WO2015172047A1 - Cyclic peptide compounds and related methods, salts and compositions - Google Patents

Abstract

This invention relates to compounds useful in the preparation of lipopeptides and related methods of preparing and using these compounds.

Description

CYCLIC PEPTIDE COMPOUNDS AND

RELATED METHODS. SALTS AND COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent application No. 61/990,129, filed on May 8, 2014, which is incorporated herein by reference in its entirety.

BACKGROUND

There is a need for antibiotics, including those that are effective against a variety of organisms. Lipopeptide antibiotics (e.g., as disclosed in WO 2010/075215, published July 1, 2010) can be obtained from deacylated BOC-protected compound obtained from daptomycin. For example, lipopeptide antibiotic compounds can be obtained by treatment of the deacylated BOC-protected compound with an activated ester, lactone or acid chloride, followed by removal of the BOC protecting group. Alternatively, lipopeptide antibiotic compounds can be obtained by the treatment of the deacylated BOC-protected compound with an isocyanate followed by removal of the BOC protecting group. Typically, synthetic intermediates in the manufacture of lipopeptide antibiotics include the use of organic solvents and are performed using dissolved intermediates in the liquid phase.

Typically, synthetic intermediates in the manufacture of lipopeptide antibiotics include the use of dissolved intermediates in the liquid phase. There remains a need for methods of obtaining lipopeptide antibiotics from a stable, solid and portable intermediate that can be derived from natural products such as the A-21978C lipopeptides (e.g., Natural Factors) obtained from fermentation of Streptomyces roseosporus (e.g., United States Patent RE 32,333; RE 32,455; RE32,311 ; RE 32,310; 4,482,487 and 4,537,717). Such a solid intermediate in a lipopeptide synthesis could be useful as a substrate to derive multiple lipopeptide antibiotics from a single fermentation product batch (e.g., A-21978C lipopeptides). SUMMARY

This disclosure provides a compound of formula (I) or a salt thereof:

as well as related methods of synthesizing lipopeptide antibiotics there from. The compound of formula (I) is an intermediate that can be used in aqueous phase manufacturing processes to produce a variety of lipopeptide antibiotics including daptomycin and the lipopeptide antibiotics disclosed in WO 2010/075215, published July 1, 2010. The compound of formula (I) can be used as a substrate to derive multiple lipopeptide antibiotics from a single fermentation product batch (e.g., A-21978C lipopeptides). The compound of formula (I) can be obtained as a free base form and salt forms (e.g., an HC1 salt of the compound formula (I)).

The compound of formula (I) has certain useful and unexpected properties. The compound of formula (I) can be converted into a salt form having unique solubility properties. For example, a hydrochloride salt of the compound of formula (I) is insoluble in both water and dioxane, yet it is soluble in a mixture of water and dioxane, e.g., a 1 : 1 (v/v) mixture of water: dioxane. Unlike certain other lipopeptides, the salt of the compound of formula (I) forms a solid precipitate in aqueous solution, instead of forming micelles at an acidic pH (e.g., a pH of about 5 or lower). The resulting salt of the compound of formula (I) forms a stable, solid and portable intermediate that can be dried, stored and later used to form a variety of lipopeptide antibiotics such as daptomycin.

The compound of formula (I), can be obtained from sulfonated

fluorenylmethyloxycarbonyl (s-FMOC) protected daptomycin or other s-FMOC protected peptide or lipopeptide compounds (e.g., the natural factors of A-21978C) under aqueous reaction conditions, without requiring an organic solvent. Methods for obtaining lipopeptide antibiotics and the compound of formula (I) in aqueous conditions can be important when synthesizing compounds on a large scale such as a kilogram scale used in manufacturing plants. The compound of formula (I) and salts thereof can be transformed into a variety of different antibiotic lipopeptide compounds such as daptomycin and the lipopeptide antibiotics disclosed in WO 2010/075215, published July 1, 2010. The conversion of compounds of formula (I) (and salts thereof) can include the acylation of an amino group followed by deprotection of the s-FMOC (as shown in Figure 1). The deprotection to remove the s-FMOC can be carried out under conditions that effectively remove the s-FMOC while minimizing the formation of undesired side products. These conditions preferably include a large excess of piperidine at a pH of from about 8 to 1 1 (e.g., most preferably at a pH of about 10.2 to 10.6).

The compounds of formula (I) may include:

or a salt thereof.

The compounds of formula (I) may include:

or a salt thereof.

This disclosure also provides the use of compounds of formula (I) for producing a variety of lipopeptide antibiotics including daptomycin and the lipopeptide antibiotics disclosed in WO 2010/075215, published July 1 , 2010. Furthermore, this disclosure provides the use of daptomycin or other peptide or lipopeptide compounds (e.g., the natural factors of A-21978C), for producing compounds of formula (I).

Any of the embodiments described herein may be used in combination with each other.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a synthetic scheme illustrating reactions leading to a compound of formula (I) and subsequent conversion of a compound of formula (I) to a representative lipopeptide antibiotic.

Figure 2 shows the structure of CB-131010 (previously identified as the β-isomer of daptomycin, LY213846).

Figure 3 shows the structure of CB-130952 (previously identified as anhydro- daptomycin, LY178480). DETAILED DESCRIPTION

Any of the embodiments described herein may be used in combination with each other. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biochemistry and microbiology and basic terminology used therein.

The term "acyl" denotes a carbonyl radical attached to an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycyl, aryl or heteroaryl group, examples including, without limitation, such radicals as acetyl and benzoyl. Subsets of the term acyl are (1) "unsubstituted alkanoyl" which is defined as carbonyl radical attached to an unsubstituted alkyl group and (2) "unsubstituted alkenoyl" which is defined as carbonyl radical attached to an unsubsituted alkenyl group.

The term "alkenyl" is defined as linear or branched radicals having two to about twenty carbon atoms, preferably three to about fifteen carbon atoms, and containing at least one carbon- carbon double bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, or ureido. The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration. Examples of alkenyl groups include, without limitation, ethylenyl or phenyl ethylenyl. A subset of term alkenyl is "unsubstituted alkenyl" which is defined as an alkenyl group that bears no substituent groups. A specific example of an exemplary alkenyl is represented by the following structure:

The term "alkyl" is defined as a linear or branched, saturated radical having one to about twenty carbon atoms unless otherwise specified. The term "lower alkyl" is defined as an alkyl group containing 1-4 carbon atoms. One or more hydrogen atoms can also be replaced by a substitutent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, or ureido. Examples of alkyl groups include, without limitation, methyl, butyl, tert- butyl, isopropyl, trifluoromethyl, nonyl, undecyl, octyl, dodecyl, methoxymethyl, 2- (2'- aminophenacyl), 3-indolylmethyl, benzyl, and carboxymethyl. Subsets of the term alkyl are (1) "unsubstituted alkyf'which is defined as an alkyl group that bears no substituent groups (2) "substituted alkyl" which denotes an alkyl radical in which one or more hydrogen atoms is replaced by a substitutent group selected from acyl, acylamino, acyloxy, alkenyl, alkoxy, alkyl, alkynyl, amino, aryl, aryloxy, carbamoyl, carboalkoxy, carboxy, carboxyamido, carboxyamino, cyano, disubstituted amino, formyl, guanidino, halo, heteroaryl, heterocyclyl, hydroxy, iminoamino, monosubstituted amino, nitro, oxo, phosphonamino, sulfinyl, sulfonamino, sulfonyl, thio, thioacylamino, thioureido, or ureido and (3) "selected substituted alkyl" which denotes an alkyl radical in which (a) one proton is replaced by a group selected from hydroxy, carboxy, Q-Cg alkoxy, or (b) one to three protons is replaced by a halo substituent.

The term "alkynyl" denotes linear or branched radicals having from two to about ten carbon atoms, and containing at least one carbon-carbon triple bond. One or more hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfmyl, sulfonyl, formyl, oxo and guanidino. An example of alkynyl group includes, without limitation, propynyl.

The term "aryl" or "aryl ring" denotes aromatic radicals in a single or fused carbocyclic ring system, having from five to fourteen ring members. In one embodiment, the ring system has from six to ten ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkylthio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl. Examples of aryl groups include, without limitation, phenyl, naphthyl, biphenyl, terphenyl. Subsets of the term aryl are (1) the term "phenyl" which denotes a compound of the formula:

(2) the term "substituted phenyl" which is defined as a phenyl radical in which one or more hydrogen atoms are replaced by a substituent group selected from acyl, amino, acyloxy, azido, alkylthio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfmyl, sulfonyl, N- sulfonylcarboxyamido, and N- acylaminosulfonyl and (3) the term "acylamino phenyl" denotes a phenyl radical in which one hydrogen atom is replaced by an acylamino group. One or more additional hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkylthio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfmyl, sulfonyl, N- sulfonylcarboxyamido, and N-acylaminosulfonyl.

The group "Fmoc" is a 9-fluorenylmethoxycarbonyl group. The group "s-Fmoc" is a sulfonated 9-fluorenylmethoxycarbonyl group. The sulfonate can be attached to one of the aryl rings in the fluorenyl portion of the molecule.

"Heteroaryl" or "heteroaryl ring" denotes an aromatic radical which contain one to four

hetero atoms or hetero groups selected from O, N, S,

or O in a single or fused heterocyclic ring system, having from five to fifteen ring members. In one embodiment, the heteroaryl ring system has from six to ten ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy!, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl.

Examples of heteroaryl groups include, without limitation, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolyl groups. Subsets of the term heteroaryl are (1) the term "pyridinyl" which denotes compounds of the formula:

(2) the term "substituted pyridinyl" which is defined as a pyridinyl radical in which one or more hydrogen atoms is replaced by a substituent group selected from acyl, amino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, N- sulfonylcarboxyamido, and N-acylaminosulfonyl and

(3) the term "acylamino pyridinyl" which denotes a pyridinyl radical in which one hydrogen atom is replaced by an acylamino group, additionally, one or more additional hydrogen atoms can also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, N- sulfonylcarboxyamido, and N-acylaminosulfonyl.

The term "cycloalkyl" or "cycloalkyl ring" is defined as a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to twelve ring members. In one embodiment, a cycloalkyl is a ring system having three to twelve ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl. Examples of a cycloalkyl group include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl.

The term "heterocyclyl," "heterocyclic" or "heterocyclyl ring" is defined as a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, NH,

, hi a single or fused heterocyclic ring system having from three to twelve ring members. In one embodiment, a heterocyclyl is a ring system having three to seven ring members. One or more hydrogen atoms may also be replaced by a substituent group selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl. Examples of a heterocyclyl group include, without limitation, morpholinyl, piperidinyl, and pyrrolidinyl.

The term "lipopeptide" refers to a molecule that comprises a lipid-like moiety covalently linked to a peptide moiety, as well as salts, esters, amides and ethers thereof. The term

"lipopeptide" also encompasses protected forms of lipopeptides in which one or more amino, carboxylate or hydroxyl groups are protected. See, e. g., "Protective Groups in Organic

Synthesis" by Theodora W. Greene, John Wiley and Sons, New York, 1981 for examples of protecting groups.

The term "micelle" refers to aggregates of amphipathic molecules. Micelle structures include, but are not limited to, spherical, laminar, cylindrical, ellipsoidal, vesicular (liposomal), lamellar and liquid crystal.

The salts of the compounds of the invention include acid addition salts and base addition salts. In a preferred embodiment, the salt is a pharmaceutically acceptable salt of the compound of formula (I). The term "pharmaceutically acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds of the invention may be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids include, without limitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.

Certain disclosed compounds can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or non- racemic mixtures thereof. The compounds of the invention can be utilized in the present invention as a single isomer or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimpo sable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid or base. Examples of appropriate acids include, without limitation, tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid. The mixture of diastereomers can be separated by crystallization followed by liberation of the optically active bases from the optically active salts. An alternative process for separation of optical isomers includes the use of a chiral

chromatography column optimally chosen to maximize the separation of the enantiomers. Still another method involves synthesis of covalent diastereoisomeric molecules by reacting compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to obtain the enantiomerically pure compound. The optically active compounds of the invention can likewise be obtained by utilizing optically active starting materials. These isomers may be in the form of a free acid, a free base, an ester or a salt.

The disclosure includes the disclosed compounds in isolated forms. An isolated compound refers to a compound which represents at least 10%, preferably at least 20%, more preferably at least 50% and most preferably at least 80% of the compound present in the mixture. In a preferred embodiment, the compound, a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the compound exhibits a detectable (i. e. statistically significant) antimicrobial activity when tested in conventional biological assays.

Compounds and Intermediates

This disclosure provides a compound that is useful as a solid intermediate in the preparation of lipopeptides that can be isolated and stored for later conversion to other compounds (e.g., antibiotic compounds). For example, the compound of formula (I) is a useful chemical intermediate that can form a free base in aqueous solution, or a variety of solid salt forms:

In some embodiments, the compound of formula (I) is:

a salt thereof.

In some embodiments, the compound of formula (I)

or a salt thereof.

In some embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the salt is an HC1 salt of the compound of formula (I).

The compound of formula (I) and its salts have useful and unexpected solubility properties. These properties are unexpected in view of the known solubility properties of other lipopeptides that are converted into micelles by lowering the pH of a lipopeptide containing aqueous solution, such as the A-21978C lipopeptides (See e.g., Comparative Example 8).

Surprisingly, it has been found that a free base of a compound of formula (I) (e.g., a non- salt form) is converted into a salt (e.g., an HC1 salt) by lowering the pH of an aqueous solution comprising a compound of formula (I), rather than forming micelles. This salt form of a compound of formula (I) can have unique solubility properties (e.g., the HC1 salt of the compound of formula (I) has little or no solubility in water). Thus, the salt form of a compound of formula (I) unexpectedly precipitates out of the aqueous medium and is readily isolated at an acidic pH (e.g., a pH of about 5 or lower). This resulting salt of the compound of formula (I) is also a stable, portable intermediate that can be dried, stored and later used to form a variety of peptide antibiotics such as lipopeptide antibiotics (e.g., daptomycin, See e.g., Example 7).

In particular, the hydrochloride salt of the compound of formula (I) is insoluble in both water and dioxane, yet it is soluble in a mixture of water and dioxane such as 1 : 1 (v/v). Also provided are hydrochloride salt forms of the compound of formula (I) that are soluble in other ratios of water and dioxane including 3:7, 1 :2, 4:7, 3:5, 2:3, 5:7, 3:4, 4:5, 5:6, 6:7, 7:6, 6:5, 5:4, 4:3, 7:5, 3:2, 5:3, 7:4, 2: 1, and 7:3 (e.g., preferably a 1 : 1 v/v mixture of water:dioxane).

Methods of Preparation and Compositions

Methods of preparing a compound of formula (I) are also disclosed herein. In one aspect, the disclosure is directed to a method comprising treating a composition of one or more compounds of formula (III) with a protecting group comprising a sulfonated FMOC moiety under aqueous conditions to form a s-FMOC-protected compound from the compound of formula (III).

(wherein R₂ is CMS alkyl or C₂-i8 alkenyl). The s-FMOC-protected compound obtained from the compound of formula (III) can be deacylated to obtain a compound of formula (I)

The compound of formula (III) can be a lipopeptide antibiotic or mixture of lipopeptides. In some embodiments, a compound of formula (III) is daptomycin. Compounds of formula (III) can be prepared via a variety of fermentation methods such as those disclosed in U.S. Patent Nos. 4,537,717, 4,885,243 and 4,874,843 and International Publication No. WO 2001/053330. For instance, Streptomyces roseosporus strains NRRL 1 1379 and NRRL 15998, a mutant strain of NRRL 1 1379, are useful A-21978C producing cultures. These cultures are part of the stock culture collection of the Northern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Peoria, 111. 61604, from which they are available to the public under the accession numbers NRRL 1 1379 and NRRL 15998. The S. roseosporus NRRL 1 1379 culture and conditions for its use in the production of the A-21978C lipopeptide antibiotics (including compounds of formula (III)) are described by Robert L. Hamill and Marvin M. Hoehn in U.S. Pat. No. 4,331 ,594, incorporated herein by reference.

The method described herein can be used with a single compound of formula (III) or a mxiture of compounds including one or more compounds of formula (III) having different structures such as those generated by the fermentation methods disclosed in U.S. Patent Nos. 4,537,717, 4,885,243 and WO 2001/053330.

A lipopeptide can be converted to a s-FMOC protected lipopeptide that can be subsequently deacylated. The step of protecting the lipopeptide such as a compound of formula (III) with a protecting group comprising a sulfonated FMOC moiety (e.g., forming a s-FMOC- protected compound from the compound of formula (III)) is preferably carried out under aqueous conditions. In some embodiments, attachment of a sulfonated FMOC can be carried out under reduced temperature (e.g., 0-10 °C, preferably about 4 °C)). In some embodiments, attachment of a sulfonated FMOC group is carried out in a pH of 7.5-8.5 (e.g., 7.8-8.2). The step of deacylating a s-FMOC-protected compound obtained from the compound of formula (III) can be an enzymatic deacylation. This deacylation can be carried out under elevated temperature (e.g., 30-40 °C, preferably about 37 °C)). The deacylation step can also be carried out at a specific pH such as a pH of 7.0-8.0 (preferably about 7.5). In some

embodiments, enzymatic deacylation can be carried out using a deacylase enzyme that is immobilized (e.g., the enzyme is bound to a support such as column). For example, the deacylation of a s-FMOC protected compound obtained from the compound of formula (III) can be performed in a bi-phase system comprising the s-FMOC protected compound obtained from the compound of formula (III) dissolved in liquid solution in contact with a deacylating agent attached to a solid support.

A variety of enzymatic deacylating agents are suitable for use with the processes disclosed herein. For example, an enzyme which is useful for deacylation of an ornithine protected compound obtained from the compound of Formula (III) (e.g., a compound of formula (Ilia)) is produced by certain microorganisms of the family Actinoplanaceae. Some of these known species and varieties of this family include Actinoplanes philippinensis, Actinoplanes arnzeniacus, Actinoplanes utahensis, Actinoplanes missouriensis, Spirillospora albida, Streptosporiaragium roseum, Streptosporangium vulgare, Streptosporangiurn roseurn var hollandensi, Streptosporangium album, Streptosporangium viridialbum, Amorphosporangium auranticolor, Ampullarielle regularis, Ampullariella campanulate, Ampullariella lobata, Aznpullariella digitata, Pilimelia terevasa, Pimelia anulata, Planomonospora parontospora, Planmonospora venezuelensis, Planobispora longispora, Planobispora rosea,

Dactylosporangium aurantiacum, and Dactylosporangium thailandende. Any natural and artificial variant or mutant obtained from the Actinoplanacea and which produce the enzyme may be used.

Preferred sources of the deacylation enzyme are Actinoplanes utahensi NRRL 12052 ;

Actinoplanes missouriensis NRRL 12053; Actinoplanes sp. : NRRL8122, Actitzoplanes sp. : NRRL 12065, Streptosporsngium roseurra var hollandetasis: NRRL 12064, Actinoplanes utahenis ATCC 14539 and Actinoplales missouriensis ATCC 14538. The more preferred source of deacylation enzyme is the species Actinoplanes utahensi. The most preferred source of deacylation enzyme is one produced from recombinant Streptomyces lividans, which expresses the Actinoplanes utahensis deacylation enzyme as described in J. Ind. Microbiol. Biotechnol. 2000, 24 (3) 173-180. This enzyme is also known as echinocandin B deacylase or ECB deacylase. Other suitable methods for enzymatic deacylation of protected compounds obtained from the compound of formula (III) (including compounds of formula (Ilia)) can be found in United States Patent 4,524,135; 4,537,717; 4,482,487; RE 32,310, and RE 32,311 , each herein incorporated by reference in its entirety.

In some embodiments, the method further comprises converting the compound of formula (I) to a salt form (e.g., an HC1 salt). This conversion can occur under aqueous conditions at acidic pH (e.g., pH 5 or less) (See e.g., Example 2).

As discussed above, preparation of a compound of formula (I) can be carried out using a variety of starting materials (e.g., a mixture of Natural Factors obtained from the fermentation process described above vs. a single compound (e.g., daptomycin)). One such procedure below is disclosed below.

Preparation of 7:

Following a fermentation procedure as outlined in Examples 1-5 of U.S. Patent No. 4,331,594, a mixture of natural factors 6 is produced and subsequently used to prepare the key intermediate disclosed herein. The position representing the variability of the natural factors is represented by Ri. In a cooled, 5L jacketed reactor equipped with mechanical stirrer, thermocouple, pH probe and bottom drain, water is cooled to 4 °C with stirring (180 rpm) and sodium bicarbonate is added. The mixture of natural factors 6 is then added in one portion, followed by slow addition of the remaining liquid to avoid excessive foaming. Ice-water bath cooled 2M NaOH (aq.) is added via addition funnel to adjust the solution to pH 7.8-8.2. To this stirred mixture is added (HS0₃)-FMOC-OSu (9-(((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)methyl)-9H-fluorene-2- sulfonic acid. The mixture is stirred, maintaining the temperature at 4 °C and pH 7.8-8.2 by continuous, slow, dropwise addition of cold 2M NaOH (aq.). The reaction is monitored by LCMS. If LCMS indicates that all of the starting material 6 has not been consumed, an additional portion of (HS0₃)-FMOC-OSu (9-(((((2,5-dioxopyrrolidin-l- yl)oxy)carbonyl)oxy)methyl)-9H-fluorene-2-sulfonic acid is then added and the mixture is stirred an additional 1.25h, maintaining the temperature and pH as noted above. Upon consumption of the starting material 6, as determined by LCMS, the reaction mixture is adjusted to pH 6.5-6.8 by the addition of cold 6M HC1 and stored at 4 °C. The yield of 7 is determined via HPLC.

Preparation of 3 (a compound of formula (I)):

Compound 7 is stored as a solution at pH 6.5 (3.4 L). The reaction is carried out on an AKTA Explorer (GE Biosciences, S/N-8-1403-00) with UNICORN software. An XK-50 jacketed column is packed with 500 mL of deacylase and equilibrated.

A fraction of the solution of compound 7 is diluted and the pH is adjusted to pH 7.5, immediately prior to loading onto a deacylase column. Each load on the column is maintained at 4 °C in an ice- water bath and loaded onto the 37 °C column. The eluted product solution is collected and immediately cooled in an ice-water bath. Periodically, the eluted material is adjusted to pH 6.0- 6.5 and stored at 4 °C. This process is repeated for subsequent loads and upon completion, an additional portion of salt strip is eluted to clean the column. LCMS analysis of the eluted fractions is used to confirm the complete deacylation. All eluted materials are combined at pH 6.0-6.5 and concentrated and desalted by nanofiltration (NF) using a Filmtec NF-2540 membrane to 5.5L. The yield of deacylation and NF is based on solution concentration of 3 followed by further concentration of the solution of compound 3. The yield is based on solution

concentration of compound 3. Also described herein is the use of a composition comprising a compound of formula

(III):

(wherein R₂ is CMS alkyl or C_2-t8 alkenyl) to obtain a compound of formula (I):

In some embodiments, the compound of formula (III) is daptomycin. In some embodiments, the composition comprises a mixture of lipopeptides of formula (III).

Also described herein are compositions comprising a compound of formula (I):

In some embodiments, the composition is a solid. In some embodiments, the composition further comprises a compound of formula (Ilia)

wherein R₂ is C MS alkyl or C2-18 alkenyl and is dissolved in an aqueous solution. In some embodiments, the pH of the solution is about 7.8-8.2. In some embodiments, the composition at a reduced temperature (e.g., 0-10 °C (e.g., 4 °C)). In some embodiments, the composition includes one or more impurities of Figures 2-3.

In some embodiments, the composition further comprises a deacylase enzyme in the aqueous solution.

Methods of Use and Compositions

Also described herein are methods of using a compound of formula (I) to prepare a number of lipopeptide antibiotics. In one embodiment, the invention is directed to a method comprising the steps of:

a. contacting a compound of formula (I) with a substrate comprising Ri to obtain a compound of formula (V)

b. deprotecting the compound of formula (V) under aqueous conditions;

to obtain a lipopeptide antibiotic of formula (II) wherein,

Ri is Ci-18 alkyl, C2-18 alkenyl or C2-19 acyl.

Also described herein is the use of a compound of formula (I) to prepare a number of lipopeptide antibiotics. In one embodiment, the disclosure is directed to the use of a compound of formula (I):

to obtain a compound of formula (II)

wherein Rj is CM S alkyl, C2-is alkenyl or C2-19 acyl. In s

Also described herein is the use of a compound of formula (I):

to obtain a compound of formu

wherein Ri is C\.i_$ alkyl, C_2-18 alkenyl or C_2-19 acyl.

In some embodiments, R_t is C_2-i₉ acyl (e.g., C_2-19 alkanoyl). In some embodiments, Ri is C_2-i9 alkanoyl (e.g., decanoyl). In some embodiments, Ri is C_2-i₉ alkenoyl (e.g.,

In some embodiments, Ri is a moiety selected from a lipopeptide disclosed in U.S. Patent No. 4,537,717, WO 2010/075215 or WO 2003/014147. A number of exemplary R₁ moieties are attached in Table 1.

Table 1

21

22

 U 2015/029927

In another embodiment, exemplary Rj moieties are represented in Table 2 below:

Table 2



In another embodiment, exemplary R₁ moieties are represented in Table 3 below: Tab

In some embodiments, step a is a coupling reaction (e.g., an acylation). This coupling (e.g., acylation) reaction can occur at the tryptophan amine with activated reagents such as activated esters, acid chlorides, imidates and/or lactones. Step a can also be another reaction such as a reductive amination. This reaction can also occur at the tryptophan amino reductively with certain aldehydes to yield lipopeptide antibiotics.

In some embodiments, step a is carried out under aqueous conditions. In some embodiments, step a is carried out in a mixture of two or more solvents (v/v). For instance, step a is carried out in a mixture of two solvents (e.g., at a ratio of 1:10; 1:9; 1:8; 1:7; 1:6; 1:5; 1:4; 1:3; 1:2; 1:1; 2:1; 3:1; 4:1; 5:1; 6:1; 7:1; 8:1 or 9:1). In some embodiments, the ratio of two solvents is 1 : 1. In one embodiment, one solvent in the mixture is dioxane and in other embodiments another solvent in the mixture is water. In some embodiments, the two solvents used are dioxane and water. In some embodiments, step a comprises heating (e.g., to 40 °C).

In some embodiments, step b is carried out under basic conditions (e.g., at a pH of 8-11 , 8-10.6, 9-10.6 or 10.2-10.6). In some embodiments, step b uses a base (e.g., an organic or inorganic base). In some embodiments, step b uses a large excess of base (e.g., 1 10 equivalents of base). In some embodiments, step b uses an organic base (e.g., piperidine, morpholine or piperazine). In some embodiments, step b is performed under reduced temperatures (e.g., 0 °C to 10 °C (e.g., 4 °C)). In some embodiments, step b is carried out under conditions such that less than 10% (by weight, e.g., less than 5%, 3%, 1%) of the reaction products comprise impurities such as the β isomer or the anhydro isomer of lipopeptides.

In some embodiments, a compound of formula (II) is daptomycin. In some

embodiments, a compound of formula (II) is a lipopeptide antibiotic disclosed in WO

2010/075215 or in WO 2003/014147.

As discussed above, use of a compound of formula (I) to produce lipopeptide antiobiotics can be carried out using a variety of starting materials and reactants. Outlined below is a representative procedure.

A 5L jacketed reactor, warmed to 40 °C and equipped with mechanical stirrer, thermocouple, pH probe, and bottom drain is charged with 1,4-dioxane, sodium bicarbonate, and an ester of Ri (e.g., an activated ester of Ri). The mixture is stirred until the internal temperature is 40 °C. To this mixture is added the aqueous solution of compound 3 at pH 6.5. The reaction mixture is stirred at 40 °C for 3h, with monitoring for starting material consumption by LCMS and HPLC. After consumption of the starting material the mixture is used without further manipulation. The reactor is cooled to 4 °C and stirred until the internal temperature of the mixture is <7 °C. To this crude mixture in a water/dioxane mixture (1 : 1), cooled to 4 °C, is added a pH adjusted, pre-mixed, cooled, solution of piperidine and piperidine hydrochloride in water. Cold 2M NaOH is added dropwise to adjust to pH 10.6 and the reaction is maintained at 3 °C and pH 10.6 until completion, as determined by HPLC. The solution is then adjusted to pH 6-6.5 by the addition of cold 6M HC1 (aq.).

Also described herein are compositions comprising a compound of formula (I):

In some embodiments, the composition further comprises one or more materials selected from the group consisting of HOBt, NaHC0₃, dioxane and piperidine. In some embodiments, the composition further comprises an ester. In some embodiments, the temperature of the composition is about 40 °C. In some embodiment, the composition further comprises a compound of formula (V):

wherein Ri is C S alkyl, C₂-i 8 alkenyl or C_{2- 1}9 acyl. In some embodiments, C_2-i9 acyl is C_{2- 1}9 alkanoyl (e.g., decanoyl). In some embodiments, R_\ is C_{2- 1}9 alkenoyl (e.g.,

In some embodiments, the composition is an aqueous composition under basic conditions (e.g., at a pH of 8-1 1, 8-10.6, 9-10.6 or 10.2-10.6). In some embodiments, the composition further comprises a compound of formula (II):

(II),

wherein Ri is C_1-18 alkyl, C₂-19 alkanoyl, C₅.₁₉ alkenoyl. In some embodiments, Rj is C2-19 alkanoyl (e.g., decanoyl).

Pharmaceutical Compounds and Compositions

In some aspects, a compound of formula (I) can be converted to a compound (e.g., a compound of formula (II)) that can be used as an antibiotic. These antibiotics may be used to treat a subject having a bacterial infection in which the infection is caused or exacerbated by any type of bacteria, such as gram-positive bacteria. In one embodiment, one or more compounds according to formula (II) or pharmaceutical compositions thereof are administered to a patient to treat an infection. In another embodiment, the bacterial infection may be caused or exacerbated by gram-positive bacteria.

Any of the embodiments described herein may be used in combination with each other.

Examples

In the examples below, the following abbreviations have the following meanings unless otherwise indicated. Abbreviations not defined below have their generally accepted meaning.

BOC = tert-butoxycarbonyl

CFU = colony-forming units

DCM = dichloromethane

IPA = isopropanol

DIPEA = diisopropylethylamine DMF = dimethylformamide

DMSO = dimethyl sulfoxide

EtOAc = ethyl acetate

HOBT = 1 -hydroxy benzotrizole

HC1 hydrochloric acid

LCMS = Liquid chromatography mass spectroscopy

HPLC = High performance liquid chromatography

MIC Minimum inhibitory concentration

MS Mass spectrometry

THF tetrahydrofuran

TFA trifluoroacetic acid

TLC thin layer chromatography

TTF Tangential flow filtration

WFI Water for in ection

Preparation of compound 2:

Compounds of formula (I) can be obtained as a free base from a protected lipopeptide such as compound 2. Compound 2 can be obtained from a lipopeptide such as compound 1 as described in this example. In a cooled, 5L jacketed reactor equipped with mechanical stirrer,

thermocouple, pH probe and bottom drain, water (440 mL) was cooled to 4 °C with stirring (180 rpm) and sodium bicarbonate (27.9 g, 333 mmol, 5 eq.) was added. Daptomycin (1) (980 mL of 0.0679M aq. Solution, 66.5 mmol, 1.0 eq.) was added in one 350 mL portion, followed by slow addition of the remaining 630 mL to avoid excessive foaming over 10 min. When the internal temperature read 4 °C, ice-water bath cooled 2M NaOH (aq.) was added dropwise via addition funnel to adjust the solution to pH 7.8-8.2. Approximately 80 mL of base was needed. To this stirred mixture was added (HS0₃)-FMOC-OSu (9-(((((2,5-dioxopyrrolidin-l- yl)oxy)carbonyl)oxy)methyl)-9H-fluorene-2-sulfonic acid (46 g, 110 mmol, 1.66 eq.) in one portion. The mixture was stirred, maintaining the temperature at 4 °C and pH 7.8-8.2 by continuous, slow, dropwise addition of cold 2M NaOH (aq.). At 1.25 h, LCMS analysis of a 100 mL aliquot diluted 50x in water indicated -15% starting material remained. An additional portion of (HS0₃)-FMOC-OSu (9-(((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)methyl)-9H- fluorene-2-sulfonic acid (7.8 g, 18.69 mmol, 0.28 eq.) was added and the mixture was stirred an additional 1.25h, maintaining the temperature and pH as noted above. Upon consumption of Daptomycin (1), as determined by LCMS, the reaction mixture was adjusted to pH 6.5-6.8 by the addition of cold 6M HC1 and stored at 4 °C. The final volume of the solution, after collecting and washing the reactor, was 3400 mL. Based on an HPLC determined concentration of 37.6 mg/mL of s-FMOC-protected-daptomycin, this corresponds to 127.87g of 2 s-FMOC-protected daptomycin (66.5 mmol, 100% yield).

Preparation of 3:

Compounds of formula (I) (e.g., compound 3) can be prepared from compound 2. Compound 2 was stored as a solution at pH 6.5 (3.4 L). Additionally, 25L of a "Strip" buffer was also prepared containing 10 mM sodium phosphate dibasic, 1M sodium chloride at pH 7.5. The reaction was carried out on an AKTA Explorer (GE Biosciences, S/N-8-1403-00) with

UNICORN software. An X -50 jacketed column packed with 500 niL of immobilized ECB deacylase resin (ACS-Dofar) was equilibrated at 37 °C with 10 mM NaHP0₄ buffer at pH 7.5 (5 CV's) at 20 mL/min.

A 900 mL fraction of the solution of compound 2 was diluted (5x) to 4.5L in 10 mM sodium phosphate and the pH was adjusted to pH 7.5, immediately prior to loading onto the immobilized deacylase column. There were three 4.5L "Loads" 1, 2 and 3, and one 3.5L load, 4 prepared similarly. Each was maintained at 4 °C in an ice- water bath and loaded onto the 37 °C column at a flow rate of 20 mL/min for a 25 minute contact time with the resin. The eluted product solution was collected and immediately cooled in an ice- water bath. Periodically (every ~5L) the eluted material was adjusted to pH 6.0-6.5 and stored at 4 °C. This process was repeated for Loads 1 , 2, 3, and 4 and upon completion, an additional 3L of salt strip was eluted to clean the column. LCMS analysis of the eluted fractions confirmed the complete deacylation. All eluted materials were combined at pH 6.0-6.5 (total volume = 26L) and concentrated and desalted by nanofiltration (NF) using a Filmtec NF-2540 membrane to 5.5L. The yield of deacylation and NF was based on solution concentration of 3 (11 1.33 g, 62.94 mmol, 91% yield). Further concentration of the solution of compound 3 to 1.52 L was achieved via tangential flow filtration (ultrafiltration, UF) using a Pall Centramate lkDa membrane system. This solution (1520 mL, 55.0 mg/mL) was adjusted to pH 6.0-6.5 and stored at 4 °C for future use. The yield was based on solution concentration of compound 3 (83.6g, 47.27mmol, 72% yield).

Example 2: Preparation of compound 3-HCl, an HC1 salt of the deacylated s-FMOC protected core:

Preparation of 3-HCl:

A compound of formula (I) can be obtained as a solid HCl salt form from a protected lipopeptide such as compound 2. Compound 2 can be obtained from a lipopeptide such as compound 1 as described in Example 2. A crude sample of 2 was used as starting material and diluted using 10L of phosphate buffer and the pH adjusted to 7.5. An XK-50 jacketed column packed with 500 mL of ECB deacylase was equilibrated at 37 °C with 10 mM NaHP0₄ buffer at pH 7.5 (5 CV's) at 20 mL/min. The solution of compound 2 was diluted to 5L in 10 mM sodium phosphate and the pH adjusted to pH 7.5 and the diluted compound 2 was loaded on the column at 9 mL/min. The load was maintained at 4 °C and concentrated using UF with a PALL IK ultrasette membrane to ~4L in 6 hours (atl 1 mL/min throughput). The remaining 4L was stored in the fridge overnight. After washing the column, the load and washes were combined. They were cone, by ultrafiltration using a PALL IK ultrasette membrane to ~1L. The mixture was cooled to 4 °C and acidified with HCl until precipitate appeared. At pH 2, the mixture was filtered through a Buchner funnel with filter paper. The precipitate was washed with 0.5N HCl (aq.) and allowed to dry overnight. Upon drying, 99g solid was obtained as a brownish yellow hard, dense powder. The solid was determined to contain 1 eq. HCl and ~7% water. No other significant impurities were detected.

Example 3: Solubility of Compounds 3 and 3-HCl:

Solubility studies of compounds 3 and 3-HCl were conducted in a variety of solvents. As described below, it was discovered that 3-HCl was not soluble in either 100% water or 100% dioxane. However, it was discovered that 3-HCl was soluble in a 1 : 1 mixture of dioxane and water. Table 6:

3-HCI in Dioxane 0.0

3 Lyo in DC 2.8

3-HCI in DCM 1.1

3 Lyo in IPA 0.0

3-HCI in IPA 0.0

3-Lyo in Water 196.0

3-HCI in Water 0.0

3 Lyo in 1: 1 Dioxane/Water 186.0

3-HCI in 1:1 Dioxane/Water 274.0

3 Lyo in EtOH 0.43

3-HCI in EtOH 2.30

3 Lyo refers to a lyophilized compound 3 prepared by the lyophilization of concentrated compound 3 prior to acid pH adjustment.

3-HCI refers to precipitation of compound 3 HC1 salt.

Example 4: Acylation and Deprotection of Compound 3:

Lipopeptide antibiotic compounds such as compound 5 can be obtained from compounds of formula (I) such as compound 3 (or compound 3-HCI). In this (non-limiting) example, compound 5 is obtained from compound 3. A 5L jacketed reactor, warmed to 40 °C and equipped with mechanical stirrer, thermocouple, pH probe, and bottom drain was charged with 1,4-dioxane (1520 mL), sodium bicarbonate (23.20 g, 276 mmol), and the lipid tail HOBt ester (41.8 g, 120 mmol, 3.3 eq.) (for preparation of the HOBt ester, See WO 2010/075215). The mixture was stirred (180 rpm) until the internal temperature was 40 °C. To this mixture was added the aqueous solution of compound 3 (1520 mL of 0.024M solution, 36.4 mmol, 1.0 eq.) at pH 6.5. The reaction mixture was stirred at 40 °C for 3h, with monitoring for starting material consumption by LCMS and HPLC, during which time the pH of the reaction rose to 7.8. After three hours, HPLC analysis of a 100 mL aliquot indicated -6% unreacted 3 remained so additional lipid tail HOBt ester (16.08 g, 46.0 mmol, 1.0 eq.) was added. After an additional 2h, HPLC analysis of a 100 mL aliquot indicated that 6% of 3 remained and the mixture was used without further manipulation. The reactor was cooled to 4 °C and was stirred until the internal temperature of the mixture was <7 °C.

To this crude mixture (36.4 mmol) in a water/dioxane mixture (4L, 1 : 1), cooled to 4 °C, was added a pH adjusted, pre-mixed, cooled, solution of piperidine (0.045L, 473 mmol) and piperidine hydrochloride (575g, 4730 mmol) in water (total volume=900 mL, pH 10.6). The pH rose from 7.5 to 9.6. Cold 2M NaOH was added dropwise (-150 mL) over ten minutes to adjust to pH 10.6 and the reaction was maintained at 3 °C and pH 10.6 until completion (2h), as determined by HPLC. The solution was then adjusted to pH 6-6.5 by the addition of cold 6M HCl (aq.). The solution of crude 5 was determined to be 5050 mL at 11.1 mg/mL (33.35mmol, 91% yield over two steps).

Example 5: Fermentation and Acidification of 1

The compounds of formula (I) can be obtained from a fermentation product containing cyclic peptide compounds such as compound 1. Compound 1 can be protected to form compound 2, which can be deacylated to form the compounds of formula (I) such as compound 3. Accordingly, compounds of formula (I) can be obtained from a fermentation culture containing compound 1 and/or other suitable lipopeptide compounds.

Fermentation of 1 and Acidification

Compound 1 can be obtained from a fermentation culture. A fermentation culture of S. roseosporus NRRL Strain 15998 is conducted in a controlled decanoic acid feed fermentation at levels that optimize the production of the antibiotic while minimizing the production of contaminants. The residual decanoic acid feed is measured by gas chromatography and the target residual level is 10 ppm decanoic acid from the start of induction (approximately at hour 30) until harvest. Centrifugation of the culture and subsequent analysis of the clarified broth are used to measure the production of 1 by HPLC. The harvest titer is typically between 1.0 and 3.0 grams per liter of fermentation broth.

The fermentation is harvested either by microfiltration using a Pall-Sep or by full commercial-scale centrifugation and depth filter. The clarified broth is applied to an anion exchange resin, Mitsubishi FP-DA 13, washed with 30 mM NaCl at pH 6.5 and eluted with 300 mM NaCl at pH 6.0-6.5. Alternatively, the FP-DA 13 column is washed with 60 mM NaCl at pH 6.5 and eluted with 500 mM NaCl at pH 6.0-6.5. The pH is adjusted to 3.0 to 4.8 and the temperature is adjusted to 2-15 °C. Under these conditions, compound 1 forms a micelle. The micellar solution is purified by washing the micellar preparation while it is retained on a ultrafilter using a 10,000 NMW filter (AG Technology Corp. UF hollow fiber or equivalent) in any configuration. The micelles of compound 1 are retained by the filter, but a large number of impurities are eliminated because they pass through the 10,000 NMW filter. Ultrafiltration of micelles of 1 increases purity from approximately 40% to 80% or greater.

The eluate is applied to a HIC resin, HP-20ss, washed with 30% acetonitrile, and eluted with 35% acetonitrile at pH 4.0-5.0. Alternatively, the HIC resin is washed with 20-30% isopropyl alcohol and eluted with 30-40% isopropyl alcohol at pH 3.5-6.5. Under these conditions of increased solvent and a higher pH of 6.0-7.5, 1 reverts to a single, non-micelle state. The eluate is applied to FP-DA 13 resin column and washed and eluted as before. The final anion exchange step reduces solvent by one third or more. Reverse osmosis diafiltration and concentration at pH 1.5-2.5 is performed using a 0.2 μηι filter and the 1 preparation is frozen. A final reverse osmosis diafiltration is conducted with Water-For-Injection (WFI) to wash 1 and adjust its concentration prior to sterile-filling. Vials or bulk quantities of 1 are then lyophilized.

Fermentation of 1 without Acidification

Compound 1 is produced by fermentation and clarified from the broth using

microfiltration as described above. The preparation is purified using hydrophobic interaction chromatography, as described in U.S. Pat. No. 4,874,843. In this method, repeated column chromatography on HP -20 and HP-20ss resin is used. The purity of 1 is 93% with visible impurities on HPLC chromatographs and measurable pyrogen. The product is diluted in water and its pH was adjusted to pH 6.5 with NaOH or the equivalent. The preparation of 1 is filtered through a 10,000 NMW ultrafiltration membrane. Under these conditions, 1 is monomeric and passes through the ultrafiltration membrane. The resulting product remains 93% pure, but several impurities that had been present at 0.1-0.2% are removed by the ultrafiltration membrane. In addition, pyrogen content is reduced to undetectable levels.

While some embodiments have been shown and described, various modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. For example, it is not intended that the claims set forth hereinafter be construed narrower than the literal language thereof, nor is it intended that exemplary embodiments from the specification be read into the claims. Accordingly, it is to be understood that the present invention has been described herein by way of illustration only, and that such descriptions do not constitute limitations on the scope of the claims.

Claims

Claims

1. A compound of formula (I) or a salt thereof:

(I)·

2. The compound of claim 1, wherein the compound is:

or a salt thereof.

3. The compound of claim 1 , wherein the compound is:

or a salt thereof.

4. The compound of any one of claims 1-3, wherein the salt is an HCl salt.

5. The compound of any one of claims claim 1-4, wherein the salt of the compound of formula (I) is insoluble in water or dioxane.

wherein Rj is C_1-18 alkyl, C₂-18 alkenyl or C2-19 acyl.

7. The use of claim 6 wherein the compound of formula (II) is selected from:

44

wherein Rj is CMS alkyl, C2-18 alkenyl or C₂.i₉ acyl.

wherein R₂ is C_1-18 alkyl or C_2-18 alkenyl.

10. The use of claim 9, wherein the compound of formula (III) is daptomycin.

1 1. The use of claim 9, wherein the composition comprises a mixture of lipopeptides of formula (III).

12. A method of making a compound of formula (II), the method comprising: a. obtaining a compound of formula (V) from a compound of formula (I) wherein Rj is Ci-18 alkyl, C_2-18 alkenyl or C2-19 acyl

(V),

b. deprotecting the compound of formula (V) under aqueous conditions;

(II),

to obtain a compound of formula (II).

13. The method of claim 12, wherein R] is C_2-19 acyl.

14. The method of claim 13, wherein Ri is C2-19 alkanoyl.

15. The method of claim 13, wherein the Ri is C_2-i9 alkenoyl.

16. The method of any one of claims 12-15, wherein the compound of formula (II) is selected from:

17. The method of any one of claims 12-16, wherein step b is carried out at a temperature of about 4-10 °C, at a pH of about 10.2-10.6 in the presence of a molar excess of piperidine relative to the compound of formula (I).

18. A compos ution:

(I).

19. The composition of claim 18, wherein the composition further comprises a deacylase enzyme.

20. The composition of any one of claims 18-19, wherein the composition is at a pH of about 7.0-8.0.

21. The composition of any one of claims 18-20, wherein the composition further comprises one or more materials selected from the group consisting of HOBt, NaHC0₃, dioxane, an ester and piperidine. A composition comprising a compound of formula (I):

wherein R_\ is C_1-18 alkyl, C₂-₁₈ alkenyl or C_2-19 acyl. The composition of claim 22, wherein the composition further comprises a compound of

(Π),

wherein Ri is CMS alkyl, C2-1& alkenyl or C2-19 acyl.

24. A method of preparing a compound of formula (I) comprising the step of:

a) providing a composition comprising a compound of formula (III)

b) treating the composition comprising a compound of formula (III) with a reagent under aqueous conditions to obtain a protected compound having a sulfonated FMOC moiety, and b) subsequently subjecting the resulting s-FMOC-protected compound to a deacylation reaction to obtain a compound of formula (I)

The method of claim 24, wherein the compound of formula (III) is daptomy

26. The method of claim 24, wherein the composition of step a comprises a mixture of lipopeptides of formula (III).

27. A bi-phase composition comprising:

a. a compound of formula (V) in an aqueous solution

(V),

wherein R₁ is CMS alkyl, C2-18 alkenyl or C2-19 acyl; and

b. a deacylase enzyme bound to a solid support in contact with the aqueous solution; and c. a compound of formula (I)

28. The bi-phasic composition of claim 27, wherein the aqueous solution has a pH of about 7.5.

29. The bi-phase composition of any one of claims 27-28, wherein the deacylase enzyme is Actinoplanes utahensi: NRRL 12052.