructose, fucose, galactose, glucose, inositol, lactose, lactulose, lixose, maltitol, maltose, maltotriose, mannitol, mannose, melezitose, melibiose, microcrystalline cellulose, palatinose, pentaerythritol, raffinose, rhamnose, ribose, sorbitol, sorbose, starch, sucrose, trehalose, xylitol, xylose and hydrates thereof. Suitable carbohydrates may also include the D and L enantiomers, as well as the alpha and beta anomers of the compounds listed above. The preferred carbohydrates are simple sugars (for example monosaccharides and disaccharides). For a better understanding, sugars (or carbohydrates) can be grouped into four classifications: insoluble, soluble, highly soluble and co-crystallizing. For illustrative purposes only, the following definitions are used for the four classifications when methanol is used as the recrystallizing solvent for the semi-synthetic echinocandin compound 6 (a) which is shown in the following examples.

Insoluble carbohydrates are defined as those that have little or no solubility in methanol (<3 equivalents) at 40-60 ° C. Insoluble carbohydrates impart little or no improvement to order, determined by X-ray powder diffraction (XRPD). Although the complexes that are formed are heterogeneous, the complexes demonstrate improved thermal stability compared to the amorphous echinocandin product. Examples of carbohydrate insoluble in methanol include D-arabinose, L-arabinose, D-cellobiose, dulcitol, L-fucose, D-galactose, aD-glucose, β-D-glucose, L-glucose, inositol, hydrate of aD- lactose, lactulose, L-lixose, maltitol, D-maltose hydrate, maltotriose hydrate, mannitol, melecitosa hydrate, aD-melibiose hydrate, microcrystalline cellulose, palatinose hydrate, L-sorbose, starch and sucrose. Soluble carbohydrates are defined as those carbohydrates that are soluble in methanol from 2 to 20 equivalents, from 40 to 60 ° C. A homogeneous product is formed with the echinocandin compound under a set of specific equivalent ranges. Carbohydrates that fall within this class show both improved order by XRPD and improved stability compared to the amorphous echinocandin product alone. The soluble carbohydrate compositions not only show improved thermal stability compared to the amorphous compound but also have improved and demonstrated water dispersion properties. Examples of carbohydrates in this class for methanol as a solvent include adonitol, L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, hydrate (S) - (+) - erythrulose, d-fructose , D- (+) - fucose, L-fucose, aD-glucose, β-D-glucose, L-glucose, D-lixose, L-lixose, D-maltose hydrate, D-mannose, L-mannose, hydrate of melecitosa, palatinose hydrate, pentaerythritol, L-rhamnose, D-ribose, L-ribulose hydrate, D-sorbitol, sucrose, D-trehalose, xylitol and D-xylose. Highly soluble carbohydrates are those that have extremely high solubility in a solution containing methanol and the echinocandin compound (> 20 equivalents) at 40-60 ° C. These show an increased order in the isolated complex, determined by XRPD, but do not contain any heterogeneous carbohydrate. The complex also shows improved thermal stability compared to the amorphous echinocandin product. Examples of highly soluble carbohydrates include 2-deoxy-D-ribose, (S) - (+) - erythrulose hydrate, L-fucose, L-rhamnose, D-ribose and L-ribulose hydrate. Co-crystallizing carbohydrates are defined as those carbohydrates that have good solubility in methanol (> 2 equivalents) at 40-60 ° C. By allowing the homogenous mixture of echinocandin compound to cool and the carbohydrate shows an improved order by XRPD and improved stability compared to the amorphous echinocandin. Examples of co-crystallising carbohydrates in methanol include adonitol, D-arabitol, L-arabitol, D-raffinose pentahydrate, D-sorbitol, D-trehalose hydrate, xylitol and L-xylose. The co-crystalline compositions not only show improved thermal stability compared to the amorphous compound, but also demonstrate improved dispersion properties in water. The co-crystalline complexes have the potential to help or improve the in vivo dispersion of the bulk drug. Each of the carbohydrates is usually found in more than one class, with the exception of some insoluble carbohydrates. For example, adonitol is very soluble in methanol; however, the addition of higher equivalents of adonitol remains soluble in the test solution, but co-crystallizes upon letting it cool. Therefore, adonitol is classified as a carbohydrate both soluble and co-crystallizing in methanol. For purposes of illustration, the semi-synthetic echinocandin compound 6 (a) (semi-ECB) is recrystallized in the presence of each of the carbohydrates that are included in Table 1 to form the corresponding semi-ECB / carbohydrate complex. Each of the complexes is then tested for thermal stability using the following general procedure.

Thermal stability stress test Before placing the sample in a voltage stability test for two weeks, each sample is retested to determine potency and total related substances (TRS) to obtain a true TO point. Samples are placed (including an amorphous ECB control) in sealed flasks, at 50 ° C for 2 weeks and then assays are performed to determine power and TRS, at the end of the test. The greater degradation of impurity is used as the basis for complete stability. The degradation rate is determined as the relative ratio of the main degradation product, peak B of the test material versus the control. The recovery is called "Rec". A decrease in the degradation rate implies a higher thermal stability of the comparative test materials. Table 1 summarizes the results compared to the control sample. The potency (Pot) and TRS are determined using high pressure liquid chromatography (HPLC) equipped with a ZorbaxMR XDB-C18 column of 15 cm x 4.6 mm, with a particle size of 3.5 microns. The samples are eluted with an aqueous solution of 0.85% phosphoric acid, w / w and a 95% aqueous acetonitrile solution using methanol as the diluent. An elution gradient scheme is used when the ratio of phosphoric acid solution to acetonitrile solution varies from 95: 5 to 59:41, to 5:95 and to 95: 5 over a period of one hour. In table 1, * Values before stress test, ** Registered in percent by weight instead of weight equivalents, *** KF = water, determined by Karl Fischer (Coulomb.

Table 1 Each of the carbohydrates tested shows an improvement in thermal stability compared to the control where carbohydrate is not added. Although insoluble carbohydrates do not work as well as the other classes, an improvement over the amorphous form of the ECB is nonetheless observed. The data also shows that thermal stability can be stabilized by using appropriate weight equivalents of the added sugar. For example, (S) - (+) - erythrulose provides a more stable complex when only 8.0 equivalents by weight are added, instead of 30.0 equivalents by weight to the methanol crystallization process. Meanwhile, 2-deoxy-D-ribose provides a more stable complex when 33.6 weight equivalents of sugar are used, instead of 8.0 weight equivalents. For an echinocandin / fructose complex, preferably the complex contains between about 7 and 14%, w / w fructose, more preferably between about 8.5 and 11% w / w fructose. In general, the carbohydrate weight percent in the echinocandin / carbohydrate complex is between 5 and 35%, based on the carbohydrate that is used. The carbohydrate crystallization process has been observed to reduce the concentration of typical degradation impurities in the order of about 80 to 90%. The impurities by fermentation are generally reduced from about 5 to 20%. The total related substances in general (TRS) are reduced by approximately 45-55%. For comparison, the crystallization process for fructose is about 6% more efficient in the rejection of impurity without comparison with direct recrystallization in methanol for compound 6 (a). Preferred carbohydrate complexes with semi-ECB crystallized from methanol include carbohydrates which are selected from L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, (S) - (+) - erythrulose, D-fructose, D- (+) -fucose, L-fucose, D-galactose, aD-glucose, β-D-glucose, L-glucose, D-lixose, L-lixose, maltitol, D-maltose, maltotriose, D.small, melecitosa, palatinose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, D-trehalose, xylitol, L-xylose and hydrates thereof. The most preferred are the semi-ECB / carbohydrate complexes wherein the carbohydrate is selected from L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, (S) - (+) - erythrulose, D-fructose, D- (+) -fucose, L-fucose, D-galactose, β-D -glucose, D-lixose, L-lixose, D-maltose, maltotriose, melecitosa, palatinose, D-raffinose, D-sorbitol, D-trehalose, xylitol, L-xylose and hydrates thereof. The cyclic peptides used in the present invention can be produced by culturing various microorganisms. Suitable initial natural product materials belonging to the echinocandin cyclic peptide family include echinocandin B, echinocandin C, echinocandin D, aculeacin A ?, mulundocandin, sporiofungin A, pneumocandin A0, F11899A and pneumocandin B0. In general, the cyclic peptides may be characterized as a cyclic hexapeptide nucleus with an amino group acylated at one of the amino acids. The amino group of the naturally occurring cyclic peptide is typically acylated with a fatty acid group that forms a side chain outside the nucleus. Examples of naturally occurring acyl groups include linoleoyl (echinocandin B, C and D), palmitoyl (aculeacin Aβ and WF11899A), stearoyl, 12-methylmiristoyl (mulundocandin), 10, 12-desmethylmyristoyl (sporiofungin A and pneumocandin). A0) and similar. Semi-synthetic derivatives can be prepared by removing the side chain of the fatty acid from the cyclic peptide core to produce a free amino group (ie, a non-pending acyl group -C (O) R). The. free amine is then reacted with a suitable acyl group. For example, the echinocandin B core has been reacted with certain side chain portions that do not occur unnaturally to provide many antifungal agents. See, for example, U.S. Patent Number 4,293,489 (Debono). Those skilled in the art will appreciate that the N-acyl side chain encompasses various side chain portions known in the art. Suitable side chain portions include substituted and unsubstituted alkyl groups, alkenyl groups, alkynyl groups, aryl groups, heteroaryl groups and combinations thereof. Preferably, the side chain contains a linearly rigid section and a flexible alkyl section to maximize the antifungal potency. Representative examples of the preferred acyl side chains include R groups having the following structures: wherein A, B, C and D are independently hydrogen, alkyl of 1 to 12 carbon atoms, alkynyl of 2 to 12 carbon atoms, alkoxy of 12 carbon atoms, alkylthio of 1 to 12 carbon atoms, halo, -O- (C ^ m- [O - (CH2) p-0- (alkyl of 12 carbon atoms) or -O- (CH gXE; m is 2, 3 or 4; n is 2, 3 or 4 p is 0 or 1, q is 2, 3 or 4, X is pyrrolidino, piperidino or piperazino, and E is hydrogen, alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 12 carbon atoms, benzyl or cycloalkylmethyl of 3 to 12 carbon atoms As indicated above, the cyclic peptides described herein can be prepared by fermentation of known microorganisms as described in the art The subsequent deacylation is typically carried out enzymatically using a deacylase enzyme by known materials and methods described in the art For example, U.S. Patent No. 3,293,482 describes the deac tion and preparation of the cyclic peptide of formula I, wherein R4, R5 and Rla are methyl, R9 is hydrogen, and R1 t R3, R6, R8 and R10 are each hydroxy. U.S. Patent No. 4,299,763 describes the deacylation and preparation of the cyclic peptide of formula I wherein R4, R5 and RX1 are methyl, R2 is hydroxy, R7 and R9 are hydrogen and Rlf R2, R3, R6, R7, R8, and R10 are each hydroxy. U.S. Patent No. 3,978,210 describes the preparation of aculeacin. U.S. Patent No. 4,304,716, describes the deacylation and preparation of the cyclic peptide of formula I wherein Rs is -CH2C (0) NH2; R1X is methyl, R4 and R9 are hydrogen; R1; R2, R3, R6, R7, R8 and R10 are each hydroxy and the acyl group with the R substituent is myristoyloyl. The cyclic peptides wherein R2 and R7 are each hydrogen, can be prepared by subjecting the corresponding compound (wherein R2 and R7 are each hydroxy, the ornithine alpha-amino group can be a free or acylated amino group) to an acid strong and a reducing agent at a temperature between -5 ° C and 70 ° C, in a suitable solvent. Suitable strong acids include trichloroacetic acid, trifluoroacetic acid or boron trifluoride heterate. A preferred strong acid is trifluoroacetic acid. Suitable reducing agents include sodium cyanoborohydride or, triethylsilane. A preferred reducing agent is triethylsilane. Suitable solvents include methylene chloride, chloroform or acetic acid, preferably methylene chloride. The strong acid is present in an amount of about 2 to 60 moles per mole of substrate, and the reducing agent is present in an amount of about 2 to 60 moles per mole of substrate. The acid reduction process selectively removes the hydroxy amine (R2) and benzylic (R7) groups. The acylation of the a-amino group in the ornithine unit can be carried out in various ways well known to those skilled in the art. For example, the amino group can be acylated by reaction with an appropriately substituted acyl halide, preferably in the presence of an acid scavenger such as tertiary amine (for example triethylamine). The reaction is typically carried out at a temperature between about -20 ° C and 25 ° C. Suitable reaction solvents include polar aprotic solvents, such as dioxane or dimethylformamide. The choice of solvent is not critical to the extent that the solvent used is inert to the reaction carried out and the "reagents are sufficiently solubilized to carry out the desired reaction.The amino group can also be acylated by reaction with an appropriately substituted carboxylic acid, in the presence of a coupling agent Suitable coupling agents include dicyclohexylcarbodiimide (DCC), N, N'-carbonyldiimidazole, bis (2-oxo-3-oxazolidin) phosphinic chloride (BOP-Cl), N ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate (PyBOP) and the like., the amino group can be acylated with an activated ester of a carboxylic acid such as p-nitrophenyl, 2,4,5-trichlorophenyl, hydroxybenzotriazole hydrate (H0BT-H20), pentafluorophenol and N-hydroxysuccinimide carboxylate esters. Preferred acylating portions are 2, 4, 5-trichlorophenyl and carboxylate esters of HOBT. The reaction is typically carried out for 1 to 65 hours at a temperature of about 0 ° C to 30 ° C in an aprotic solvent. The reaction is generally complete after about 24 to 48 hours when carried out at a temperature between about 15 ° C and 30 ° C. Suitable solvents include tetrahydrofuran and dimethylformamide or mixtures thereof. The amino group is generally present in equimolar proportions relative to the activated ester or with a slight excess of the amino group. The precursor acids R-COOH are prepared by hydrolyzing a nitrile of the formula R-CN or an ester of the formula (R-COO (alkyl of 1 to 4 carbon atoms) The nitrile and ester intermediates can be prepared using known in the art For example, the nitrile and ester intermediates, wherein R is an alkoxyaryl moiety, can be prepared using process A or process B.

Procedure A An equivalent of an alkyl bromide, iodide or p-toluenesulfonate is added to a mixture containing one equivalent of a base, such as potassium t-butoxide or potassium carbonate (K, C03) and one equivalent of a compound hydroxyaryl, in 200-300 ml of acetonitrile (CH3CN). The reaction mixture is refluxed for 6 h and then concentrated in vacuo to provide a residue which is dissolved in a mixture of Et20 / 2N NaOH. The resulting layers are separated and the organic layer is dried over magnesium sulfate (MgSO4), filtered and dried to provide an alkoxyaryl product.

Procedure B The equivalent of diethyl azodicarboxylate is added dropwise to a mixture containing 1 equivalent of a hydroxyaryl compound, 1 equivalent of alkyl alcohol and 1 equivalent of triphenylphosphine in 200-300 ml of THF. After 17 h the solvent is removed in vacuo to give a residue which is dissolved in Et20. The resulting mixture is washed with a 2N NaOH solution, dried over MgSO4, filtered and concentrated to give a product which is then crystallized from a mixture of Et20 / pentane or, if the product contains a tertiary amine, forms the hydrochloride salt and crystallizes from a mixture of methanol (MeOH) / EtOAc. The nitrile and ester intermediates, wherein R is an alkynylaryl moiety, can be prepared using the following procedure C.

Procedure C A mixture containing two equivalents of Et20, 0.05 equivalents of palladium dichloride, 0.1 equivalent of triphenylphosphine, 0.025 equivalents of cuprous iodide and 1 equivalent of an alkyne is added to one equivalent of aryl bromide, iodide or trifluoromethanesulfonate in CH3CN ( 600 ml / 0.1 mol of aryl reagent) under nitrogen (N2). The resulting mixture is refluxed for 17 h and then the solvent is removed in vacuo to provide a residue which forms a suspension in 300 mL of Et20 and is then filtered. The filtrate is washed with a solution of IN HCl, dried over MgSO4, filtered and then dried to provide the product. The ester intermediates wherein R is a terphenyl portion, can be prepared using the following procedure D.

Procedure D 1. Formation of boronic acid reagent 1.2 equivalents of butyllithium are added to one equivalent of a cold aryl halide (-78 ° C) in THF. After 15 minutes, 2 equivalents of triisopropyl borate are added. After 10 minutes, the reaction mixture is warmed to room temperature and suspended by the addition of water (H20), followed by the addition of IN HCl. The resulting layers are separated and the organic layer is concentrated in vacuo to provide a solid which is collected by filtration and washed with hexane. 2. Formation of the terphenyl ester 0.03 equivalents of tetrakis- (triphenylphosphine) palladium are added to a mixture containing 1 equivalent of arylboronic acid, 1.5 equivalents of K2C03 and 1 equivalent of methyl 4-iodobenzoate (or the trichlorophenyl ester of iodobenzoate) in toluene purged with N2. The reaction mixture is refluxed for 7 h and then decanted to remove K2C03 and dried in vacuo to provide a residue. This residue is triturated in CH3CN and filtered to provide the product. The aryl nitriles and esters described above can be converted to the corresponding carboxylic acids by hydrolysis using the following procedure E or process F.

Procedure E Dissolve aryl nitrile in ethanol (EtOH) and an excess of a 50% NaOH solution and reflux for 2 h. Water is added to the reaction mixture until a solid precipitates. This solid is collected by filtration, added to a mixture of dioxane / 6N HCl and the resulting mixture is refluxed for 17 h. When the reaction is essentially complete, the carboxylic acid product crystallizes by the addition of H20 and is then collected by filtration and dried in vacuo.

Method F An excess of 2N NaOH is added to an aryl ester in MeOH, and the resulting solution is refluxed for 5 h and then acidified by the addition of excess HCl. Water is added to the reaction mixture until a solid precipitates (carboxylic acid). The carboxylic acid is collected by filtration and dried in vacuo.

The carboxylic acids can be converted to the corresponding 2, 4, 5-trichlorophenyl esters using the following procedure G. The activated esters are then used to acylate the amino nucleus.

Procedure G A mixture containing 1 equivalent of arylcarboxylic acid, 1 equivalent of 2,4,5-trichlorophenol and 1 equivalent of DCC in CH-C 12 is stirred for 17 h and then filtered. The filtrate is concentrated to provide a residue which is dissolved in Et20, filtered and then pentane is added until crystallization begins. The crystals are collected by filtration and dried in vacuo. Alternatively, the carboxylic acid can be activated by conversion to the corresponding hydroxybenzotriazole ester using the following procedure H.

Process H 1 equivalent of arylcarboxylic acid and a slight excess of 1.2 equivalents of hydroxybenzotriazole substituted with N-mesylate are reacted in the presence of a slight excess of a base such as 1.3 equivalents of triethylamine (Et3N) (1.3 equivalents of triethylamine ) in DMF, under N2. When the reaction is complete, the mixture is diluted with toluene and washed with H20. The organic portion is diluted with H20 and then filtered using t-butyl methyl ether (MTBE). The resulting solid is washed with MTBE and then dried in vacuo. The echinocandin compound can be isolated and used per se or in the form of its pharmaceutically acceptable salt or hydrate in the preparation of the carbohydrate complexes. The carbohydrate complex with the echinocandin compound is prepared as described above. The term "pharmaceutically acceptable salt" refers to non-toxic acid addition salts derived from inorganic and organic acids. Suitable salt derivatives include halides, thiocyanates, sulfates, bisulphates, sulphites, bisulfites, aryisulfonates, alkyl sulfates, phosphonates, monoacid phosphates, diacid phosphates, metaphosphates, pyrophosphonates, alkanoates, cycloalkylalkanadates, arylalkates, adipates, alginates, aspartates, benzoates, fumarates, glycoheptanoates, glycerophosphates, lactates, maleates, nicotinates, oxalates, palmitates, pectinates, picrates, pivalates, succinates, tartrates, citrates, canforates, camphorsulfonates, digluconates, trifluoroacetates and the like. A typical solution formulation is prepared by mixing the echinocandin / carbohydrate complex and a surfactant (preferably a micelle-forming surfactant) in a solvent. The formulation may optionally include one or more of a buffer, a stabilizing agent or a tonicity agent. Solvents are generally selected on the basis of what is recognized as safety (GRAS) to be administered parenterally to a mammal. In general, harmless solvents are non-toxic aqueous solvents such as water or other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (for example PEG400, PEG300), etc., and mixtures thereof. A preferred solvent is water. A typical lyophilization formulation includes the echinocandin / carbohydrate complex, a surfactant (preferably a micelle-forming surfactant) a bulking agent or a stabilizing agent, or both. The addition of a micelle-forming surfactant not only optimizes the reconstitution of the lyophilized formulation in an aqueous solvent but also provides improved stability to lyophilized materials. The formulation may optionally include one or more buffering agents. Some examples of suitable parenteral solution and lyophilization formulations include their preparations and can be found in the United States patent application serial number 60/122., 623. Both the solution and freeze-dried formulations can optionally contain a stabilizing agent. A stabilizing agent is generally present at a concentration in the range of about 0.5% to about 40% (w / vol), more preferably at a concentration in the range of about 1% to about 6%. The term "stabilizing agent" refers to a pharmaceutically acceptable excipient that improves the chemical and physical stability of the active ingredient in the formulation. Suitable stabilizing agents include polyols (for example polyethylene glycols and propylene glycols, and carbohydrates such as sucrose, trehalose, fructose, lactose and mannitol), amino acids and surfactants such as polysorbates and bile salts. Preferred stabilizing agents for lyophilized formulations include mannitol, sucrose, trehalose, fructose, lactose and combinations thereof. In solution, the most preferred stabilizing agents are bile salts, polyethylene glycols and propylene glycols. Formulations for solution and lyophilization may optionally also contain a buffer. The buffer is present at a concentration in the range of about 0.03% to about 5% (w / v), more preferably at a concentration in the range of about 0.1% to about 1%. The term "buffer" refers to a pharmaceutically acceptable excipient which maintains the pH of the solution within a particular range specific to the buffer system. A suitable pH range is from pH 3.0 to 7.0. The preferred range is from 4.0 to 5.5, more preferably from 4.0 to 5.0. Suitable buffers include acetates, citrates, phosphates, tartrates, lactates, succinates, amino acids and the like. Preferred buffers for the solution formulation include acetate, citrate, tartrates, phosphate salts and combinations thereof. In the lyophilized formulation, the preferred buffer is tartaric acid. The solution formulation optionally may contain one or more tonicity agents. The tonicity agent is generally present in a concentration in the range of about 1 to about 100 mg / ml, more preferably in the range of about 9 to about 50 mg / ml. The term "tonicity agent" refers to a pharmaceutically acceptable excipient that returns to the solution compatible with the blood. Tonicity agents are particularly desirable in injectable formulations. Suitable tonicity agents include glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol and the like. Preferred tonicity agents include mannitol, sorbitol, lactose, sodium chloride and combinations thereof. When lyophilized, the formulations may optionally contain a bulking agent. The bulking agent is present in a formulation at a concentration in the range of about 2% to about 10% (w / v), more preferably at a concentration in the range of about 3% to about 6%. The term "bulk agent" refers to a pharmaceutically acceptable excipient that adds bulk to a formulation resulting in a well-formed cake when lyophilized. Suitable bulking agents include mannitol, glycine, lactose, sucrose, trehalose, dextran, hydroxyethyl starch, ficoll and gelatin. Preferred volume agents include mannitol, sucrose, trehalose, lactose and combinations thereof. The formulations can be prepared using conventional mixing dissolution procedures. For example, the volume drug substance is dissolved (e.g. the echinocandin / carbohydrate complex) in a suitable solvent in the presence of a surfactant and optionally one or more bulking agents, buffers, stabilizing agents or tonicity agents, or any of them in any combination. The resulting solution is sterilized by filtration and preferably lyophilized to provide the desired formulation. Prior to lyophilization, the surfactant is generally present in an amount greater than 1% by weight per volume of solution. A suitable method for freeze-drying is described in Nail et al., Freeze Dryng Principles and Practice, in Pharmaceutical Dosage Forms, 2nd Ed., Marcel Dekker, Inc. NY, pp. 163-233 (1993). In general, lyophilization formulations contain a bulking agent and non-lyophilized formulations contain one or more tonicity agents. When applied, the formulations are typically diluted or reconstituted (if lyophilized) and further diluted, if necessary, before administration. An example of reconstitution instructions for the lyophilized product is to add ten ml of water for injection (WFI) to the bottle and shake gently to dissolve. Typical times of reconstitution are less than one minute. The resulting solution is then further diluted in an infusion solution such as 5% dextrose in water (D5W) before its administration. The active ingredient is typically formulated in pharmaceutical dosage forms to provide an easily controllable dosage of the medicament and to provide the patient with an elegant and easily manageable product. The formulations may comprise from 0.1% to 99.9% by weight of active ingredient, more generally from about 10% to about 30% by weight. As used herein, "unit dose" or "unit dosage" refers to physically separate units that contain a predetermined amount of active ingredient calculated to produce the desired therapeutic effect. When a unit dose is administered orally or parenterally, it is typically provided in the form of a tablet, capsule, pill, powder pack, topical composition, suppository, wafer, units measured in ampoules or in multi-dose containers, etc. Alternatively, a unit dose in the form of a dry or liquid aerosol can be administered which can be inhaled or sprayed. The dosage to be administered may vary based on the physical characteristics of the patient, the severity of the patient's symptoms and the means used to administer the medication. The specific dose for a given patient is usually established based on the judgment of the attending physician. Suitable carriers, diluents and excipients are well known in the art and include materials such as carbohydrates, waxes, soluble or expandable polymers in water, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The carrier, diluent or particulate excipient used will depend on the medium and purpose for which the active ingredient is applied. The formulations may also include wetting agents, lubricating agents, emulsifiers, suspension-improving agents, preservatives, sweeteners, perfume-providing agents, flavoring agents, and combinations thereof. A pharmaceutical composition can be administered using various methods. Suitable methods include topical (eg ointments or sprays), oral, injection and inhalation. The particular treatment method will depend on the type of infection to be corrected. Echinocandin type compounds have been shown to exhibit antifungal and antiparasitic activities such as inhibition of growth of various infectious fungi including Candida spp. (ie C. albicans, C. parapsilosis, C. krusei, C. glabrata, C. tropicalie or C. lusi taniaw), - Torulopus spp. (ie, T. glabrata), Aspergillus spp. (ie, A. igatus); Histoplasma spp (ie H. capsulatum); Cryptococcus spp (ie, C. neoformans); Blastomyces spp (ie, B. der ati ti dis); Fusarium spp .; Trichophyton spp., Pseudalleecheria boydii, Coccidioides i mi ts, Sporothrix schenckii, etc. Compounds of this type also inhibit the growth of certain organisms mainly responsible for opportunistic infections in immunosuppressed individuals such as inhibition of the growth of Pneumocystis carinii (the causative organism of pneumocyst pneumonia (PCP) in AIDS and other immunocompromised patients.) Other protozoa which are inhibited by compounds of the echinocandin type include Plasmodium spp., Leishmania spp., Trypanosoma spp., Cryptosporidium spp., Isospora spp., Cyclospora spp., Trichomnas spp., Microsporidiosis spp., etc.

Consequently, the formulations generated are useful for combating systemic fungal infections or fungal skin infections. Accordingly, the formulations and processes of the present invention can be used in the manufacture of a medicament for the therapeutic applications described herein. For example, fungal activity can be inhibited (preferably Candida albicans or Aspergillus fumigatis activity) or parasitic activity can be inhibited by contacting the echinocandin / carbohydrate complex of the present invention with a fungus or parasite, respectively. The term "contacting" includes a joint or joint, or apparent mutual tangential contact of a compound of the invention with a parasite or fungus. The term does not imply any additional limitation to the process, for example by a mechanism of inhibition. The methods are defined to encompass the inhibition of parasitic and fungal activity by the action of the compounds and their inherent antiparasitic and antifungal properties. A method for treating a fungal infection which comprises administering an effective amount of a pharmaceutical formulation prepared by the present invention to a host in need of such treatment is also provided. A preferred method includes treating an infection with Candida albicans or Aspergillus fumigatis. The term "effective amount" refers to an amount of active compound which is capable of inhibiting fungal activity. The dose administered will vary depending on factors such as the nature and severity of the infection, the age and general health of the host and the tolerance of the host to the antifungal agent. The particular dose regimen can likewise vary according to these factors. The medication can be administered in a single daily dose or in multiple doses during the day. The regimen can last from approximately 2-3 days to approximately 2-3 weeks or more. A typical daily dose (administered in single or divided dose) contains a dosage concentration between about 0.01 mg / kg and 100 mg / kg of body weight of an active compound. Preferred daily doses are generally between about 0.1 mg / kg and 60 mg / kg, and more preferably between about 2.5 mg / kg and 40 mg / kg. The following examples are provided to illustrate, but not to limit, the invention. All references mentioned herein are incorporated herein by reference.

EXAMPLES The echinocandin compound used to exemplify the formulations of the present invention is prepared as described in the following preparations. Specifically, the following sequence describes the preparation of a complex carbohydrate (fructose) with an echinocandin of compound 6 (a) having the following structure: It will be understood by those skilled in the art that the following serves as an illustrative example and that other semi-synthetic echinocandin compounds useful as antifungal agents can be synthesized using similar procedures or procedures described in the references mentioned above in the specification. Materials used in the following preparations are available from Aldrich Chemicals (Milwaukee, Wisconsin) unless otherwise designated.

Compound preparations Preparation of 4-bromo-4'-pentyl-1-oxy-phenyl-Ka): Anhydrous K2C03 (416 g, 3 moles) is added to a mixture of 4-bromo-4'-hydroxybiphenyl (300 g, 1.2 moles), 1-iodopentane (234 ml, 1.79 mmoles) and 600 ml of 2-butanone. The reaction mixture is refluxed for 44 h until the CCD (85:15 hexanes / EtOAc) completes the bromoalcohol consumption. This mixture is cooled to about 30 ° C, diluted with 600 ml of CH2C12 and then filtered. The filtrate is washed twice with H20 and twice with a saturated aqueous solution of NaCl, dried over anhydrous Na2SO4, filtered and then dried under reduced pressure to provide a solid. This solid is isolated by filtration, washed repeatedly with a total of 2 1 of ice-cold heptane to remove all traces of iodopentane and after drying overnight under high vacuum. Performance; 340 g (88%) of a white powder.

Preparation to the ternativa of 4-bromo-4 '-pent iloxibi phenyl Ka): 4-Bromo-4'-hydroxybiphenyl (12.5 g, 50.2 mmol) was added to a solution of NaOH (2.28 g, 97% pure, 55.2 mmol) in 150 mL of deionized HjO, followed by the addition of l-iodopentane (11.9 g). g, 60.2 mmoles) and tetrabutylammonium bromide (0.82 g, 2.51 mmoles). The mixture is stirred at 90 ° C for 3.75 h until the solids are placed in solution. Subsequently, as the reaction progresses, the desired product begins to precipitate. The mixture is cooled slowly and then filtered to provide a solid which is washed with deionized water until the pH of the filtrate is neutral and then dried for 16 h in a vacuum oven at 30 ° C. Yield: 15.41 g (96%) of 5a, Rf 0.5 (97: 3 hexanes / EtOAc). 1 H NMR: d 0.93 (t, 3H, J = 6.9 Hz), 1.41 (m, 4H); 1.79 (m, 2H); 3.97 (t, 2H, J = 6.6 Hz); 6.98 (m, 2H); 7.23 (m, 6H). 13 C NMR: d 14.03; 22.43; 28.22; 28.98; 68.12; 114.91; 120.71; 127.93; 128.27; 131.77; 132.24; 139.82; 159.03; MS (FAB +): m / z 320. IR (CHC113): 2960, 2936, 2874, 1608, 1518, 1485, 1475 cpr1. Analysis for C17H19BrO: Calculated: C, 63.96; H. 6.00; Br, 25.0; Found: C, 64.10; H. 5.97; Br. 25.28.

Preparation of 4'-pentyloxybhenyl phenyl 4 -boronic acid 2 (a) -.

To a cold mixture at -20 ° C of compound Ka) (100 g, 0.31 mol) in 1 1 of t-butyl methyl ether (MTBE) is slowly added dropwise under N, n-butyllithium (150 ml of a 2.5 M solution in hexanes, 0.37 moles), while maintaining the internal temperature between -19 ° and -18 ° C. The resulting mixture is stirred for 3.5 h between -17 ° and -16 ° C, resulting in a light yellow-green solution. This solution is cooled to -78 ° C and diluted with 100 ml of anhydrous THF, resulting in a white precipitate. Then a cold (-78 ° C) solution of triisopropyl borate (145 ml, 0.62 mol) in 200 ml of MTBE under nitrogen is added dropwise, during 1.5 h, while maintaining the reaction temperature between -78 ° and - 74 ° C. The resulting reaction mixture is stirred for 1.5 h at -78 ° C and then allowed to warm to -50 ° C for 1 h, at which time the cooling bath is removed and the mixture is stirred overnight (16-). 21 h) resulting in a white precipitate. The mixture is stirred vigorously with 1000 ml of 2 M HCl for 5 minutes and then the resulting layers are separated and the organic layer is dried under reduced pressure to provide a residue. This residue is diluted with 100 ml of MTBE, followed by 800 ml by heptane to give a white powder which is isolated by suction filtration and washed three times with 300 ml of heptane.

Yield: 88 g (98%). Rf 0.45 (95: 5 CH2Cl2 / MeOH). X H NMR: 0.92 (m, 3H); 1.41 (m, 4H); 1.80 (m, 2H); 4.00 (m, 2H); 6.99 (m, 2H); 7.45-7.63 (m, 3H); 7.67 (m, 2H); 8.24 (d, 1H, J = 8.3 Hz). 13 C NMR: 1401; 22.26; 28.03; 28.77; 39.61; 39.89; 40.17; 40.45; 67.82; 114.77; 125.32; 127.83; 132.93; 134.84; 141.88; 158.71. MS (FD +): m / z 284. IR (CHC13): 2959, 2952, 2874, 1606, 1526, 1500 cpf1.

Preparation of compound 3 (a): 3 (a) A solution of 174 ml of toluene and 20 ml of propanol is degassed 3 times by applying vacuum to the solution for 20-30 seconds, followed by purging with N2. A 2M solution of Na 2 CO 3 is also degassed. 97 ml of toluene / propanol solution is added to a mixture of methyl 4-iodobenzoate (14.12 g, 53.9 mmol) and of compound 2 (a) (15.0 g, 52.8 mmol), followed by a degassed 2M aqueous Na2CO3 solution. (29 ml, 58.0 mmol). The resulting mixture is degassed twice for 20-30 seconds each under a positive pressure of N2, followed by the addition of palladium (II) acetate (0.24 g, 1.1 mmol) and triphenylphosphine (0.84 g, 3.2 mmol) and then it degass twice more. The reaction mixture is then refluxed under N2 for 5 h, resulting in a light yellow mixture. This mixture is cooled to 23 ° C, which results in the formation of a precipitate which is collected by filtration, washed successively with 123 ml of toluene, 143 ml of 2: 1 MTBE / EtOAc, 123 ml of deionized water and 42 ml of 2: 1 MTBE / EtOAc and then dried for 16 h in a vacuum oven at 35 ° C. Yield: 18.7 g (94%). Rf 0.48 (benzene) XH NMR: d 0.93 (t 3H, J = 6.80 Hz); 1.42 (m, 4H); 1.81 (m, 2H), 3.95 (s, 3H); 4.00 (t, 2H, J = 6.48 Hz); 6.97 (d, 2H, J = 8.52 Hz); 7.55 (d, 2H, J = 8.52 Hz); 7.66 (m, 6H); 8.10 (d, 2H, J = 8.20 Hz). MS (FD +): m / z 374. IR (KBr); 2938, 1723 crn "1. Analysis calculated for C25H2603: Calculated: C, 80.18; H, 7.00; Found: C, 79.91; H. 6.94.

Preparation of compound 4 (a): 4 (a) A mixture of compound 3 (a) (80 g, 0.21 mol), 160 ml of 5M KOH and cetyltrimethylammonium bromide (4.8 g, 0.013 mol) in 800 ml of xylene is refluxed for 3 h and then cool to 10 ° C and filter to give a white solid. This solid is washed three times with 500 ml each of H20 to remove the catalyst and most of the base. The resulting material is treated with 500 ml of DME. The pH of the solution is adjusted to pH by the addition of 100 ml of 6M HCl. The resulting mixture is refluxed for 30 minutes while the pH is periodically checked to ensure that acid remains, then cooled and filtered. The resulting solid is washed successively with 400 ml of MTBE and water (4 x 400 ml) until the washes are neutral with litmus. Yield: 76 g (98% yield). XH NMR 0.89 (t, 3H, J = 6.82 Hz), 1.38 (m, 4H), 1.73 (m, 2H), 3.96 (t, 2H, J = 6.3 Hz), 6.95 (d, 2H, J = 8.56 Hz), 7.57 (d, 2H, J = 8.54 Hz), 7.64-7.74 (m, 6H), 8.00 (d, 2H, J = 8.21 Hz), 8.09 (s, 1H). MS / FD +) m / z 360. IR (KBr): 2958, 2937, 2872, 1688 cm "1. Analysis for C24H2403: Calculated: C, 79.97; H, 6.71; Found: C, 80.50; H. 6.77.

Preparation of the HOBT ester of compound 4 (a): TO . HOBT mesylate formation To a cold (0 ° C) mixture of hydroxybenzotriazole hydrate (200 g, 1.48 moles) in 1.5 1 of anhydrous CH-C12, anhydrous Et3N (268 ml, 1.92 moles) is slowly added while maintaining a temperature of 0-10. ° C, followed by the addition of methanesulfonyl chloride (126 ml, 1.63 moles) while maintaining a temperature of 0-5 ° C. The resulting mixture is stirred for 3 h at 0 ° C and washed successively with cold water (2 x 1.2 1) and 1.2 1 of brine. The combined organic extracts are concentrated under reduced pressure to provide a solid. This solid recrystallizes from 100 ml of CH2C12 and 1 l of heptane. The crystals are collected by suction filtration and washed repeatedly with a total of 1 l of heptane and then dried overnight under high vacuum (0.5 mmHg). Yield: 245 g (78%) Rf 0.55 (1: 1 hexanes / CH2C12). NMR? E: d 3.58 (s, 3H), 7.46 (t, 1H, J = 7.60 Hz), 7.60 (d, 1H, J = 8.28 Hz), 7.65 (d, 1H, J = 8.56 Hz), 7.68 ( d, 1H, J = 8.20 Hz), 8.05 (d, 1H, J = 8.41 Hz).

B. Formation of the HOBT ester A mixture of compound 4 (a) (50 g, 0.14 mole) and the material described above in part A (36 g, 0.17 mole) in 650 ml of DMF is treated dropwise with Et ^ N (25 ml, 0.18 mole) ) under N2. The resulting mixture is stirred for 4 h at room temperature until all the acid is consumed, determined by CCD (95: 5 CH2Cl2 / MeOH). When all the acid is consumed, an aliquot of the reaction mixture (~ 3 drops of tube) provides a clear homogenous solution when diluted with 3 ml of 1: 1 CH2C12 / THF. The reaction mixture is then diluted with 500 ml of toluene, washed with 500 ml of water. The organic layer (containing solid product) is diluted with 500 ml of water and filtered using MTBE for transfer. The solid is rinsed with MTBE (2 x 400 ml) and dried under vacuum to provide green-white flake material. NOTE: This material can be dissolved in THF and filtered to remove any remaining metal contamination. Yield: 61 g (92%). Rf 0.68 (1: 1 CH2C12 / hexanes). XH NMR: d 0.93 (t, 3H, J = 7.0 Hz), 1.42 (m, 4H), 1.81 (m, 2H), 4.00 (t, 2H, J = 6.53 Hz), 6.99 (d, 2H, J = 8.6 Hz), 7.42-7.59 (m, 5H), 7.71 (dd, 4H, J = 13.91 Hz, 8.40 Hz), 7.86 (d, 2H, J = 8. 30 Hz), 8.11 (d, 1H, J = 8.31 Hz), 8.35 (d, 2H, J = 8.33 Hz). 13 C NMR: d 14.03, 22.44, 28.18, 28.94, 40.10, 40.37, 68.11, 108.45, 110.11, 114.95, 118.71, 120.48, 123.04, 124.94, 124.99, 127.00, 127.23, 127.51, 127.73, 128.06, 128.82, 128.86, 131.35, 132.30, 137.15, 141.43, 143.54, 147.85, 159.15, 162.73. MS (FD +): m / z 477. IR (CHC13): 2960, 2936, 2874, 1783, 1606 cnT1. Analysis for C 30 H 27 N 3 O 3: Calculated: C, 75.45; H, 5.70; N, 8.80: Found: C, 75.69; H, 5.58; N, 8.92.

Preparation of antifungal compound 6 (a): Deionized water is used throughout the procedure. A mixture of compound 5 (a) (11 g, 23 mmoles) = and the core of compound 6 (a) (wherein R is hydrogen -92% pure by HPLC, 19.25 g, 22.2 mmoles) in 275 ml of anhydrous DMF it is stirred under N2 for 4 h (until the CLAR shows complete consumption of the cyclic peptide of the initial material). The mixture is filtered through a pad of Celite and the filtrate is concentrated under reduced pressure at 35 ° C to provide a paste that can be stirred. This paste is poured into 500 ml of MTBE which results in the precipitation of a fine powder which is collected by vacuum filtration and dried to provide 27 g of crude material. This material is crushed to a powder with a mortar and pestle, a suspension is formed for 5 minutes in 200 ml of toluene, filtered by suction (slow filtration), rinsed with 100 ml of MTBE and then dried in vacuo to provide a yellow solid. Yield: 23 g (95% pure by HPLC, retention time = 7.79 min). Alternatively, the conversion can be carried out using an excess of the cyclic core (1.1 equivalents). When the reaction is substantially complete, as indicated by HPLC, 10 g of a powder of crude material is added in portions to a vigorously stirred mixture of 9: 1 acetone / water (60 ml). To the resulting suspension is added Celite (2.5 g, previously washed with 91 of an acetone / water mixture). After stirring for 2 minutes, the mixture is filtered through a pad of Celite (previously washed with 9: 1 acetone / water) and the cake is rinsed twice with 10 ml of 9: 1 acetone / water. The filtrate is poured into a beaker with 200 ml of deionized water while gently stirring the mixture resulting in the formation of a precipitate. This precipitate is collected by suction filtration, rinsed with H20 (4 x 25 ml) and then dried in vacuo at room temperature. Yield: 6.81 g (97% pure by HPLC). The product is further purified using preparative HPLC chromatography. Rf 0.29 (80:20 CHCl3 / MeOH). MS (FAB +): m / z for C 58 H 74 N 707, Calculated: 1140.5141; Found: 1140.5103. IR (KBr): 3365, 2934, 1632, 1518 cm "1.

Preparation of a fructose complex with a compound 6 (a): A jacket reactor is charged with 1 equivalent of compound 6 (a), 8 equivalents of fructose and a sufficient amount of methanol to make 58 mg / ml of compound l (a). The mixture is heated to 50-55 ° C until the solution is complete. The solution is cooled to 45 ° C. After seeding at 45 ° C, the seeded solution is cooled to 25 ° C at a cooling rate of -2o / hour. The mixture is further cooled to 0 ° C for 2 hours (cooling speed = -12.5 ° / hour) and then stirred at 0 ° C for 12 hours. The product is isolated by vacuum filtration, washed with cold methanol containing 1% fructose, on a weight / weight basis, and then dried for 24 hours in a 30 ° C vacuum oven. The assays are performed in a gradient HPLC system equipped with a particle size of 15 cm × 4.6 mm, 3.5 μm Zorbaz ™ with an analytical column SB-C18 or XDB-C18. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (27)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An echinocandin / carbohydrate complex, characterized in that it comprises a carbohydrate and an echinocandin compound represented by the following structure: where: R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or combinations thereof, - R1 # R2, R3, R6, R7 and R10 are independently hydroxy or hydrogen, - R4 is hydrogen, methyl or -CH2C (0) NH2; Rs and Rn are independently methyl or hydrogen, - R8 is -OH, -OS03H, -OP03H2, -OP03HRa, or -OP02HRa, wherein Ra is hydroxy, alkyl of 1 to 6 carbon atoms, alkoxy of 1 to 6 carbon atoms, carbon, phenyl, phenoxy, p-halophenyl, p-halofenoxy, p-nitrophenyl, p-nitrophenoxy, benzyl, benzyloxy, p-halobenzyl, p-halobenzyloxy, p-nitrobenzyl or p-nitrobenzyloxy, - R9 is -H, -OH or -0S03H; and pharmaceutically acceptable salts or hydrates thereof.

2. The complex according to claim 1, characterized in that R4, R5 and RX1 are each methyl, - R2 and R7 are independently hydrogen or hydroxy; R1 t R3, R6 and R10 are each hydroxy; Ra is -OH, -OP03HRa or -OP02HRa wherein Ra is methyl, -R is linoleoyl, palmitoyl, stearoyl, myristoyl, 12-methylmiristoyl, 10, 12-dimethylmiristoyl or a group having the general structure: wherein A, B, C and D are independently hydrogen, alkyl of 1 to 12 carbon atoms, alkynyl of 2 to 12 carbon atoms, alkoxy of 12 carbon atoms, alkylthio of 1 to 12 carbon atoms, halo, or -O- (CH-) m- [O- (CH 2) p-0- (alkyl of 12 carbon atoms) or -O- (CH 2) qXE; m is 2, 3 or 4; n is 2, 3 or 4; p is 0 or 1; q is 2, 3 or 4, - X is pyrrolidino, piperidino or piperazino; E is hydrogen, alkyl of 1 to 12 carbon atoms, cycloalkyl of 3 to 12 carbon atoms, benzyl or cycloalkylmethyl of 3 to 12 carbon atoms.

3. The complex according to claim 2, characterized in that: R2 and R7 are each hydroxy; R8 is hydroxy; Y

4. The complex according to claim 1, characterized in that the carbohydrate is selected from the group consisting of adonitol, arabinose, arabitol, ascorbic acid, chitin, D-cellobiose, 2-deoxy-D-ribose, dulcitol (S) - (+ ) -eritrulose, fructose, fucose, galactose, glucose, inositol, lactose, lactulose, lixose, maltitol, maltose, maltotriose, mannitol, mannose, melezitose, melibiose, microcrystalline cellulose, palatinos pentaerythritol, raffinose, rhamnose, ribose, sorbitol, sorbose, starch, sucrose, trehalose, xylitol, xylose and hydrates thereof.

5. The complex according to claim 3, characterized in that the carbohydrate is selected from the group consisting of L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, (S) - (+) - erythrulose , D-fructose, D- (+) -fucose, L-fucose, D-galactose, aD-glucose, β-D-glucose, L-glucose, D-lixose, L-lixose, maltitol, D-maltose, maltotriose , D-mannose, melezitose, palatinose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, D-trehalose, xylitol, L-xylose and hydrates thereof.

6. The complex according to claim 5, characterized in that the carbohydrate is selected from the group consisting of L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, (S) - (+) - erythrulose, D-fructose, D- (+) -fucose, L-fucose, D-galactose, β-D-glucose, D-lixose, L-lixose, D-maltose, maltotriose, melecitosa, palatinose, D-refining, D- sorbitol, D-trehalose, xylitol, L-xylose and hydrates thereof.

7. An echinocandin / carbohydrate complex, characterized in that it is prepared by the steps of: (a) providing an echinocandin compound represented by the following structure wherein: R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group or combinations thereof, Rn 2 / R31 R6? And R? O are independently hydroxy or hydrogen; R4 is hydrogen, methyl or -CH2C (0) NH2, - Rs and Rn are independently methyl or hydrogen, - R8 is -OH, -OSO3H, -OP03H2, -OP03HRa, or -OP02HRa, wherein Ra is hydroxy, 1 to 6 carbon atoms, alkoxy of 6 carbon atoms, phenyl, phenoxy, p-halophenyl, p-halofenoxy, p-nitrophenyl, p-nitrophenoxy, benzyl, benzyloxy, p-halobenzyl, p-halobenzyloxy, p-nitrobenzyl or p-nitrobenzyloxy; R9 is -H, -OH or -0S03H; and pharmaceutically acceptable salts or hydrates thereof, - (b) mixing together the echinocandin compound of step (a) with a carbohydrate in a solvent to form a mixture, - (c) heating the mixture for solubility the echinocandin compound and to solubilize or disperse the carbohydrate; (d) allowing the mixture to cool to produce the echinocandin / carbohydrate complex; and (e) isolating the echinocandin / carbohydrate complex.

8. The complex according to claim 7, characterized in that: R 4, R 5 and R a, are each methyl, - R 2 and R 7 are independently hydrogen or hydroxy; R1 (R3, R6 and R10 are each hydroxy, Ra is -OH, -OP03HRa or -OP02HRa wherein Ra is methyl, R is linoleoyl, palmitoyl, stearoyl, myristoyl, 12-Methylmuristoyl, 10, 12-dimethylmystroyl or a group having the general structure: wherein A, B, _C and __ are independently hydrogen, alkyl of 1 to 12 carbon atoms, alkynyl of 2 to 12 carbon atoms, alkoxy of 1 to 12 carbon atoms, alkylthio of. The 12 carbon atoms, halo, or -0- (Ot,) ,,, - [0- (CH2) p-0- (alkyl of 1 to 12 carbon atoms) or -O- (CR qXE; m is 2, 3 or 4, n is 2, 3 or 4, p is 0 or 1, q is 2, 3 or 4, X is pyrrolidino, piperidino or piperazino, E is hydrogen, alkyl of 1 to 12 carbon atoms, cycloalkyl from 3 to 12 carbon atoms, benzyl or cycloalkylmethyl of 3 to 12 carbon atoms.

9. The complex according to claim 8, characterized in that: R2 and R7 are each hydroxy; Rβ is hydroxy; Y

10. The complex according to claim 7, characterized in that the carbohydrate is selected from the group consisting of adonitol, arabinose, arabitol, ascorbic acid, chitin, D-cellobiose, 2-deoxy-D-ribose, dulcitol (S) - (+ ) -eritrulose, fructose, fucose, galactose, glucose, inositol, lactose, lactulose, lixose, maltitol, maltose, maltotriose, mannitol, mannose, melecitosa, melibiose, microcrystalline cellulose, palatinose, pentaerythritol, raffinose, rhamnose, ribose, sorbitol, sorbose , starch, sucrose, trehalose, xylitol, xylose and hydrates thereof.

11. The complex according to claim 9, characterized in that the carbohydrate is selected from the group consisting of L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, (S) - (+) - erythrulose, D-fructose, D- (+) -fucose, L-fucose, D-galactose, aD-glucose, β-D-glucose, L-glucose, D-lixose, L-lixose, maltitol, D-maltose, maltotriose, D-mannose, melezitose, palatinose, D-raffinose, L-rhamnose, D-ribose, D-sorbitol, D-trehalose, xylitol, L-xylose and hydrates thereof.

12. The complex according to claim 9, characterized in that the carbohydrate is selected from the group consisting of L-arabinose, D-arabitol, L-arabitol, 2-deoxy-D-ribose, (S) - (+) - erythrulose, D-fructose, D- (+) -fucose, L-fucose, D-galactose, β-D-glucose, D-lixose, L-lixose, D-maltose, maltotriose, melecitosa, palatinose, D-refining, D- sorbitol, D-trehalose, xylitol, L-xylose and hydrates thereof.

13. The complex according to claim 7, characterized in that the solvent is selected from the group consisting of methanol, ethanol, benzyl alcohol, mixtures of benzyl alcohol with methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-butanol, t-butanol, 2-pentanol, 2-methyl-l-propanol, MEK, acetone, ethyl acetate, toluene, acetonitrile, fluorobenzene, methylene chloride, nitromethane, cielopentanone and cyclohexanone.

14. The complex according to claim 13, characterized in that the solvent is selected from the group consisting of methanol, ethanol, benzyl alcohol and mixtures of benzyl alcohol with methyl ethyl ketone, ethyl acetate and acetonitrile.

15. The complex according to claim 14, characterized in that the solvent is methanol.

16. The complex according to claim 15, characterized in that the carbohydrate is soluble in methanol when heated to about 40 ° to 60 ° C.

17. The complex according to claim 15, characterized in that the carbohydrate is highly soluble in methanol when heated to about 40 ° to 60 ° C.

18. The complex according to claim 15, characterized in that the carbohydrate is insoluble in methanol when heated to about 40 ° to 60 ° C.

19. The complex according to claim 7, characterized in that the carbohydrate co-crystallizes with an echinocandin compound.

20. A process for preparing a parenteral formulation, characterized in that it comprises the step of: (i) mixing the echinocandin / carbohydrate complex according to claim 1 in an aqueous solvent.

21. The process in accordance with the claim 20, characterized in that it further comprises the steps of (ii) sterilizing by filtration and (iii) lyophilizing.

22. A pharmaceutical formulation, characterized in that it comprises the echinocandin / carbohydrate complex according to claim 1, and a pharmaceutically acceptable excipient.

23. The pharmaceutical formulation, according to claim 22, characterized in that the excipient is selected from the group consisting of tonicity agents, stabilizing agents, buffers, bulking agents, surfactants and combinations thereof.

24. A method for treating a fungal infection in a mammal in need thereof, characterized in that it comprises administering to the mammal the echinocandin / carbohydrate complex, according to claim 1.

25. The method according to claim 24, characterized in that the fungal infection occurs from the activity of Candida albicans or Aspergillus fumigatis.

26. A method for treating a fungal infection in a mammal in need thereof, characterized in that it comprises contacting an echinocandin / carbohydrate complex according to claim 1 with body fluids of the mammal, wherein the complex collapses to an amorphous form when gets in contact with such bodily fluids.

27. The method according to claim 26, characterized in that the fungal infection arises from the activity of Candida albicans or Aspergillus fumigatis.