Novel lipopeptides as antibacterial agents

FIELD OF THE INVENTION

[0001] The present invention relates to novel lipopeptide compounds The invention also relates to pharmaceutical compositions of these compounds and methods of using these compounds as antibacterial compounds. The invention also relates to methods of producing these novel lipopeptide compounds and intermediates used in producing these compounds.

BACKGROUND OF THE INVENTION

[0002] The rapid increase in the incidence of gram-positive infections—including those caused by resistant bacteria—has sparked renewed interest in the development of novel classes of antibiotics. A class of compounds which have shown potential as useful antibiotics includes the A-21978C lipopeptides described in, for example, U.S. Pat. Nos. RE 32,333; RE 32,455; RE 32,311; RE 32,310, 4,482,487; 4,537,717; and 5,912,226. Daptomycin, a member of this class, has potent bactericidal activity in vitro and in vivo against clinically relevant gram-positive bacteria that cause serious and life-threatening diseases. These bacteria include resistant pathogens, such as vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide intermediate susceptible Staphylococcus aureus (GISA), coagulase-negative staphylococci (CNS), and penicillin-resistant Streptococcus pneumoniae (PRSP), for which there are few therapeutic alternatives. See, e.g., Tally et al., 1999, Exp. Opin. Invest. Drugs 8.1223-1238

[0003] Despite the promise that antibacterial agents such as daptomycin offer, the need for novel antibiotics continues. Many pathogens have been repeatedly exposed to commonly-used antibiotics. This exposure has led to the selection of variant antibacterial strains resistant to a broad spectrum of antibiotics The loss of potency and effectiveness of an antibiotic caused by resistant mechanisms renders the antibiotic ineffective and consequently can lead to life-threatening infections that are virtually untreatable. As new antibiotics come to market pathogens may develop resistance or intermediate resistance to these new drugs, effectively creating a need for a stream of new antibacterial agents to combat these emerging strains. In addition compounds that exhibit bacteriacidal activity would offer advantages over present bacteriastatic compounds. Thus, novel synthetic antibacterial agents would be expected to be useful to treat not only “natural” pathogens, but also intermediate drug resistant and drug resistant pathogens because the pathogen has never been exposed to the novel antibacterial agent. Additionally, new antibacterial agents may exhibit differential effectiveness against different types of pathogens.

SUMMARY OF THE INVENTION

[0004] The present invention addresses this problem by providing novel lipopeptide compounds which have antibacterial activity against a broad spectrum of bacteria, including drug-resistant bacteria. Further, the compounds of the present invention exhibit bacteriacidal activity.

[0005] The present invention comprises, in one aspect, antibacterial compounds of Formula I:

[0006] wherein X and X″ are independently selected from C═O, C═S, C═NH, C═NRX, S═O or SO2;

[0007] wherein n is 0 or 1;

[0008] wherein RX is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, hydroxyl, alkoxy, carboxy or carboalkoxy;

[0009] wherein B is X″RY, H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;

[0010] wherein RY is selected from hydrido, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or hydroxyl;

[0011] wherein A is H, NH2, NHRA, NRARB, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, aryl, heteroaryl, cycloalkyl or heterocyclyl;

[0012] wherein RA and RB are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or carboalkoxy;

[0013] wherein when n is 0, then A is additionally selected from:

[0014] wherein each of R50-R53 is independently selected from C1-C15 alkyl,

[0015] alternatively, wherein B and A together form a 5-7 membered heterocyclic or heteroaryl ring.

[0016] Wherein R1 is

[0017] wherein X′ and X′″ are independently selected from C═O, C═S, C═NH, C═NRX′, S═O or SO2;

[0018] wherein m is 0 or 1;

[0019] wherein RX′ is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, hydroxyl, alkoxy, carboxy or carboalkoxy;

[0020] wherein B′ is X′″RY′, H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and

[0021] wherein RY′ is selected from hydrido, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or hydroxyl.

[0022] In one aspect of the invention, A′ is H, NH2, NHRA′, NRA′RB′, heteroaryl, cycloalkyl or heterocyclyl;

[0023] wherein RA′ and RB′ are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or carboalkoxy,

[0024] wherein when m is 0, then A′ is additionally selected from:

[0025] wherein each of R50-R53 is independently selected from C1-C15 alkyl;

[0026] provided that when B′ is H and X′ is C═O, then A′ is other than

[0027] (a) a pyridinyl ring substituted with one substitutent NHC(O)RD or

[0028] (b) a C5-C6 saturated cycloalkyl ring substituted with one substitutent NHC(O)RD,

[0029] wherein RD is C1-C17 unsubstituted alkyl or C2-C17 unsubstituted alkenyl; and when B′ is H and m=0, then A′ is not H

[0030] In another aspect of the invention, A′ is aryl;

[0031] provided that when B′ is H and X′ is C═O, then A′ is other than a phenyl ring substituted with substitutent NHC(O)RD, wherein RD is defined as above, which may be further optionally substituted on the phenyl ring with 1-2 substituents independently selected from amino, nitro, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, halo, mercapto, C1-C3 alkylthio, carbamyl or C1-C3 alkyl carbamyl.

[0032] In a third aspect of the invention, A′ is alkyl, alkenyl, alkynyl, alkoxy or aryloxy;

[0033] provided that when B′ is H and X′ is C═O, then A′ is other than

[0034] (a) —(C1-C16 unsubstituted alkyl)—NH2;

[0035] (b) —(C1-C10 unsubstituted alkyl)—NHC(O)RD, wherein RD is defined as described above;

[0036] (c) —C1-C18 alkyl, optionally substituted with up to one hydroxyl, carboxyl or C1-C3 alkoxy, or one to three halo substituents;

[0037] (d) —C4-C18 unsubstituted alkenyl;

[0038] wherein R54 is selected from C1-C17-unsubstituted alkyl or C2-C17-unsubstituted alkenyl; wherein R55 is selected from hydroxyethyl, hydroxymethyl, mercaptomethyl, mercaptoethyl, methylthioethyl, 2-thienyl, 3-indolemethyl, phenyl optionally substituted with a group selected from halo, nitro, C1-C3-unsubstituted alkyl, hydroxy, C1-C3-unsubstituted alkoxy, C1-C3-unsubstituted alkylthio, carbamyl or C1-C3 unsubstituted alkylcarbamyl; or benzyl optionally substituted with a group selected from halo, nitro, C1-C3-unsubstituted alkyl, hydroxy, C1-C3-unsubstituted alkoxy, C1-C3-unsubsituted alkylthio, carbamyl or C1-C3 unsubstituted alkylcarbamyl; wherein t is 0 or 1 and wherein u is an integer from 1-3; and

[0039] when B′ is H and X′ is C═O, then X′, together with A′, does not form a carbamate amino protecting group; and

[0040] when B′ is H and m is 0, then A′ is other than C4-C14 unsubstituted alkyl.

[0041] In a fourth aspect of the invention, B′ and A′ together form a 5-7 membered heterocyclic or heteroaryl ring.

[0042] Wherein R2 is

[0043] wherein K and K′ together form a C3-C7 cycloalkyl or heterocyclyl ring or a C5-C10 aryl or heteroaryl ring;

[0044] wherein J is selected from the group consisting of hydrido, amino, NHRJ, NRJRK, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, hydroxyl, thio, alkylthio, alkenylthio, sulfinyl, sulfonyl, azido, cyano, halo,

[0045] wherein each of R24, R25, and R26 is independently selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R24 and R25 together form a 5-8 membered heterocyclyl ring;

[0046] wherein RJ and RK are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; or

[0047] alternatively, wherein J, together with R17, forms a 5-8 membered heterocyclyl or cycloalkyl ring; or

[0048] alternatively, wherein J, together with both R17 and R18, forms a 5-8 membered aryl, cycloalkyl, heterocyclyl or heteroaryl ring; and

[0049] wherein each of R17 and R18 is independently selected from the group consisting of hydrido, halo, hydroxyl, alkoxy, amino, thio, sulfinyl, sulfonyl and

[0050] or

[0051] wherein R17 and R18 taken together can form a group consisting of ketal, thioketal,

[0052] wherein each of R22 and R23 is independently selected from the group consisting of hydrido and alkyl.

[0053] In another embodiment, the invention also provides pharmaceutical compositions comprising compounds of Formula I and methods of use thereof.

[0054] In a further embodiment, the invention provides methods of making compounds of Formula I and pharmaceutical compositions thereof

[0055] In a further embodiment, the invention provides compounds useful as intermediates for the preparation of compounds of Formula I.

[0056] In a still further embodiment, the invention provides methods of use of the compounds of Formula I to treat bacterial infections in humans.

DETAILED DESCRIPTION OF THE INVENTION

[0057] Definitions

[0058] Molecular terms, when used in this application, have their common meaning unless otherwise specified.

[0059] The term “hydrido” denotes a single hydrogen atom (H).

[0060] The term “acyl” is defined as 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.

[0061] The term “amino” denotes a nitrogen radical containing two substituents independently selected from the group consisting of hydrido, alkyl, cycloalkyl, carboalkoxy, heterocyclyl, aryl, heteroaryl and sulfonyl Subsets of the term amino are (1) the term “unsubstituted amino” which denotes an NH2 radical, (2) the term “mono substituted amino” which is defined as a nitrogen radical containing a hydrido group and a substituent group selected from alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and (3) the term “disubstituted amino” which is defined as a nitrogen radical containing two substituent groups independently selected from, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl. Preferred mono substituted amino radicals are “lower mono substituted amino” radicals, whereby the substituent group is a lower alkyl group. Preferred disubstituted amino radicals are “lower disubstituted amino” radicals, whereby the substituent groups are lower alkyl.

[0062] The term “acyloxy” denotes an oxygen radical adjacent to an acyl group.

[0063] The term “acylamino” denotes a nitrogen radical adjacent to an acyl group.

[0064] The term “carboalkoxy” is defined as a carbonyl radical adjacent to an alkoxy or aryloxy group.

[0065] The term “carboxyamido” denotes a carbonyl radical adjacent to an amino group.

[0066] The term “halo” is defined as a bromo, chloro, fluoro or iodo radical.

[0067] The term “thio” denotes a radical containing a substituent group independently selected from hydrido, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, attached to a divalent sulfur atom, such as, methylthio and phenylthio.

[0068] The term “alkyl” is defined as a linear or branched, saturated radical having one to about twenty carbon atoms unless otherwise specified. Preferred alkyl radicals are “lower alkyl” radicals having one to about five carbon atoms. One or more hydrogen atoms can also be replaced by a substitutent 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, oxo, guanidino, formyl and an amino acid side chain. Examples of alkyl groups include, without limitation, methyl, tert-butyl, isopropyl, and methoxymethyl. Subsets of the term alkyl are (1) “unsubstituted alkyl” which is defined as an alkyl group that bears no substituent groups (2) “substituted alkyl” which denotes an alkyl radical in which (a) one or more hydrogen atoms is replaced by a substitutent group selected from acyl, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, N-sulfonylcarboxyamido, N-acylaminosulfonyl or (b) two or more hydrogen atoms are each replaced by a substituent group independently selected from hydroxyl, carboxy, C1-C3 alkoxy, amino, acylamino, oxo or guanidino; and (3) the term “selected substituted alkyl” which denotes an alkyl radical in which (a) one proton is replaced by a group selected from hydroxyl, carboxy C1-C3 alkoxy, unsubstituted amino, acylamino, or acylamino phenyl or (b) one to three protons is replaced by a halo substituent.

[0069] The term “alkenyl” is defined as linear or branched radicals having two to about twenty carbon atoms, preferably three to about ten 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, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino. 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.

[0070] 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, sulfinyl, sulfonyl, formyl, oxo and guanidino An example of alkynyl group includes, without limitation, propynyl

[0071] 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 a preferred 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

[0072] (2) the term “substituted phenyl” which is defined as a phenyl radical in which one or more protons 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, sulfinyl, 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, sulfinyl, sulfonyl, N-sulfonylcarboxyamido, and N-acylaminosulfonyl.

[0073] “Heteroaryl” or “heteroaryl ring” denotes an aromatic radical which contain one to four hetero atoms or hetero groups selected from O, N, S,

[0074] in a single or fused heterocyclic ring system, having from five to fifteen ring members. In a preferred 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, hydroxyl, 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:

[0075] (2) the term “substituted pyridinyl” which is defined as a pyridinyl radical in which one or more protons 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.

[0076] 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 a preferred embodiment, a cycloalkyl 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, 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.

[0077] 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,

[0078] wherein RZ is as defined for

[0079] in a single or fused heterocyclic ring system having from three to twelve ring members. In a preferred 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.

[0080] The term “alkoxy” denotes oxy-containing radicals substituted with an alkyl, cycloalkyl or heterocyclyl group. Examples include, without limitation, methoxy, tert-butoxy, benzyloxy and cyclohexyloxy.

[0081] The term “aryloxy” denotes oxy-containing radicals substituted with an aryl or heteroaryl group. Examples include, without limitation, phenoxy.

[0082] The term “amino acid side chain” denotes any side chain (R group) from a naturally-occurring or a non-naturally occurring amino acid

[0083] The term “sulfinyl” is defined as a tetravalent sulfur radical substituted with an oxo substituent and a second substituent selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group.

[0084] The term “sulfonyl” is defined as a hexavalent sulfur radical substituted with two oxo substituents and a third substituent selected from alkyl, cycloalkyl, heterocyclyl aryl, or heteroaryl.

[0085] The term “carbamate amino protecting group” is defined as a recognized amino protecting group that when bound to an amino group forms a carbamate. Examples of carbamate amino protecting groups can be found in “Protective Groups in Organic Synthesis” by Theodora W. Greene, John Wiley and Sons, New York, 1981. Examples of carbamate amino protecting groups include benzyloxycarbonyl, t-butoxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, adamantyloxycarbonyl, chlorobenzyloxycarbonyl, nitrobenzyloxycarbonyl or the like.

[0086] The salts of the compounds of the invention (preferably a compound of Formula I) 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 (preferably a compound of Formula I) 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 Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include, without limitation, formic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactic, and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts of compounds of the invention (preferably a compound of Formula I) include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine and procaine. All of these salts may be prepared by conventional means from the corresponding compound of the invention (preferably a compound of Formula I) by treating, for example, the compound of the invention (preferably a compound of Formula I) with the appropriate acid or base.

[0087] The compounds of the invention (preferably compounds of Formula I) 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 (preferably compounds of Formula I) can be utilized in the present invention as a single isomer or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable 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 these 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 available method involves synthesis of covalent diastereoisomeric molecules by reacting compounds of the invention (preferably compounds of Formula I) 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 (preferably compounds of Formula I) 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.

[0088] The invention also embraces isolated compounds. 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 such as those described herein.

[0089] Lipopeptide Compounds

[0090] A compound of the formula (I):

[0091] and salts thereof,

[0092] wherein R is:

[0093] wherein X and X″ are independently selected from C═O, C═S, C═NH, C═NRX, S═O or SO2;

[0094] wherein n is 0 or 1;

[0095] wherein RX is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, hydroxyl, alkoxy, carboxy or carboalkoxy;

[0096] wherein B is X″RY, H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or heterocyclyl;

[0097] wherein RY is selected from hydrido, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or hydroxyl;

[0098] wherein A is H, NH2, NHRA, NRARB, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, aryl, heteroaryl, cycloalkyl or heterocyclyl;

[0099] wherein RA and RB are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or carboalkoxy;

[0100] wherein when n is 0, then A is additionally selected from:

[0101] wherein each of R50-R53 is independently selected from C1-C15 alkyl;

[0102] alternatively, wherein B and A together form a 5-7 membered heterocyclic or heteroaryl ring.

[0103] Wherein R1 is

[0104] wherein X′ and X′″ are independently selected from C═O, C═S, C═NH, C═NRX′, S═O or SO2;

[0105] wherein m is 0 or 1;

[0106] wherein RX′ is selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, hydroxyl, alkoxy, carboxy or carboalkoxy;

[0107] wherein B′ is X′″RY′, H, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; and

[0108] wherein RY′ is selected from hydrido, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or hydroxyl.

[0109] In one aspect of the invention, A′ is H, NH2, NHRA′, NRA′RB′, heteroaryl, cycloalkyl or heterocyclyl;

[0110] wherein RA′ and RB′ are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocyclyl or carboalkoxy,

[0111] wherein when m is 0, then A′ is additionally selected from

[0112] wherein each of R50-R53 is independently selected from C1-C15 alkyl;

[0113] provided that when B′ is H and X′ is C═O, then A′ is other than

[0114] (a) a pyridinyl ring substituted with one substitutent NHC(O)RD or

[0115] (b) a C5-C6 saturated cycloalkyl ring substituted with one substitutent NHC(O)RD;

[0116] wherein RD is C1-C17 unsubstituted alkyl or C2-C17 unsubstituted alkenyl; and when B′ is H and m=0, then A′ is not H.

[0117] In another aspect of the invention, A′ is aryl;

[0118] provided that when B′ is H and X′ is C═O, then A′ is other than a phenyl ring substituted with substitutent NHC(O)RD, wherein RD is defined as above, which may be further optionally substituted on the phenyl ring with 1-2 substituents independently selected from amino, nitro, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, halo, mercapto, C1-C3 alkylthio, carbamyl or C1-C3 alkyl carbamyl.

[0119] In a third aspect of the invention, A′ is alkyl, alkenyl, alkynyl, alkoxy or aryloxy;

[0120] provided that when B′ is H and X′ is C═O, then A′ is other than

[0121] (a) —(C1-C16 unsubstituted alkyl)—NH2;

[0122] (b) —(C1-C10 unsubstituted alkyl)—NHC(O)RD, wherein RD is defined as described above;

[0123] (c) —C1-C18 alkyl, optionally substituted with up to one hydroxyl, carboxyl or C1-C3 alkoxy, or one to three halo substituents;

[0124] (d) —C4-C18 unsubstituted alkenyl;

[0125] wherein R54 is selected from C1-C17-unsubstituted alkyl or C2-C17-unsubstituted alkenyl; wherein R55 is selected from hydroxyethyl, hydroxymethyl, mercaptomethyl, mercaptoethyl, methylthioethyl, 2-thienyl, 3-indolemethyl, phenyl optionally substituted with a group selected from halo, nitro, C1-C3-unsubstituted alkyl, hydroxy, C1-C3-unsubstituted alkoxy, C1-C3-unsubstituted alkylthio, carbamyl or C1-C3 unsubstituted alkylcarbamyl; or benzyl optionally substituted with a group selected from halo, nitro, C1-C3-unsubstituted alkyl, hydroxy, C1-C3-unsubstituted alkoxy, C1-C3-unsubsituted alkylthio, carbamyl or C1-C3 unsubstituted alkylcarbamyl; wherein t is 0 or 1 and wherein u is an integer from 1-3; and

[0126] when B is H and X is C═O, then X, together with A, does not form a carbamate amino protecting group; and

[0127] when B′ is H and m is 0, then A′ is other than C4-C14 unsubstituted alkyl

[0128] In a fourth aspect of the invention, B′ and A′ together form a 5-7 membered heterocyclic or heteroaryl ring.

[0129] Wherein R2 is

[0130] wherein K and K′ together form a C3-C7 cycloalkyl or heterocyclyl ring or a C5-C10 aryl or heteroaryl ring;

[0131] wherein J is selected from the group consisting of hydrido, amino, NHRJ, NRJRK, alkyl, alkenyl, alkynyl, alkoxy, aryloxy, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, hydroxyl, thio, alkylthio, alkenylthio, sulfinyl, sulfonyl, azido, cyano, halo,

[0132] wherein each of R24, R25, and R26 is independently selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R24 and R25 together form a 5-8 membered heterocyclyl ring;

[0133] wherein RJ and RK are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl or heterocyclyl; or

[0134] alternatively, wherein J, together with R17, forms a 5-8 membered heterocyclyl or cycloalkyl ring; or

[0135] alternatively, wherein J, together with both R17 and R18, forms a 5-8 membered aryl, cycloalkyl, heterocyclyl or heteroaryl ring; and

[0136] wherein each of R17 and R18 is independently selected from the group consisting of hydrido, halo, hydroxyl, alkoxy, amino, thio, sulfinyl, sulfonyl and

[0137] or

[0138] wherein R17 and R18 taken together can form a group consisting of ketal, thioketal,

[0139] wherein each of R22 and R23 is independently selected from the group consisting of hydrido and alkyl.

[0140] In a preferred embodiment of the invention, R is selected from

[0141] wherein each of R3, R4 R5, and R6 is independently selected from the group consisting of hydrido, alkyl, aryl, heterocyclyl and heteroaryl, and wherein R44 is selected from the group consisting of alkyl, aryl, heterocyclyl and heteroaryl.

[0142] In a more preferred embodiment of the invention R is selected from

[0143] wherein R4′ is selected from the group consisting of alkyl, aryl-substituted alkyl, substituted phenyl, heteroaryl, heterocyclyl, optionally substituted (C8-C14)-straight chain alkyl and

[0144] wherein R7 is an alkyl group.

[0145] In an even more preferred embodiment of the invention, R is

[0146] wherein X3 is chloro or trifluoromethyl and wherein q is 0 or 1.

[0147] In a preferred embodiment of the invention, R1 is selected from the group consisting of:

[0148] wherein R8 is selected from an amino acid side chain, wherein said amino acid side chain may be one that is naturally occurring or one that is not naturally occurring, wherein each of R9, R10 and R11 is selected from hydrido, alkyl, aryl, heterocyclyl and heteroaryl; wherein R12 is selected from the group consisting of heterocyclyl, heteroaryl, aryl, and alkyl and wherein R13 is selected from (C1-C3)-alkyl and aryl.

[0149] In a more preferred embodiment of the invention, R1 is selected from the group consisting of

[0150] wherein R8 is selected from tryptophan side chain and lysine side chain, wherein each of R10 and R11 is independently selected from hydrido and alkyl; wherein R12 is selected from imidazolyl, N-methylimidazolyl, indolyl, quinolinyl, benzyloxybenzyl, and benzylpiperidenylbenzyl; and wherein X4 is selected from fluoro and trifluoromethyl.

[0151] In a preferred embodiment of R2, J is selected from the group consisting of hydrido, amino, azido and

[0152] wherein R17 and R18 taken together form a group selected from the group consisting of ketal,

[0153] alternatively, R17 is hydroxyl when R18 is hydrido. Alternatively, wherein J, together with R17, forms a heterocyclyl ring.

[0154] In a more preferred embodiment of the invention, R2 is selected from

[0155] wherein R17 and R18 taken together form a group selected from

[0156] wherein R22 is selected from the group consisting of H and alkyl; wherein R19 is selected from the group consisting of hydrido, amino, azido and

[0157] In an even more preferred embodiment of the invention R2 is

[0158] Another aspect of the present invention provides compounds of formula (I), wherein R is selected from NHCO-[(C6-C14)-alkyl]CH3, and R1 and R2 are selected from Table A below. More preferably, R is selected from NHCO-[(CH2)6-14]-CH3.

1TABLE AR1R2

NHSO2Ph

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

NH(CH2)2OH

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

[0159]

2

TABLE I

Table I provides exemplary compounds of Formula I:

Cpd #
R
R1
R2
Mass Spec
Synth Ex #

1
NHCO(CH2)8CH3

667

668

1863
6

2
NHCO(CH2)8CH3

669

670

1663
6

3
NHCO(CH2)8CH3
NHSO2Ph

671

1762
5

4
NHCO(CH2)8CH3

672

673

1792
4

5
NHCO(CH2)8CH3

674

675

1694
4

6
NHCO(CH2)8CH3

676

677

1722
4

7
NHCO(CH2)8CH3

678

679

1764
4

8
NHCO(CH2)8CH3

680

681

1720
4

9
NHCO(CH2)8CH3

682

683

1775
4

10
NHCO(CH2)8CH3

684

685

1740
2

11
NHCO(CH2)8CH3

686

687

1775
2

12
NHCO(CH2)8CH3

688

689

1820
2

13
NHCO(CH2)8CH3

690

691

1755
2

14
NHCO(CH2)8CH3

692

693

1755
2

15
NHCO(CH2)8CH3

694

695

1771
2

16
NHCO(CH2)8CH3

696

697

1771
2

17
NHCO(CH2)8CH3

698

699

1775
2

18
NHCO(CH2)8CH3

700

701

1812
3b

19
NHCO(CH2)8CH3

702

703

1785
2

20
NHCO(CH2)8CH3

704

705

1755
2

21
NHCO(CH2)8CH3

706

707

1756
3b

22
NHCO(CH2)8CH3

708

709

1757
2

23
NHCO(CH2)8CH3

710

711

1742
2

24
NHCO(CH2)8CH3

712

713

1790
2

25
NHCO(CH2)8CH3

714

715

1758
2

26
NHCO(CH2)8CH3

716

717

1758
2

27
NHCO(CH2)8CH3

718

719

1758
2

28
NHCO(CH2)8CH3

720

721

1726
3b

29
NHCO(CH2)8CH3

722

723

1728
3b

30
NHCO(CH2)8CH3

724

725

1741
3b

31
NHCO(CH2)8CH3

726

727

1741
3b

32
NHCO(CH2)8CH3

728

729

1771
3b

33
NHCO(CH2)8CH3

730

731

1851
3b

34
NHCO(CH2)8CH3

732

733

1767
3b

35
NHCO(CH2)8CH3

734

735

1782
3b

36
NHCO(CH2)8CH3

736

737

1780
8

37
NHCO(CH2)8CH3

738

739

1873
8

38
NHCO(CH2)8CH3

740

741

1729
1

39
NHCO(CH2)8CH3

742

743

1838
3b

40
NHCO(CH2)8CH3

744

745

1741
1

41
NHCO(CH2)8CH3

746

747

1908
3

42
NHCO(CH2)8CH3

748

749

1865
3

43
NHCO(CH2)8CH3

750

751

1893
3

44
NHCO(CH2)8CH3

752

753

1908
3

45
NHCO(CH2)8CH3

754

755

1808
3

46
NHCO(CH2)8CH3

756

757

1764
3

47
NHCO(CH2)8CH3

758

759

1750
3

48
NHCO(CH2)8CH3

760

761

1736
3

49
NHCO(CH2)8CH3

762

763

2004
3a

50
NHCO(CH2)8CH3

764

765

1712
1

51
NHCO(CH2)8CH3

766

767

1904
3a

52
NHCO(CH2)8CH3

768

769

1725
1

54
NHCO(CH2)8CH3

770

771

1749
3a

55
NHCO(CH2)8CH3

772

773

1884
3

56
NHCO(CH2)8CH3

774

775

1785
3

57
NHCO(CH2)8CH3

776

777

1853
3

58
NHCO(CH2)8CH3

778

779

1847
3

60
NHCO(CH2)8CH3

780

781

1778
3

61
NHCO(CH2)8CH3

782

783

1792
3

62
NHCO(CH2)8CH3

784

785

1826
3

63
NHCO(CH2)8CH3

786

787

1826
3

64
NHCO(CH2)8CH3

788

789

1838
3

65
NHCO(CH2)8CH3

790

791

1812
3

66
NHCO(CH2)8CH3

792

793

1808
3

67
NHCO(CH2)8CH3

794

795

1769
3

68
NHCO(CH2)8CH3

796

797

1824
3

69
NHCO(CH2)8CH3

798

799

1775
3

70
NHCO(CH2)8CH3

800

801

1820
3

72
NHCO(CH2)8CH3

802

803

1707
3

73
NHCO(CH2)8CH3

804

805

1758
3

74
NHCO(CH2)8CH3

806

807

1959
3

75
NHCO(CH2)8CH3

808

809

1810
3

76
NHCO(CH2)8CH3

810

811

1787
1g

77
NHCO(CH2)8CH3
NH(CH2)2OH

812

1665
1

78
NHCO(CH2)8CH3

813

814

1820
1

79
NHCO(CH2)8CH3

815

816

1750
1

80
NHCO(CH2)8CH3

817

818

1779
1

81
NHCO(CH2)8CH3

819

820

1767
1e

82
NHCO(CH2)8CH3

821

822

1763
1

83
NHCO(CH2)8CH3

823

824

1869
1

84
NHCO(CH2)8CH3

825

826

1764
1

85
NHCO(CH2)8CH3

827

828

1714
1c

86

829

830

831

1935
9

87
NHCO(CH2)8CH3

832

833

1863
1

88
NHCO(CH2)8CH3

834

835

2151
1

89
NHCO(CH2)8CH3

836

837

1887
1

90
NHCO(CH2)8CH3

838

839

2046
1

91
NHCO(CH2)8CH3

840

841

1996
1

92
NHCO(CH2)8CH3

842

843

1809
1

93
NHCO(CH2)8CH3

844

845

1783
1

94
NHCO(CH2)8CH3

846

847

1770
1

95
NHCO(CH2)8CH3

848

849

1836
1

96
NHCO(CH2)8CH3

850

851

1792
1

97
NHCO(CH2)8CH3

852

853

1847
1

98
NHCO(CH2)8CH3

854

855

1838
1

99
NHCO(CH2)8CH3

856

857

1837
1

100
NHCO(CH2)8CH3

858

859

1817
1

101

860

861

862

1867
9

102
NHCO(CH2)11CH3

863

864

1849
9

103
NHCO(CH2)8CH3

865

866

1885
1

104
NHCO(CH2)8CH3

867

868

2150
1

105
NHCO(CH2)8CH3

869

870

1756
1

106
NHCO(CH2)8CH3

871

872

1833
1

107
NHCO(CH2)8CH3

873

874

1871
1

108
NHCO(CH2)8CH3

875

876

1873
1

109
NHCO(CH2)8CH3

877

878

1872
1

110
NHCO(CH2)8CH3

879

880

2014
1

111
NHCO(CH2)8CH3

881

882

1817
1

112
NHCO(CH2)8CH3

883

884

2121
1

113
NHCO(CH2)8CH3

885

886

2036
1

114
NHCO(CH2)8CH3

887

888

1826
1

115
NHCO(CH2)8CH3

889

890

1736
1

116
NHCO(CH2)8CH3

891

892

1797
1

117
NHCO(CH2)8CH3

893

894

1860
1

118
NHCO(CH2)8CH3

895

896

2055
1

119
NHCO(CH2)8CH3

897

898

1837
1

120
NHCO(CH2)8CH3

899

900

2104
1

121
NHCO(CH2)8CH3

901

902

1803
1

122
NHCO(CH2)8CH3

903

904

1755
1

123
NHCO(CH2)8CH3

905

906

1812
1

124
NHCO(CH2)8CH3

907

908

2002
1

125
NHCO(CH2)8CH3

909

910

1946
1

126
NHCO(CH2)8CH3

911

912

1918
1

127
NHCO(CH2)8CH3

913

914

1811
1

128
NHCO(CH2)8CH3

915

916

2050
1

129
NHCO(CH2)8CH3

917

918

1756
1

130
NHCO(CH2)8CH3

919

920

1762
1

131
NHCO(CH2)8CH3

921

922

1904
1

132
NHCO(CH2)8CH3

923

924

1962
1

133
NHCO(CH2)8CH3

925

926

1726
1

134
NHCO(CH2)8CH3

927

928

2074
1

135
NHCO(CH2)8CH3

929

930

1729
1

136
NHCO(CH2)8CH3

931

932

1729
1

137
NHCO(CH2)8CH3

933

934

2014
1

138
NHCO(CH2)8CH3

935

936

1762
1

139
NHCO(CH2)8CH3

937

938

1751
1

140
NHCO(CH2)8CH3

939

940

1881
1

141
NHCO(CH2)8CH3

941

942

1914
1

142
NHCO(CH2)8CH3

943

944

1753
1

143
NHCO(CH2)8CH3

945

946

1803
1

144
NHCO(CH2)8CH3

947

948

1813
1

145
NHCO(CH2)8CH3

949

950

2006
1

146
NHCO(CH2)8CH3

951

952

1701
1

147
NHCO(CH2)8CH3

953

954

1799
1

148
NHCO(CH2)8CH3

955

956

1978
1

149
NHCO(CH2)8CH3

957

958

1834
1

150
NHCO(CH2)8CH3

959

960

1777
1

151
NHCO(CH2)8CH3

961

962

1847
1

152
NHCO(CH2)8CH3

963

964

2074
1

153
NHCO(CH2)8CH3

965

966

1895
1

154
NHCO(CH2)8CH3

967

968

1867
1

155
NHCO(CH2)8CH3

969

970

1839
1

156
NHCO(CH2)8CH3

971

972

1781
1

157
NHCO(CH2)8CH3

973

974

1780
1

158
NHCO(CH2)8CH3

975

976

1781
1

159
NHCO(CH2)8CH3

977

978

1805
1

160
NHCO(CH2)8CH3

979

980

1990
1

161
NHCO(CH2)8CH3

981

982

1785
1

162
NHCO(CH2)8CH3

983

984

2092
1

163
NHCO(CH2)8CH3

985

986

1944
1

164
NHCO(CH2)8CH3

987

988

1817
1

165
NHCO(CH2)8CH3

989

990

2014
1

166
NHCO(CH2)8CH3

991

992

1747
1

167
NHCO(CH2)8CH3

993

994

1853
1

168
NHCO(CH2)8CH3

995

996

1762
1

169
NHCO(CH2)8CH3

997

998

1829
1

171
NHCO(CH2)8CH3

999

1000

1914
1

172
NHCO(CH2)8CH3

1001

1002

1767
1

173
NHCO(CH2)8CH3

1003

1004

1736
1

174
NHCO(CH2)8CH3

1005

1006

1718
1

175
NHCO(CH2)8CH3

1007

1008

1808
1

176
NHCO(CH2)8CH3

1009

1010

1781
1

177
NH2

1011

1012

1632
1

178
NHCO(CH2)8CH3

1013

1014

1783
3

179
NHCO(CH2)8CH3

1015

1016

1884
3

180
NHCO(CH2)8CH3

1017

1018

1905
3

181
NHCONH(CH2)10CH3

1019

1020

1851
9

182
NHCO(CH2)8CH3

1021

1022

1801
3b

183
NHCO(CH2)8CH3

1023

1024

1833
1

184
NHCO(CH2)8CH3

1025

1026

1727
1

185
NHCO(CH2)8CH3

1027

1028

1743
1

186
NHCO(CH2)8CH3

1029

1030

1890
1

187
NHCO(CH2)8CH3

1031

1032

1756
1

189
NHCO(CH2)8CH3

1033

1034

1717
3b

190
NHCO(CH2)8CH3

1035

1036

1805
2

192
NHCO(CH2)8CH3

1037

1038

1811
8

193
NHCO(CH2)8CH3

1039

1040

1836
3

194
NHCO(CH2)8CH3

1041

1042

1795
1

195
NHCO(CH2)8CH3

1043

1044

1862
1

196
NHCO(CH2)8CH3

1045

1046

1780
1

197
NHCO(CH2)8CH3

1047

1048

1746
1

198
NHCO(CH2)8CH3

1049

1050

1754
1

199
NHCO(CH2)8CH3

1051

1052

1780
1

200
NHCO(CH2)8CH3

1053

1054

1792
8a

201
NHCO(CH2)8CH3

1055

1056

1821
1

202
NHCO(CH2)8CH3

1057

1058

1

203
NHCO(CH2)8CH3

1059

1060

1793
1

204
NHCO(CH2)8CH3

1061

1062

1893

205
NH(CH2)8CH3

1063

1064

1779
9a

206
NHCO(CH2)8CO2Me

1065

1066

1851
9

207
NHCO(CH2)6CO2Me

1067

1068

1823
9

208
NHCO(CH2)8CH3

1069

1070

1878
1

209
NHCO(CH2)8CH3

1071

1072

1880
1h

210
NHCO(CH2)8CH3

1073

1074

1851
1

211
NHCO(CH2)8CH3

1075

1076

1924
1

212
NHCO(CH2)8CH3

1077

1078

1701
1d

213
NHCO(CH2)6NHBoc

1079

1080

1980
9

214
NHCO(CH2)7NHBoc

1081

1082

1994
9

215
NHCO(CH2)10NHBoc

1083

1084

2036
9

216
NHCO(CH2)11NHBoc

1085

1086

2050
9

217
NHCO(CH2)10NH2

1087

1088

1836
9

218
NHCO(CH2)11NH2

1089

1090

1850
9

219
NHCO(CH2)6CH(CH3)2

1091

1092

1807
9

220
NHCONH(CH2)11CH3

1093

1094

1865
9

221
NHCO(CH2)8CH3

1095

1096

1807
6

222
NHCO(CH2)8CH3

1097

1098

1935
1

223
NHCO(CH2)8CH3

1099

1100

1779
1

224
NHCO(CH2)8CH3

1101

1102

1936
1

225
NHCO(CH2)8CH3

1103

1104

1735
1

226
NHCO(CH2)8CH3

1105

1106

1958
1

227
NHCO(CH2)8CH3

1107

1108

1899
1

228
NHCO(CH2)8CH3

1109

1110

1917
1

229
NHCO(CH2)8CH3

1111

1112

1914
1

230
NHCO(CH2)8CH3

1113

1114

1969
1

231
NHCO(CH2)8CH3

1115

1116

1990
1

232
NHCO(CH2)8CH3

1117

1118

1940
1

233
NHCO(CH2)8CH3

1119

1120

1902
1

234
NHCO(CH2)8CH3

1121

1122

1901
1

235
NHCO(CH2)8CH3

1123

1124

1934
1

236
NHCO(CH2)8CH3

1125

1126

1984
1

237
NHCO(CH2)8CH3

1127

1128

1926
1

238
NHCO(CH2)8CH3

1129

1130

1944
1

239
NHCO(CH2)8CH3

1131

1132

1940
1

240
NHCO(CH2)8CH3

1133

1134

1995
1

241
NHCO(CH2)8CH3

1135

1136

2016
1

242
NHCO(CH2)8CH3

1137

1138

1928
1

243
NHCO(CH2)8CH3

1139

1140

1927
1

244
NHCO(CH2)8CH3

1141

1142

1960
1

245
NHCO(CH2)8CH3

1143

1144

1790
3

246

1145

1146

1147

1807
9

247

1148

1149

1150

1841
9

248

1151

1152

1153

1864
9

249

1154

1155

1156

1843
9

250

1157

1158

1159

1882
9

251

1160

1161

1162

1823
9

252
NHCO(CH2)8CH3

1163

1164

1931
1

253
NHCO(CH2)8CH3

1165

1166

1886
1f

254
NHCO(CH2)7CH3

1167

1168

1650
7

255
NHCO(CH2)9CH3

1169

1170

1678
7

256
NHCO(CH2)10CH3

1171

1172

1692
7

257
NHCO(CH2)11CH3

1173

1174

1706
7

258
NHCO(CH2)12CH3

1175

1176

1720
7a

259
NHCO(CH2)8CH3

1177

1178

1706
6

260
NHCO(CH2)9CH3

1179

1180

1678
7

261
NHCO(CH2)11CH3

1181

1182

1705
7

262
NHCO(CH2)12CH3

1183

1184

1719
7a

263

1185

1186

1187

1738
7

264

1188

1189

1190

1862
9

265
NHCO(CH2)8CH3

1191

1192

1890
1

266
NHCO(CH2)8CH3

1193

1194

1841
1

267
NHCO(CH2)8CH3

1195

1196

1910
1

268
NHCO(CH2)8CH3

1197

1198

1940
9

269

1199

1200

1201

1862
6

270
NHCO(CH2)8CH3

1202

1203

1706
6

271
NHCO(CH2)8CH3

1204

1205

1851
1

272
NHCO(CH2)8CH3

1206

1207

2081
1

273
NHCO(CH2)8CH3

1208

1209

1964
1

274
NHCO(CH2)8CH3

1210

1211

1793
1

275
NHCO(CH2)8CH3

1212

1213

1797
1

276
NHCO(CH2)8CH3

1214

1215

1973
1

277
NHCO(CH2)8CH3

1216

1217

1778
1

278
NHCO(CH2)8CH3

1218

1219

1780
1

279
NHCO(CH2)8CH3

1220

1221

1940
1

280
NHCO(CH2)8CH3

1222

1223

1797
1

281
NHCO(CH2)8CH3

1224

1225

1974
1

282
NHCO(CH2)8CH3

1226

1227

1807
1a

283
NHCO(CH2)8CH3

1228

1229

1797
1

284
NHCO(CH2)8CH3

1230

1231

1973
1

285
NHCO(CH2)8CH3

1232

1233

1796
1b

286
NHCO(CH2)8CH3

1234

1235

1898
1

287
NHCO(CH2)8CH3

1236

1237

1806
1

288
NHCO(CH2)8CH3

1238

1239

1812
1

289
NHCO(CH2)8CH3

1240

1241

1806
1

290
NHCO(CH2)8CH3

1242

1243

1806
1

291
NHCO(CH2)8CH3

1244

1245

1848
1

292

1246

1247

1248

1738
7

293
NHCO(CH2)10CH3

1249

1250

1692
7

294
NHCO(CH2)7CH3

1251

1252

1650
7

295
NHCO(CH2)11CH3

1253

1254

1991
10b

296
NHCO(CH2)10CH3

1255

1256

1978
10b

297
NHCO(CH2)9CH3

1257

1258

1964
10b

298
NHCONH(CH2)7CH3

1259

1260

1950
10b

299
NHCONH(CH2)10CH3

1261

1262

1992
10b

300
NHCONH(CH2)11CH3

1263

1264

2006
10b

301
NHCO(CH2)11CH3

1265

1266

1791
10b

302
NHCO(CH2)10CH3

1267

1268

1778
10b

303
NHCO(CH2)9CH3

1269

1270

1764
10b

304
NHCONH(CH2)7CH3

1271

1272

1750
10b

305
NHCONH(CH2)10CH3

1273

1274

1792
10b

306
NHCONH(CH2)11CH3

1275

1276

1806
10b

307
NHCO(CH2)9CH3

1277

1278

1922
10b

308
NHCO(CH2)10CH3

1279

1280

1936
10b

309
NHCO(CH2)10CH3

1281

1282

1836
10b

310
NHCO(CH2)9CH3

1283

1284

1821
10b

311
NHCONH(CH2)7CH3

1285

1286

1808
10b

312
NHCONH(CH2)7CH3

1287

1288

1759
10b

313
NHCONH(CH2)7CH3

1289

1290

1665
7

314
NHCONH(CH2)10CH3

1291

1292

1707
7

315
NHCONH(CH2)7CH3

1293

1294

1779
10a

316
NHCONH(CH2)7CH3

1295

1296

1700
10a

317
NHCONH(CH2)7CH3

1297

1298

1806
10a

318
NHCO(CH2)9CH3

1299

1300

1793
10a

319
NHCO(CH2)9CH3

1301

1302

1714
10a

320
NHCO(CH2)11CH3

1303

1304

1821
10a

321
NHCO(CH2)11CH3

1305

1306

1848
10a

322
NHCO(CH2)11CH3

1307

1308

1742
10a

323
NHCO(CH2)8CH3

1309

1310

1943
1

324
NHCO(CH2)8CH3

1311

1312

2010
1

325
NHCO(CH2)8CH3

1313

1314

1893
1

326
NHCO(CH2)8CH3

1315

1316

956
1

327
NHCO(CH2)8CH3

1317

1318

1875
1

328
NHCO(CH2)8CH3

1319

1320

1919
1

329
NHCO(CH2)8CH3

1321

1322

1987
1

330
NHCO(CH2)8CH3

1323

1324

1909
1

331
NHCO(CH2)8CH3

1325

1326

1998
1

332
NHCO(CH2)10CH3

1327

1328

1807
10a

333
NHCO(CH2)10CH3

1329

1330

1834
10a

334
NHCO(CH2)10CH3

1331

1332

1728
10a

335
NHCONH(CH2)11CH3

1333

1334

1757
10a

336
NHCONH(CH2)11CH3

1335

1336

1864
10a

337
NHCONH(CH2)11CH3

1337

1338

1836
10a

338
NHCO(CH2)12CH3

1339

1340

1963
10b

339
NHCO(CH2)12CH3

1341

1342

1863
10b

340
NHCO(CH2)12CH3

1343

1344

2006
10b

341
NHCO(CH2)12CH3

1345

1346

1805
10b

342
NHCO(CH2)9CH3

1347

1348

1773
10b

343
NHCO(CH2)10CH3

1349

1350

1786
10b

344
NHCO(CH2)12CH3

1351

1352

1814
10b

345
NHCO(CH2)12CH3

1353

1354

1756
10a

346
NHCO(CH2)12CH3

1355

1356

1836
10a

347
NHCO(CH2)7CH3

1357

1358

1765
10a

348
NHCO(CH2)7CH3

1359

1360

1686
10a

349
NHCO(CH2)7CH3

1361

1362

1792
10a

350

1363

1364

1365

1832
10b

351
NHCO(CH2)11CH3

1366

1367

1801
10b

352
NHCONH(CH2)10CH3

1368

1369

1801
10b

355
NHCONH(CH2)10CH3

1370

1371

1743
10a

356
NHCONH(CH2)10CH3

1372

1373

1822
10a

358
NHCO(CH2)8CH3

1374

1375

1893
1

359
NHCO(CH2)8CH3

1376

1377

948
1

360
NHCO(CH2)8CH3

1378

1379

938
1

361
NHCO(CH2)8CH3

1380

1381

952
1

362
NHCO(CH2)8CH3

1382

1383

969
1

363
NHCO(CH2)8CH3

1384

1385

970
1

364
NHCO(CH2)8CH3

1386

1387

976
1

365
NHCO(CH2)8CH3

1388

1389

976
1

366
NHCO(CH2)8CH3

1390

1391

984
1

367
NHCO(CH2)8CH3

1392

1393

984
1

368
NHCO(CH2)8CH3

1394

1395

986
1

369
NHCO(CH2)8CH3

1396

1397

987
1

370
NHCO(CH2)8CH3

1398

1399

978
1

371
NHCO(CH2)8CH3

1400

1401

998
1

372
NHCO(CH2)8CH3

1402

1403

1003
1

373
NHCO(CH2)8CH3

1404

1405

1003
1

374
NHCO(CH2)8CH3

1406

1407

970
1

375
NHCO(CH2)8CH3

1408

1409

950
1

376
NHCO(CH2)8CH3

1410

1411

950
1

377
NHCO(CH2)8CH3

1412

1413

950
1

378
NHCO(CH2)8CH3

1414

1415

955
1

379
NHCO(CH2)8CH3

1416

1417

957
1

380
NHCO(CH2)8CH3

1418

1419

958
1

381
NHCO(CH2)8CH3

1420

1421

959
1

382
NHCO(CH2)8CH3

1422

1423

959
1

383
NHCO(CH2)8CH3

1424

1425

965
1

384
NHCO(CH2)8CH3

1426

1427

965
1

385
NHCO(CH2)8CH3

1428

1429

975
1

386
NHCO(CH2)8CH3

1430

1431

975
1

387
NHCO(CH2)8CH3

1432

1433

975
1

388
NHCO(CH2)8CH3

1434

1435

957
1

389
NHCO(CH2)8CH3

1436

1437

976
1

390
NHCO(CH2)8CH3

1438

1439

976
1

391
NHCO(CH2)8CH3

1440

1441

976
1

392
NHCO(CH2)8CH3

1442

1443

983
1

393
NHCO(CH2)8CH3

1444

1445

983
1

394
NHCO(CH2)8CH3

1446

1447

948
1

395
NHCO(CH2)8CH3

1448

1449

941
1

398
NHCO(CH2)8CH3

1450

1451

1

399
NHCO(CH2)8CH3

1452

1453

1

400
NHCO(CH2)8CH3

1454

1455

1

401
NHCO(CH2)8CH3

1456

1457

1

402
NHCO(CH2)8CH3

1458

1459

1

403
NHCO(CH2)8CH3

1460

1461

1

404
NHCO(CH2)8CH3

1462

1463

1

405
NHCO(CH2)8CH3

1464

1465

1

406
NHCO(CH2)8CH3

1466

1467

1

407
NHCO(CH2)8CH3

1468

1469

1

408
NHCO(CH2)8CH3

1470

1471

1

409
NHCO(CH2)8CH3

1472

1473

1

410
NHCO(CH2)8CH3

1474

1475

[0160] Preferred compounds of the present invention are compounds 45, 54, 76, 81, 85, 102, 209, 212, 253, 260, 262, 282, 285, 319, 322, 333, 334, 335, 336, 344 and 355.

[0161] According to a preferred embodiment, the present invention provides one or more crystalline forms of compounds of formula (I) and salts thereof

[0162] Lipopeptide Intermediates

[0163] The present invention also provides compounds that are particularly useful as intermediates for the preparation of the compounds of Formula I. These compounds may also have antibacterial properties, as discussed above. In one aspect of the invention, compounds of Formula II are provided.

1476

[0164] wherein R14 is selected from the group consisting of

1477

[0165] wherein R56 is an optionally substituted straight-chain C8-C14 alkyl group and wherein q′ is 0-3.

[0166] In another aspect of the invention, compounds of Formula III are provided as useful intermediates for the preparation of compounds of Formula I and/or as antibacterial compounds:

1478

[0167] wherein R15 is selected from hydrido and a carbamate amino protecting group, preferably a tert-butoxycarbonyl group, wherein R16 is selected from the group consisting of

1479

[0168] wherein R57 is a halo or halo substituted alkyl group, preferably a fluoro or trifluoromethyl group; wherein, R20 is an amino acid side chain, preferably a lysine or tryptophan side chain.

[0169] Compounds 2, 10, 25, 38, 45, 50, 54, 76, 78, 79, 80, 81, 82, 84, 85, 103, 105, 107, 111, 115, 130, 138, 139, 146, 147, 150, 158, 164, 168, 174, 210, 212, 227, 253, 274, 275, 280, 283, 285, 317, 372 and 386 are useful both as antibacterial compounds and as intermediates in the synthesis of compounds of this invention.

[0170] Lipopeptide Compound Pharmaceutical Compositions and Methods of Use Thereof

[0171] Another object of the instant invention is to provide lipopeptide compounds or salts thereof, as well as pharmaceutical compositions or formulations comprising lipopeptide compounds or its salts.

[0172] Lipopeptide compounds, or pharmaceutically acceptable salts thereof, can be formulated for oral, intravenous, intramuscular, subcutaneous or parenteral administration for the therapeutic or prophylactic treatment of diseases, particularly bacterial infections. For oral or parenteral administration, lipopeptide compounds of this invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The compositions comprising a compound of this invention will contain from about 0.1 to about 99% by weight of the active compound, and more generally from about 10 to about 30%.

[0173] The pharmaceutical preparations disclosed herein are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent or eliminate the infection (See, e g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y., the contents of which are incorporated herein by reference, for a general description of the methods for administering various antimicrobial agents for human therapy) The compositions of the invention (preferably of Formula I) can be delivered using controlled ( e.g., capsules) or sustained release delivery systems (e.g., bioerodable matrices). Exemplary delayed release delivery systems for drug delivery that are suitable for administration of the compositions of the invention (preferably of Formula I) are described in U.S. Pat. Nos. 4,452,775 (issued to Kent), 5,239,660 (issued to Leonard), 3,854,480 (issued to Zaffaroni).

[0174] The pharmaceutically-acceptable compositions of the present invention comprise one or more compounds of the invention (preferably compounds of Formula I) in association with one or more nontoxic, pharmaceutically-acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as “carrier” materials, and if desired other active ingredients. The compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The compositions may contain croscarmellose sodium, microcrystalline cellulose, corn starch, sodium starch glycolate and alginic acid.

[0175] Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.

[0176] Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicone fluid, talc, waxes, oils and colloidal silica.

[0177] Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more aesthetic in appearance or to help identify the product.

[0178] For oral use, solid formulations such as tablets and capsules are particularly useful. Sustained release or enterically coated preparations may also be devised For pediatric and geriatric applications, suspensions, syrups and chewable tablets are especially suitable. For oral administration, the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically-effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally are in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

[0179] For intravenous (IV) use, a lipopeptide compound according to the invention can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline or Ringer's solution. Intravenous administration may be accomplished by using, without limitation, syringe, minipump or intravenous line.

[0180] Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions can be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration. The compounds can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or various buffers.

[0181] For intramuscular preparations, a sterile formulation of a lipopeptide compound or a suitable soluble salt form of the compound, for example the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g., an ester of a long chain fatty acid such as ethyl oleate.

[0182] A dose of an intravenous, intramuscular or parental formulation of a lipopeptide compound may be adminstered as a bolus or by slow infusion. A bolus is a dose that is administered in less than 30 minutes. In a preferred embodiment, a bolus is administered in less than 15 or less than 10 minutes. In a more preferred embodiment, a bolus is administered in less than 5 minutes. In an even more preferred embodiment, a bolus is administered in one minute or less. An infusion is a dose that is administered at a rate of 30 minutes or greater. In a preferred embodiment, the infusion is one hour or greater. In another embodiment, the infusion is substantially constant.

[0183] For topical use the compounds of the present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.

[0184] For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

[0185] For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.

[0186] Alternatively, the compounds of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the unit dosage form of the compound can be a solution of the compound or preferably a salt thereof in a suitable diluent in sterile, hermetically sealed ampoules or sterile syringes. The concentration of the compound in the unit dosage may vary, e.g. from about 1 percent to about 50 percent, depending on the compound used and its solubility and the dose desired by the physician. If the compositions contain dosage units, each dosage unit preferably contains from 1-500 mg of the active material. For adult human treatment, the dosage employed preferably ranges from 5 mg to 10 g, per day, depending on the route and frequency of administration.

[0187] In another aspect, the invention provides a method for inhibiting the growth of microorganisms, preferably bacteria, comprising contacting said organisms with a compound of the invention, preferably a compound of Formula I, under conditions which permit entry of the compound into said organism and into said microorganism. Such conditions are known to one skilled in the art and are exemplified in the Examples. This method involves contacting a microbial cell with a therapeutically-effective amount of compound(s) of the invention, preferably compound(s) of Formula I, in vivo or in vitro.

[0188] According to this aspect of the invention, the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (preferably a human) in accordance with known methods of drug delivery. In general, the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the invention (preferably compounds of Formula I) for the drugs in the art-recognized protocols. Likewise, the methods for using the claimed composition for treating cells in culture, for example, to eliminate or reduce the level of bacterial contamination of a cell culture, utilize art-recognized protocols for treating cell cultures with antibacterial agent(s) with the only substantial procedural modification being the substitution of the compounds of the invention (preferably compounds of Formula I) for the agents used in the art-recognized protocols.

[0189] In one embodiment, the invention provides a method for treating an infection, especially those caused by gram-positive bacteria, in a subject with a therapeutically-effective amount of a lipopeptide compound according to Formula I. Exemplary procedures for delivering an antibacterial agent are described in U.S. Pat. No. 5,041,567, issued to Rogers and in PCT patent application number EP94/02552 (publication no. WO 95/05384), the entire contents of which documents are incorporated in their entirety herein by reference. As used herein the phrase “therapeutically-effective amount” means an amount of a compound of the present invention that prevents the onset, alleviates the symptoms, or stops the progression of a bacterial infection. The term “treating” is defined as administering, to a subject, a therapeutically-effective amount of a compound of the invention (preferably a compound of Formula I) both to prevent the occurrence of an infection and to control or eliminate an infection. The term “subject”, as described herein, is defined as a mammal, a plant or a cell culture. In a preferred embodiment, a subject is a human or other animal patient in need of lipopeptide compound treatment.

[0190] The method comprises administering to the subject an effective dose of a compound of this invention. An effective dose is generally between about 0.1 and about 100 mg/kg of a lipopeptide compound of Formula I or a pharmaceutically acceptable salt thereof. A preferred dose is from about 0.1 to about 50 mg/kg of a lipopeptide compound of Formula I or a pharmaceutically acceptable salt thereof A more preferred dose is from about 1 to 25 mg/kg of a lipopeptide compound of Formula I or a pharmaceutically acceptable salt thereof. An effective dose for cell culture is usually between 0.1 and 1000 μg/mL, more preferably between 0.1 and 200 μg/mL.

[0191] The compound of Formula I can be administered as a single daily dose or in multiple doses per day The treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the infection, the age and general health of the patient, the tolerance of the patient to the compound and the microorganism or microorganisms involved in the infection. A method of administration to a patient of daptomycin, another member of the lipopeptide compound class, is disclosed in U.S. Ser. No. 09/406,568, filed Sep. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,828, filed Sep. 25, 1998, and 60/125,750, filed Mar. 24, 1999.

[0192] A lipopeptide compound according to this invention may also be administered in the diet or feed of a patient or animal. If administered as part of a total dietary intake, the amount of compound employed can be less than 1% by weight of the diet and preferably no more than 0.5% by weight. The diet for animals can be normal foodstuffs to which the compound can be added or it can be added to a premix.

[0193] The methods of the present invention comprise administering a lipopeptide compound of Formula I or a pharmaceutical composition thereof to a subject in need thereof in an amount that is efficacious in reducing or eliminating the bacterial infection. The compound may be administered orally, parenterally, by inhalation, topically, rectally, nasally, buccally, vaginally, or by an implanted reservoir, external pump or catheter. The compound may be prepared for opthalmic or aerosolized uses. The compounds of the present invention can be administered as an aerosol for the treatment of pneumonia or other lung-based infections. A preferred aerosol delivery vehicle is an anhydrous or dry powder inhaler. Lipopeptide compounds of Formula I or a pharmaceutical composition thereof also may be directly injected or administered into an abscess, ventricle or joint. Parenteral administration includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, cisternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion. In a preferred embodiment, lipopeptide compounds are administered intravenously, subcutaneously or orally. In a preferred embodiment for administering a lipopeptide compound according to Formula I to a cell culture, the compound may be administered in a nutrient medium.

[0194] The method of the instant invention may be used to treat a subject having a bacterial infection in which the infection is caused or exacerbated by any type of bacteria, particularly gram-positive bacteria. In one embodiment, a lipopeptide compound or a pharmaceutical composition thereof is administered to a patient according to the methods of this invention. In a preferred embodiment, the bacterial infection may be caused or exacerbated by gram-positive bacteria. These gram-positive bacteria include, but are not limited to, methicillin-susceptible and methicillin-resistant staphylococci (including Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and coagulase-negative staphylococci), glycopeptide intermediary- susceptible S. aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. avium, S. bovis, S. lactis, S. sangius and Streptococci Group C, Streptococci Group G and viridans streptococci), enterococci (including vancomycin-susceptible and vancomycin-resistant strains such as Enterococcus faecalis and E. faecium), Clostridium dfficile, C. clostridiiforme, C. innocuum, C. perfringens, C. ramosum, Haemophilus influenzae, Listeria monocytogenes, Corynebacterium jeikeium, Bifidobacterium spp., Eubacterium aerofaciens, E. lentum, Lactobacillus acidophilus, L. casei, L. plantarum, Lactococcus spp., Leuconostoc spp., Pediococcus, Peptostreptococcus anaerobius, P. asaccarolyticus, P. magnus, P. micros, P. prevotii, P. productus, Propionibacterium acnes, Actinomyces spp., Moraxella spp. (including M. catarrhalis) and Escherichia spp. (including E. coli).

[0195] In a preferred embodiment, the antibacterial activity of lipopeptide compounds of Formula I against classically “resistant” strains is comparable to that against classically “susceptible” strains in in vitro experiments. In another preferred embodiment, the minimum inhibitory concentration (MIC) value for lipopeptide compounds according to this invention against susceptible strains is typically the same or lower than that of vancomycin. Thus, in a preferred embodiment, a lipopeptide compound of this invention or a pharmaceutical composition thereof is administered according to the methods of this invention to a patient who exhibits a bacterial infection that is resistant to other compounds, including vancomycin or daptomycin. In addition, unlike glycopeptide antibiotics, lipopeptide compounds exhibits rapid, concentration-dependent bactericidal activity against gram-positive organisms. Thus, in a preferred embodiment, a lipopeptide compound according to this invention or a pharmaceutical composition thereof is administered according to the methods of this invention to a patient in need of rapidly acting antibiotic therapy.

[0196] The method of the instant invention may be used for any bacterial infection of any organ or tissue in the body. In a preferred embodiment, the bacterial infection is caused by gram-positive bacteria. These organs or tissue include, without limitation, skeletal muscle, skin, bloodstream, kidneys, heart, lung and bone. The method of the invention may be used to treat, without limitation, skin and soft tissue infections, bacteremia and urinary tract infections. The method of the invention may be used to treat community acquired respiratory infections, including, without limitation, otitis media, sinusitis, chronic bronchitis and pneumonia, including pneumonia caused by drug-resistant S. pneumoniae or H. influenzae. The method of the invention also may be used to treat mixed infections that comprise different types of gram-positive bacteria, or which comprise both gram-positive and gram-negative bacteria. These types of infections include intra-abdominal infections and obstetrical/gynecological infections. The method of the invention also may be used to treat an infection including, without limitation, endocarditis, nephritis, septic arthritis, intra-abdominal sepsis, bone and joint infections. and osteomyelitis. In a preferred embodiment, any of the above-described diseases may be treated using lipopeptide compounds according to this invention or pharmaceutical compositions thereof.

[0197] The method of the instant invention may also be practiced while concurrently administering one or more other antimicrobial agents, such as antibacterial agents (antibiotics) or antifungal agents. In one aspect, the method may be practiced by administering more than one lipopeptide compounds according to this invention. In another embodiment, the method may be practiced by administering a lipopeptide compound according to this invention with another lipopeptide compound, such as daptomycin.

[0198] Antibacterial agents and classes thereof that may be co-administered with a compound of the present invention include, without limitation, penicillins and related drugs, carbapenems, cephalosporins and related drugs, aminoglycosides, bacitracin, gramicidin, mupirocin, chloramphenicol, thiamphenicol, fusidate sodium, lincomycin, clindamycin, macrolides, novobiocin, polymyxins, rifamycins, spectinomycin, tetracyclines, vancomycin, teicoplanin, streptogramins, anti-folate agents including sulfonamides, trimethoprim and its combinations and pyrimethamine, synthetic antibacterials including nitrofurans, methenamine mandelate and methenamine hippurate, nitroimidazoles, quinolones, fluoroquinolones, isoniazid, ethambutol, pyrazinamide, para-aminosalicylic acid (PAS), cycloserine, capreomycin, ethionamide, prothionamide, thiacetazone, viomycin, eveminomycin, glycopeptide, glycylcylcline, ketolides, oxazolidinone; imipenen, amikacin, netilmicin, fosfomycin, gentamicin, ceftriaxone, Ziracin, LY 333328, CL 331002, HMR 3647, Linezolid, Synercid, Aztreonam, and Metronidazole, Epiroprim, OCA-983, GV-143253, Sanfetrinem sodium, CS-834, Biapenem, A-99058.1, A-165600, A-179796, KA 159, Dynemicin A, DX8739, DU 6681; Cefluprenam, ER 35786, Cefoselis, Sanfetrinem celexetil, HGP-31, Cefpirome, HMR-3647, RU-59863, Mersacidin, KP 736, Rifalazil; Kosan, AM 1732, MEN 10700, Lenapenem, BO 2502A, NE-1530, PR 39, K130, OPC 20000, OPC 2045, Veneprim, PD 138312, PD 140248, CP 111905, Sulopenem, ritipenam acoxyl, RO-65-5788, Cyclothialidine, Sch-40832, SEP-132613, micacocidin A, SB-275833, SR-15402, SUN A0026, TOC 39, carumonam, Cefozopran, Cefetamet pivoxil, and T 3811.

[0199] In a preferred embodiment, antibacterial agents that may be co-administered with a compound according to this invention include, without limitation, imipenen, amikacin, netilmicin, fosfomycin, gentamicin, ceftriaxone, teicoplanin, Ziracin, LY 333328, CL 331002, R3647, Linezolid, Synercid, Aztreonam, and Metronidazole.

[0200] Antifungal agents that may be co-administered with a compound according to this invention include, without limitation, Caspofungen, Voriconazole, Sertaconazole, IB-367, FK-463, LY-303366, Sch-56592, Sitafloxacin, DB-289 polyenes, such as Amphotericin, Nystatin, Primaricin; azoles, such as Fluconazole, Itraconazole, and Ketoconazole; allylamines, such as Naftifine and Terbinafine; and anti-metabolites such as Flucytosine. Other antifungal agents include without limitation, those disclosed in Fostel et al., Drug Discovery Today 5.25-32 (2000), herein incorporated by reference. Fostel et al. disclose antifungal compounds including Corynecandin, Mer-WF3010, Fusacandins, Artrichitin/LL 15G256γ, Sordarins, Cispentacin, Azoxybacillin, Aureobasidin and Khafrefungin.

[0201] Lipopeptide compounds may be administered according to this method until the bacterial infection is eradicated or reduced In one embodiment, a lipopeptide compound is administered for a period of time from 3 days to 6 months. In a preferred embodiment, a lipopeptide compound is administered for 7 to 56 days. In a more preferred embodiment, a lipopeptide compound is administered for 7 to 28 days. In an even more preferred embodiment, a lipopeptide compound is administered for 7 to 14 days. Lipopeptide compounds may be administered for a longer or shorter time period if it is so desired.

[0202] General Procedures for Lipopeptide Compound Synthesis

[0203] Lipopeptide compounds of Formula I may be produced as described below. The lipopeptide compounds of the instant invention may be produced semi-synthetically using daptomycin as a starting point or may be produced by a total synthesis approach.

[0204] For the semi-synthetic approach according to the present invention, daptomycin may be prepared by any method known in the art. See, e.g., U.S. Pat. Nos. 4,885,243 and 4,874,843. Daptomycin may be used in its acylated state or it may be deacylated prior to its use as described herein. Daptomycin may be deacylated using Actinoplanes utahensis as described in U.S. Pat. No. 4,482,487. Alternatively, daptomycin may be deacylated as follows:

[0205] Daptomycin (5.0 g) was dissolved in water (25 ml) and adjusted to pH 9 with 5M sodium hydroxide. Ditert-butyldicarbonate (1.5 g) was added and the mixture was adjusted to maintain pH 9 with 5 M sodium hydroxide until the reaction was complete (4 hours). The pH was adjusted to 7 and the mixture was loaded onto a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted from the column with methanol. Evaporation of the methanol gave BOC-protected daptomycin as a yellow powder.

[0206] A preparation of deacylase enzyme was produced from recombinant Streptomyces lividans, which expresses the Actinoplanes utahensis deacylase enzyme. The enzyme in ethylene glycol (400 μl) was added to BOC-protected daptomycin (1 g) in water (100 ml) at pH 7-8 After incubation for 72 hours, the mixture was loaded on a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted from the column with 10% acetonitrile in water. The product was evaporated to give deacylated BOC-protected daptomycin as a yellow powder.

[0207] Kynurenine Derivatives

1480

[0208] Daptomycin can be converted into analogs bearing modifications at the R2 position by converting the aromatic amino group to the diazonium salt compound I with reagents such as sodium nitrite/hydrochloric acid or isoamylnitrite. Using chemistry known to those skilled in the art and following the teachings of the disclosure, the diazonium group can then be displaced by reagents such as sodium azide, potassium ethylxanthate or copper chloride to yield derivative compounds II, wherein R19 is as previously defined.

1481

[0209] Additionally, compound I can be converted to the azide compound III by reaction with an azide source, typically sodium azide. Modifications to the ketone group can then be undertaken using chemistry known to those having ordinary skill in the art, such as reduction, oxime formation, ketalization conversion to a leaving group and displacement to give compounds of formula IV, wherein R17 and R18 are as previously defined.

1482

[0210] Compound IV may also be converted to compound V by reducing the azide group to the amine using chemistry known to those having ordinary skill in the art, and following the teachings of the disclosure, such as reaction with triphenyl phosphine and water, or reducing agents such as sodium borohydride wherein R17 and R18 are as previously defined.

1483

[0211] Additionally compound I can be converted into compound VI by reduction with hypophosphorus acid. Modifications to the ketone group can then be undertaken using chemistry known to those having ordinary skill in the art similar to those used in scheme 2, wherein R17 and R18 are as previously defined.

[0212] Ornithine Derivatives

1484

[0213] Daptomycin can be converted into analogs bearing modifications at the R1 position by treating the aromatic amino group of the ornithine with reagents such as isocyanates, isothiocyanates, activated esters, acid chlorides, sulfonylchlorides or activated sulfonamides, heterocycles bearing readily displaceable groups, imidates, lactones or reductively with aldehydes to yield compound VIII, wherein R1 is as previously defined.

[0214] Tryptophan Amine Derivatives

1485

[0215] Daptomycin can be converted into compound IX by first protecting the ornithine amine with an appropriate amino protecting group (P) known to those skilled in the art and following the teachings of the disclosure. The decyl side chain on the tryptophan is then removed using an enzyme capable of deacylating daptomycin, such as that described above.

1486

[0216] Compound IX can be modified at the tryptophan amine with reagents such as isocyanates, isothiocyanates, activated esters, acid chlorides, sulfonylchlorides or activated sulfonamides, heterocycles bearing readily displaceable groups, imidates, lactones or reductively with aldehydes to yield compound X. Compound X can be deprotected to give compound XI according to procedures known to those skilled in the art following the disclosure of this invention, wherein R is as previously defined.

[0217] The above modifications to the ornithine amine R1, tryptophan amine R or kynurenine side chain R2 may be independently combined to yield additional compounds that are modified at up to all three sites. In order to achieve these modifications, it may be necessary to protect certain functionalities in the molecule. Protecting these functionalities should be within the expertise of one skilled in the art following the disclosure of this invention. See, e.g., Greene, supra.

[0218] Solid Support Synthesis of Lipopeptide Compounds

[0219] In an alternative embodiment of the invention, the lipopeptide compounds of Formula I may be synthesized on a solid support as outlined below. In step 1, a suitably-N-protected-βMeGlu(OH)-OAllyl ester is coupled to a suitable resin to give Compound XII. Deprotection of the amino group of Compound XII, followed by coupling of the amino group with a suitably protected seryl derivative (Al) gives Compound XIII, wherein P is a suitable protecting group. This peptide coupling process, i.e., deprotection of the alpha-amino group, followed by coupling to a suitably protected amino acid, is repeated until the desired number of amino acids have been coupled to the resin. In the scheme shown below, eleven amino acids have been coupled to give Compound XIV. Addition of an activated R group, R*, is added to Compound XIV to give Compound XV. In step 4, Compound XV is cyclized to give Compound XVI. Subsequently, in step 5, Compound XVI is removed from the resin to give the lipopeptide Compound XVII.

1487

1488

[0220] wherein A1, is a suitably protected serine derivative, wherein R31 is a suitable, cleavable hydroxyl protecting group as outlined below.

1489

[0221] wherein A2 and A7, are suitably protected glycine derivatives as outlined below.

1490

[0222] wherein A3, A5 and A9 are suitably protected aspartic acid derivatives as outlined below, wherein 28R, 29R and 30R are cleavable protecting groups, preferably t-butyl groups.

1491

[0223] wherein A4 is a suitably protected alanine derivative as outlined below

1492

[0224] wherein A6 is a suitably protected ornithine derivative as outlined below, or derivatized ornithine wherein *R1 is R1 as previously described or alternatively a protected form of R1 that would yield R1 upon subsequent deprotection.

1493

[0225] wherein A8 is a suitably protected depsipeptide as outlined below, Y is a protecting group that is cleavable under conditions that leave other protecting groups intact to the others used, i.e., Alloc; and wherein *R2 is R2 as previously described or alternatively a protected form of R2 that would yield R2 upon subsequent deprotection. Preferably 2*R is a kynurenine, or substituted kynurenine side chain, most preferably

1494

[0226] wherein A10 is a suitably protected asparagine derivative as outlined below.

1495

[0227] wherein A11 is a suitably protected tryptophan derivative as outlined below, wherein R*37 is hydrido or a suitable protecting group, preferably t-butoxy carbonyl.

[0228] It will be understood by those skilled in the art that both the amino and the side chain functional groups must be suitably protected prior to attaching them to the growing peptide chain. Suitable protecting groups can be any group known in the art to be useful in peptide synthesis. Such pairings of protecting groups are well known. See, e.g., “Synthesis Notes” in the Novabiochem Catalog and Peptide Synthesis Handbook (1999), pages S1-S93 and references cited therein. Following the disclosure of the present application, the selection of protecting groups and method of use thereof will be known to one skilled in the art.

[0229] It will also be understood by those skilled in the art that the choice of protecting group on the side chain functional groups will either result or not result in the protecting group being cleaved concomitantly with the peptide's final cleavage from the resin, which will give the natural amino acid functionality or a protected derivative thereof, respectively.

[0230] The following general procedures serve to exemplify the solid support synthesis of compounds of Formula I.

[0231] Step 1: Coupling Suitably-N-protected-βMeGlu(OH)-OAllyl Ester to a Resin

[0232] Five molar equivalents each, with respect to the resin, of a suitably-N-protected-βMeGlu(OH)-OAllyl ester, 1,3-Diisopropylcarbodiimide (DIC) and 1-Hydroxy-7-azabenzotriazole (HOAt) are stirred for 30 mins in dimethylformamide (DMF; 5 ml/g resin). A suitably functionalised resin or solid support, such as, but not limited to, Wang, Safety Catch, Rink, Knorr, PAL, or PAM resin, is added and the resulting suspension is stirred for 16 hrs. The resin-N-protected-βMeGlu(OH)-OAllyl ester is then filtered, dried and the coupling is repeated. The N-protecting group is then removed using the appropriate conditions given in the coupling steps below

[0233] Step 2: (A) General Coupling Cycle For Amino Acids With an N-9-Fluorenylmethoxycarbonyl (Fmoc) Protecting Group

[0234] Five molar equivalents each, with respect to the resin-AA(wherein resin-AA is defined as the resin attached the the growing amino acid chain), of a suitably protected Fmoc amino acid, DIC, and HOAt (0.5 molar solution in DMF) are added to the resin-AA, along with sufficient DMF to give a working volume. The mixture is shaken for one hour, filtered, and the coupling is repeated. After the second coupling the resin is washed twice with DMF, twice with methanol, and twice again with DMF. The Fmoc group of the newly coupled amino acid A1-11 is deprotected by stirring the resin product in one working volume of a solution of 20% piperidine in N-methyl pyrolidine for five minutes, filtering the resin, and stirring the resin in 20% piperidine in N-methyl pyrolidine again for 20 minutes. The resin is washed twice with DMF, twice with methanol, and twice again with DMF.

[0235] Step 2 (B): General Coupling Cycle of Amino Acids With an N-tert-Butoxy-carbonyl (N-Boc) Protecting Group

[0236] Five molar equivalents each, with respect to the resin-AA, of a suitably protected N-Boc amino acid, DIC, and HOAt (0.5 molar solution in DMF) are added to the resin-AA, along with sufficient DMF to give a working volume The mixture is shaken for one hour, filtered, and the coupling is repeated. After the repeated coupling the resin is washed twice with DMF, twice with methanol, and twice again with DMF. The Boc group of the newly coupled amino acid A1-11, is then deprotected by stirring the resin in one working volume of CH2Cl2:trifluoroacetic acid (TFA) 1:1 for 15 minutes, filtering, and stirring in one working volume of CH2Cl2:TFA 1:1 for another 15 minutes. The resin is neutralized by washing with excess diisopropylethylamine (DIPEA) in CH2Cl2 and then washed twice with DMF, twice with methanol, and twice again with DMF.

[0237] Step 3: Terminal Amine Capping Reaction

[0238] Ten molar equivalents, with respect to the resin XV, of a suitable reagent containing R* such as an activated ester, isocyanate, thioisocyanate, anhydride, acid chloride, chloroformate, or reactive salt thereof, in one working volume of DMF is added to the resin XIV and agitated for 25 hours. The resulting resin XV is washed twice with DMF, twice with methanol, and twice again with DMF.

[0239] Step 4: Cyclization

[0240] The dried resin XV is placed under an argon atmosphere, and treated with a solution of Pd(PPh3)4 125 mgs/0.1 mmol peptide substrate, in CH2Cl2: Acetic acid. N-Methylmorpholine, 40:2:1, 1 ml/0.1 mmol peptide substrate. The mixture is stirred for 3 hours at ambient temperature, filtered, and washed twice with DMF, twice with methanol, and twice again with DMF. Five molar equivalents each, with respect to the resin, of DIC, and HOAt (0.5 molar solution in DMF) are added to the resin, along with sufficient DMF to give a working volume. The reaction is shaken for 17 hours, filtered, and washed twice with DMF, twice with methanol, and twice again with DMF to give resin XVI.

[0241] Step 5: Cleavage and Isolation of the Lipopeptide

[0242] The desired lipopeptide is cleaved from resin XVI and isolated, resulting in a compound in which R27 is OH or NH2. If Fmoc chemistry is used, the dried resin is suspended in 1 ml/0.1 mmol peptide substrate of CH2Cl2:TFA:Ethanedithiol (EDT) Triisopropylsilane (TIS), 16:22:1:1, and stirred for 6-8 hours at ambient temperature. The resin is filtered, washed with 1 equal volume of cold TFA, and the combined filtrates are evaporated under reduced pressure. Crude product XVII is then precipitated by the addition of diethyl ether, and isolated by centrifugation. This product may be further purified by preparative reverse phase HPLC.

[0243] If N-Boc chemistry is used, the dried resin is suspended in hydrogen flouride (HF):anisole:dimethylsulfide (DMS), 10:1:1, and stirred for 2 hours at 0° C. The volitiles are evaporated under a stream of nitrogen. The resin is then extracted with TFA, filtered and washed twice with TFA, and the combined TFA filtrates evaporated under reduced pressure. Crude product is then precipitated by the addition of diethyl ether, and isolated by centrifugation. This product may be further purified by preparative reverse phase HPLC.

[0244] If the resin is a Safety Catch resin, then R27=OR or NRH. The dried resin XVI is suspended in N-methylpyrolidine (NMP) or dimethylsulphoxide (DMSO) (8 ml/g resin), Five equivalents of DIPEA (with respect to resin substitution) and 24 equivalents of iodo or bromoacetonitrile (with respect to resin substitution) are added. The suspension is stirred for 24 hours at ambient temperature under inert atmosphere. The resin is filtered, washed with tetrahydrofuran (THF) and DMSO. For an ester, the resin is then treated with an alcohol, hydroxide or alkoxide (20 equivalents with respect to resin substitution) in THF for 20 hours. The resin is filtered, washed with THF and water, and the combined filtrates are evaporated under reduced pressure. Crude product is precipitated by the addition of diethyl ether, and isolated by centrifugation. The product may be further purified by preparative reverse phase HPLC. For amides the resin is then treated with a primary or secondary amine (20 equivalents with respect to resin substitution) in THF for 12-40 hours, at a gentle reflux under inert atmosphere. The resin is filtered, washed with THF and water, and the combined filtrates are evaporated under reduced pressure. Crude product is then precipitated by the addition of diethyl ether, and isolated by centrifugation. This product may be further purified by preparative reverse phase HPLC.

[0245] In order that this invention may be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

EXAMPLE1

Preparation of Compounds 38, 40, 50, 52, 77-80, 82-84, 87-100, 103-169, 171-176, 183-187, 194-199, 201-204, 208, 210-211, 222-244, 252, 265-267, 271-281, 283-284, 286-291, 323-331, 358-395 and 398-410

[0246] A suspension of daptomycin in dry dimethylformamide (0.6 ml) was treated with a solution of 4-Fluorobenzaldehyde (0.2 ml) and a suspension of sodium triacetoxyborohydride (0.2 ml, 1.5M in dry dimethylformamide). After 24 hours, the reaction mixture was diluted with water/acetonitrile (1:1; 0.4 ml) and purified by preparative HPLC. The reaction mixture was loaded onto an IBSIL-C8 5μ 250×20.2 mm column and eluted at 20 ml/min with 30-60% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing product were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 38 as a pale yellow solid (23 mg).

[0247] In an analogous manner, compounds 40, 50, 52, 77-80, 82-84, 87-100, 103-169, 171-176, 183-187, 194-199, 201-204, 208, 210-211, 222-244, 252, 265-267, 271-281, 283-284, 286-291, 323-331, 358-395 and 398-410 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those skilled in the art following the teachings of the disclosure.

EXAMPLE 1a

Preparation of Compound 282

[0248] 2-Methyl-6-nitroquinoline (0.4 ml, 0.5M solution in dioxane) was treated with selenium dioxide (0 2 ml, 0.9M solution in 9/1 dioxane/water) and heated to 90° C. overnight. The mixture was cooled to room temperature and diluted with water (1 ml). The mixture was then extracted with ethyl acetate (3×2 ml). The organic extract was then dried in vacuo to give 6-nitro-2-quinolinecarboxaldehyde which was carried forward without further purification. Daptomycin (1 ml, 0.1 M in dry dimethylformamide) was treated successively with 6-nitro-2-quinolinecarboxaldehyde prepared above in dry dimethylformamide (0.2 ml) and sodium triacetoxyborohydride (0.4 ml, 1.5M solution in dry dimethylformamide). The mixture was capped and shaken briefly. After 24 h, the mixture was treated with water (0.2 ml) and loaded onto an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 25 ml/min under the gradient conditions of 30-60% acetonitrile in 5 mM ammonium phosphate buffer over 25 min followed by holding at 60% acetonitrile in 5 mM ammonium phosphate buffer for another 10 min. The desired fractions were collected and the acetonitrile was removed by evaporation. The residue was applied to a Bondesil 40μ C8 resin column, washed with water and the product was eluted with methanol. Evaporation of the methanol gave compound 282 as a pale yellow solid.

EXAMPLE 1b

Preparation of Compound 285

[0249] 4-Chloro-2-methylquinoline (0.4 ml, 0.5M solution in dioxane) was treated with selenium dioxide (0.2 ml, 0.9M solution in 9/1 dioxane/water) and heated to 90° C. overnight. The mixture was cooled to room temperature and diluted with water (1 ml). The mixture was then extracted with ethyl acetate (3×2 ml). The organic extract was then dried in vacuo to give 4-chloro-2-quinolinecarboxaldehyde which was carried forward without further purification. Daptomycin (1 ml, 0 1 M in dry dimethylformamide) was treated successively with 4-chloro-2-quinolinecarboxaldehyde prepared above and diluted in dry dimethylformamide (0.2 ml) and sodium triacetoxyborohydride (0.4 ml, 1.5M in dry dimethylformamide) The mixture was capped and shaken briefly. After 24 h the mixture was treated with water (0.2 ml) and loaded on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 25 ml/min under the gradient conditions of 30-60% acetonitrile in 5 mM ammonium phosphate buffer over 25 min followed by holding at 60% acetonitrile in 5 mM ammonium phosphate buffer for another 10 min. The desired fractions were collected and the acetonitrile was removed by evaporation. The residue was applied to a Bondesil 40μ C8 resin column, washed with water and the product eluted off with methanol. Evaporation of the methanol gave compound 285 as a yellow solid.

EXAMPLE 1c

Preparation of Compound 85

[0250] Daptomycin (1 ml, 0.1M in dry dimethylformamide) was treated successively with 1-methyl-2-imidazolecarboxaldehyde (0.2 ml, 0.5M solution in dry dimethylformamide) and sodium triacetoxyborohydride (0.4 ml, 1.5M solution in dry dimethylformamide). The mixture was capped and shaken briefly. After 24 h the mixture was treated with water (0.2 ml) and loaded onto an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 30 ml/min under the gradient conditions of 35-40% acetonitrile in 5 mM ammonium phosphate buffer over 30 min The desired fractions were collected and the acetonitrile was removed by evaporation. The residue was applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. This mixture was then loaded on a Prodigy ODS 10μ 250×21.2 mm column eluted at 50 ml/min at 33% acetonitrile in 5 mM ammonium phosphate buffer adjusted to pH 3.2. The desired fractions were collected and the acetonitrile was removed by evaporation. The residue was applied to a Bondesil 40μ C8 resin column, washed with water and the product was eluted with methanol. Evaporation of the methanol gave compound 85 as a pale yellow solid.

EXAMPLE 1d

Preparation of Compound 212

[0251] Daptomycin (1 ml, 0.1 M in dry dimethylformamide) was treated successively with 2-imidazolecarboxaldehyde (0.2 ml, 0.5M solution in dry dimethylformamide) and sodium triacetoxyborohydride (0.4 ml, 1.5M solution in dry dimethylformamide). The mixture was capped and shaken briefly. After 24 h, the mixture was treated with water (0.2 ml) and the mixture was loaded on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 30 ml/min under the gradient conditions of 35-40% acetonitrile in 5 mM ammonium phosphate buffer over 30 min. The desired fractions were collected and the acetonitrile was removed by evaporation. The residue was applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. This mixture was then loaded on a Prodigy ODS 10μ 250×21.2 mm column and eluted at 50 ml/min at 33% acetonitrile in 5 mM ammonium phosphate buffer adjusted to pH 3.2. The desired fractions were collected and the acetonitrile was removed by evaporation. The residue was applied to a Bondesil 40μ C8 resin column, washed with water and the product eluted with methanol. Evaporation of the methanol gave compound 212 as a yellow solid.

EXAMPLE 1e

Preparation of Compound 81

[0252] Daptomycin (1 ml, 0.1M in dry dimethylformamide) was treated successively with 5-fluoroindole-3-carboxaldehyde (0.2 ml, 0.5M solution in dry dimethylformamide) and sodum triacetoxyborohydride (0.4 ml, 1.5M solution in dry dimethylformamide) The mixture was capped and shaken briefly. After 24 h the mixture was treated with water (0.2 ml) and loaded on an IBSIL-C8 5μ 250×20.2 mm column The column was eluted at 25 ml/min under the gradient conditions of 30-60% acetonitrile in 5 mM ammonium phosphate buffer over 25 min followed by holding at 60% acetonitrile in 5 mM ammonium phosphate buffer for another 10 min. The desired fractions were collected, the acetonitrile was removed by evaporation and the residue applied to a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted with methanol. Evaporation of the methanol gave compound 81 as a pale yellow solid.

EXAMPLE 1f

Preparation of Compound 253

[0253] p-N,N-Bis(2-chloroethyl)aminobenzaldehyde (0.3 g) was dissolved in acetone (2.5 ml) and treated with sodium iodide (0.4 g). The mixture was warmed to 40° C. for 3 h then treated with benzylamine (0.2 ml) and triethylamine (0.4 ml). The mixture was diluted to 7 ml with acetonitrile and then heated to 60° C. After 24 h, the mixture was cooled to room temperature and the solvent was removed by evaporation 4-(4-Benzylpiperazino)benzaldehyde was purified by silica gel chromatography eluting with (10% triethylamine/methanol/dichloromethane).

[0254] Daptomycin (1 ml, 0.1 M in dry dimethylformamide) was treated successively with the 4-(4-benzylpiperazino)benzaldehyde prepared above diluted in dry dimethylformamide (0.2 ml), and sodium triacetoxyborohydride (0.4 ml, 1.5M solution in dry dimethylformamide). The mixture was capped and shaken briefly After 24 h the mixture was treated with water (0.2 ml) and loaded on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 25 ml/min under the gradient conditions of 30-60% acetonitrile in 5 mM ammonium phosphate buffer over 25 min followed by holding at 60% acetonitrile in 5 mM ammonium phosphate buffer for another 10 min. The desired fractions were collected, the acetonitrile was removed by evaporation and the residue was applied to a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted with methanol. Evaporation of the methanol gave compound 253 as a pale yellow solid.

EXAMPLE 1g

Preparation of Compound 79 and 177

[0255] Daptomycin (1 ml, 0.1 M in dry dimethylformamide) was treated successively with 4-phenylbenzaldehyde (0.2 ml, 0.5M in dry dimethylformamide) and sodium triacetoxyborohydride (0.4 ml, 1.5M in dry dimethylformamide). The reaction mixture was capped and shaken briefly to mix the solution. After 24 h the mixture was treated with water (0.2 ml) and loaded on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 25 ml/min under the gradient conditions of 30-60% acetonitrile in 5 mM ammonium phosphate buffer over 25 min followed by holding at 60% acetonitrile in 5 mM ammonium phosphate buffer for another 10 min. The desired fractions were collected, the acetonitrile was removed by evaporation and the residue was applied to a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted with methanol. Evaporation of the methanol gave compound 76 as a pale yellow solid. Compound 177 was obtained by deacylation of compound 76 according to Example 7.

EXAMPLE 1h

Preparation of Compound 209

[0256] 4-Hydroxy-3-nitrobenzaldehyde (0 4 ml, 0.2M in acetone) was successively treated with potassium hydroxide (0 1 ml, 1M in water) and 4-fluorobenzylbromide (0 4 ml, 0.2M in acetone). After 24 h the mixture was dried in vacuo to give 4-(4-fluorobenzyloxy)-3-nitro-benzaldehyde which was carried forward without further purification.

[0257] Daptomycin (1 ml, 0.1 M in dry dimethylformamide) was treated successively with, 4-(4-fluorobenzyloxy)-3-nitro-benzaldehyde previously prepared above diluted in dry dimethylformamide (0.2 ml), and sodium triacetoxyborohydride (0.4 ml, 1.5M in dry dimethylformamide). The mixture was capped and shaken briefly. After 24 h the mixture was treated with water (0.2 ml) and loaded onto an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 25 ml/min under the gradient conditions of 30-60% acetonitrile in 5 mM ammonium phosphate buffer over 25 min followed by holding at 60% acetonitrile in 5 mM ammonium phosphate buffer for another 10 min. The desired fractions were collected, the acetonitrile was removed by evaporation and the residue was applied to a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted with methanol. Evaporation of the methanol gave compound 209 as a pale yellow solid.

EXAMPLE 2

Preparation of Compounds 10, 11-17, 19-20, 22-27 and 190

[0258] Daptomycin (972 mg) was dissolved in dry dimethylformamide (20 ml), and isatoic anhydride (979 mg) was added. The mixture was stirred at ambient temperature for 10 days, then quenched by the addition of water (20 ml). The mixture was loaded onto a Bondesil 40μ C8 resin column (25 g), which had been previously washed with methanol (50 ml) and water (100 ml). The column was then eluted with water (200 ml), 15% methanol/water (1200 ml), 20% methanol/water (200 ml), 30% methanol/water (200 ml) and 40% methanol/water (200 ml). The product bearing fractions were combined and freeze dried to give compound 10 as a white solid (870 mg)

[0259] In an analogous manner, compounds 11-17, 19-20, 22-27 and 190 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those skilled in the art following the teachings of the disclosure.

EXAMPLE 3

Preparation of Compounds 44, 45, 41-43, 46-48, 55-58, 60-75, 178-180, 193 and 245

[0260] Daptomycin (500 mg) and Boc-tryptophan-p-nitrophenyl ester (157.5 mg) were stirred at room temperature in dry dimethylformamide (30 ml) for 3 days. Water (30 ml) was added and the mixture was purified on a Bondesil 40μ C8 resin column (25 g). The column was eluted with 20% acetonitrile in water (200 ml), 40% acetonitrile in water (200 ml) and finally with methanol. Evaporation of the solvent from the product-containing fractions gave compound 44 as a pale yellow solid (450 mg).

[0261] Compound 44 (200 mg) was cooled to 0° C. and a 0° C. solution of 5% thioanisole in trifluoroacetic acid (10 ml) was added. After 3 hours at 0° C. the mixture was evaporated to dryness and the residue was purified by preparative HPLC on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 20 ml/min with 38% acetonitrile in 5 mM ammonium phosphate buffer. The product containing fractions were freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 45 as a pale yellow solid.

[0262] In an analogous manner, compounds 41-43, 46-48, 55-58, 60-75, 178-180, 193 and 245 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those skilled in the art following the teachings of the disclosure.

EXAMPLE 3a

Preparation of Compounds 54, 49 and 51

[0263] Daptomycin (400 mg) and N, N-bis(tert-butoxycarbonyl)-L-lysine-4-nitrophenyl ester (173 mg) were stirred in dry dimethylformamide (5 ml) at room temperature for two days. The mixture was loaded onto an IBSIL-C8 5μ 250×20.2 mm column and was eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave the Boc protected intermediate as a pale yellow solid (370 mg).

[0264] Boc protected intermediate (200 mg) was stirred in trifluoroacetic acid (5 ml) and anisole (0.25 ml) at room temperature for 2 hours. Removal of the solvents under reduced pressure gave a residue which was loaded on an IBSIL-C8 5μ 250×20.2 mm column and eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave compound 54 as a pale yellow solid (100 mg).

[0265] In an analogous manner, compounds 49 and 51 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those skilled in the art following the teachings of the disclosure.

EXAMPLE 3b

Preparation of Compounds 32, 18, 21, 28-31, 33-35, 39, 182 and 189

[0266] Daptomycin (162 mg) and 2-methylthiobenzoic acid pentafluorophenol ester (37 mg) were stirred at room temperature in dry dimethylformamide (10 ml) for 5 days. The dimethylformamide was evaporated under reduced pressure and the residue was purified by preparative HPLC on an IBSIL-C8 5μ 250×20.2 mm column The column was eluted at 20 ml/min with 36% acetonitrile in 5 mM ammonium phosphate buffer. Fractions collected at 7.3 minutes were freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 32 as a pale yellow solid (47 mg).

[0267] In an analogous manner, compounds 18, 21, 28-31, 33-35, 39, 182 and 189 can be prepared as detailed in the above example by appropriate substitutions of reagents by one having ordinary skill in the art following the teachings of the disclosure.

EXAMPLE 4

Preparation of Compounds 5, 4, 6-8 and 9

[0268] Daptomycin (16 mg) was dissolved in dry dimethylformamide (0.5 ml) and methyl isothiocyanate (37 mg) was added. The mixture was stirred at ambient temperature for 24 hours, then quenched by the addition of 5% ammonium phosphate buffer (1 ml). The mixture was purified by preparative HPLC on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 20 ml/min with 36% acetonitrile in 5 mM ammonium phosphate buffer. The product bearing fractions were combined and freeze dried. The freeze-dried residue was dissolved in water (1.5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 5 as a pale yellow solid (5.2 mg).

[0269] In an analogous manner, compounds 4, 6-8 and 9 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those having ordinary skill in the art.

EXAMPLE 5

Preparation of Compound 3

[0270] Daptomycin (16 mg) and N-benzotriazole phenylsulfonamide (2.6 mg) were stirred at room temperature in dry pyridine for 6 days. The solvent was evaporated and the residue was purified by preparative HPLC using an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 20 ml/min with 36% acetonitrile in 5 mM ammonium phosphate buffer and product containing fractions were freeze-dried. The freeze dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 3 as a pale yellow solid (4 mg).

EXAMPLE 6

Preparation of Compounds 1, 2, 221, 259 and 270

[0271] Daptomycin (32 mg) was dissolved in dry dimethylformamide (20 ml), and N,N′-bis-Boc-1-guanidinylpyrazole (31 mg) was added. The mixture was stirred at ambient temperature for 5 days, then quenched by the addition of water (3 ml). The resultant mixture was loaded onto a Bondesil 40μ C8 resin (900 mg) that had been previously washed with methanol and water. The column was eluted with water (30 ml) followed by methanol. The product-bearing fractions were combined and evaporated to give compound 1 as a white solid.

[0272] Compound 1 (30 mg) was dissolved in trifluoroacetic acid/dichloromethane/tri-isopropylsilane/ethane dithiol (11/8/0.5/0.5, 3 ml) and stirred at ambient temperature for 90 minutes. The mixture was evaporated to dryness and the residue was precipitated by the addition of diethyl ether (10 ml). The residue was purified by preparative HPLC on an IBSIL-C8 5μ 250×20.2 mm column. The column was eluted at 20 ml/min with 38% acetonitrile in 5 mM ammonium phosphate buffer The product-bearing fractions were combined and freeze dried. The freeze-dried residue was dissolved in water (1.5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 2 as a white solid (6.4 mg).

[0273] In an analogous manner, compounds 221, 259 and 270 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those having ordinary skill in the art following the teachings of the disclosure.

EXAMPLE 7

Preparation of Compounds 255, 260, 254, 256-257, 261, 263, 292-294 and 313-314

[0274] Daptomycin (10 g) was dissolved in dry dimethylformamide (100 ml). N,N′-bis-Boc-guanidinylpyrazole (2.3 g) in dry dimethylformamide (5 ml) was added. The mixture was stirred under nitrogen at room temperature overnight. The mixture was purified on a Bondesil 40μ C8 resin column. The product containing fractions were freeze-dried to give compound 1 (7.4 g) as pale yellow fluffy solid.

[0275] Compound 1 (2.6 g) was added to a preparation of deacylase enzyme produced from recombinant Streptomyces lividans, which expresses the Actinoplanes utahensis deacylase enzyme in ethylene glycol (1.2 ml) and water (25 ml). The pH of the solution was adjusted to 9 with 1.0M sodium hydroxide solution and stirred at room temperature. After 24 hours the mixture was purified on a Bondesil 40μ C8 resin column by eluting with 10% acetonitrile/water, then 40% acetonitrile/water. The product-containing fractions were freeze dried to give deacylated bis-Boc-guanidinylated daptomycin (0.69 g) as a pale yellow solid.

[0276] Undecanoyl pentafluorophenol ester (40.3 mg) was added to deacylated bis-Boc-guanidinylated daptomycin (171.5 mg) in dry dimethylformamide (2 ml). The mixture was stirred overnight at room temperature before being concentrated to give compound 255 (105 mg) as a yellow solid.

[0277] Compound 255 was dissolved in trifluoroacetic acid (5.5 ml), dichloromethane (4 ml), ethane dithiol (0.25 ml) and triisopropylsilane (0.25 ml). The mixture was stirred for 4 hours at room temperature before being concentrated and purified by preparative HPLC on an IB-SIL 5μ 250×20.2 mm column. The column was eluted at 25 ml/min with acetonitrile and ammonium phosphate buffer 30%-60% gradient for 40 min. The desired fractions were collected at 21 minutes and freeze dried. The freeze-dried residue was dissolved in water and applied to a Bondesil C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 260 (27.8 mg) as a pale yellow solid.

[0278] In an analogous manner, compounds 254, 256-257, 261, 263, 292-294 and 313-314 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those having ordinary skill in the art following the disclosure of the invention.

EXAMPLE 7a

Preparation of Compounds 258 and 262

[0279] Tetradecanoyl pentafluorophenol ester (35.5 mg) and deacylated bis-Boc-guanidinylated daptomycin (102.5 mg) in dry dimethylformamide (2 ml). The mixture was stirred overnight at room temperature before being concentrated to give compound 258 (38.8 mg) as a yellow solid.

[0280] Compound 258 (38.8 mg) was dissolved in trifluoroacetic acid (5.5 ml), dichloromethane (4 ml), ethane dithiol (0.25 ml) and triisopropylsilane (0.25 ml). The mixture was stirred for 4 hours at room temperature before being concentrated and purified by preparative HPLC on an IB-SIL 5μ 250×20.2 mm column The column was eluted at 25 ml/min with acetonitrile and ammonium phosphate buffer 30%-60% gradient for 40 min. The desired fractions were collected at 21minutes and freeze dried. The freeze-dried residue was dissolved in water and applied to a Bondesil C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave compound 262 (2.1 mg) as a pale yellow solid

EXAMPLE 8

Preparation of Compound 37, 36 and 192

[0281] Daptomycin (162 mg) was stirred in 0.1 M hydrochloric acid (5 ml) at 0° C. for 10 minutes before sodium nitrite (8 mg) in water (0.2 ml) was added dropwise. Sulfamic acid (11 mg) was added after 15 minutes, followed by sodium azide (8 mg) 10 minutes later. The mixture was maintained at 0° C. for 4 hours and then neutralized with a saturated sodium bicarbonate solution and purified by preparative HPLC. An IBSIL-C8 5μ 250×20.2 mm column was loaded with the mixture and eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions were collected at 6.9 minutes and freeze dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave the azido daptomycin as a pale yellow solid (60 mg).

[0282] The azido daptomycin (69 mg) was dissolved in dry dimethylformamide (4 ml) and iminobiotin-N-hydroxysuccinimide ester (53 mg) was added. The mixture was covered to exclude light and stirred at ambient temperature for 3 days. The mixture was quenched by the addition of water (20 ml). The resultant mixture was loaded onto a Bondesil 40μ C8 resin (25 g) column, which had been previously washed with methanol and water, and the column was eluted with water. The product-bearing fractions were combined and freeze dried to give Compound 37 as a white solid (49 mg).

[0283] In an analogous manner, compounds 36 and 192 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those having ordinary skill in the art by following the disclosure of the invention

EXAMPLE 8a

Preparation of Compound 200

[0284] Daptomycin (1.62 g) in 50% wt aqueous solution of hypophosphorus acid (10 ml) was stirred at 0° C. for 30 minutes before adding dropwise a solution of sodium nitrite (76 mg) in water (0.5 ml). The mixture was allowed to come to room temperature and stirred for 24 hours. The mixture was purified by preparative HPLC by loading the mixture on an IBSIL-C8 5μ 250×20.2 mm column and eluting the column at 20 ml/min with 32% acetonitrile in 5 mM ammonium phosphate buffer The desired fractions were collected at 30 minutes and freeze dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column. The column was washed with water and eluted with methanol. Evaporation of the methanol gave desamino daptomycin as a pale yellow solid (200 mg).

[0285] To desamino daptomycin (80 mg) in dry dimethylformamide (2 ml) was added N-t-butoxycarbonyl-L-tryptophan-p-nitrophenyl ester (32 mg). The mixture was stirred at room temperature for 24 hours before being purified by preparative HPLC. The mixture was loaded on an IBSIL-C8 5μ 250×20.2 mm column and eluted at 20 ml/min with 40% acetonitrile in 5 mM ammonium phosphate buffer. The desired fractions were collected at 19 minutes and freeze-dried. The freeze-dried residue was dissolved in water (2 ml) and applied to a plug of Bondesil 40μ C8 resin (500 mg). The Bondesil resin was washed with water (10 ml) and then the product was eluted with methanol (10 ml). Evaporation of the methanol gave Boc protected compound 200 as a pale yellow solid (20 mg).

[0286] To Boc protected compound 200 (20 mg) in 60% trifluoroacetic acid in dichloromethane (0.5 ml) was added anisole (10 μL). The mixture was stirred at room temperature for 6 hours before being evaporated to dryness. Preparative HPLC purification of the residue was done on an IBSIL-C8 5μ 250×20.2 mm column and eluted at 20 ml/min with 38% acetonitrile in 5 mM ammonium phosphate buffer. The desired fractions were collected at 15 minutes and freeze-dried. The freeze-dried residue was dissolved in water (2 ml) and applied to a plug of Bondesil 40μ C8 resin (500 mg). The Bondesil resin was washed with water (10 ml) and the product was eluted with methanol (10 ml). Evaporation of the methanol gave compound 200 as a pale yellow solid (4 mg).

EXAMPLE 9

Preparation of Compounds 181, 86, 101-102, 206-207, 213-220, 246-251, 264 and 269

[0287] Daptomycin (250 mg) and N-tBoc-L-tryptophan-p-nitrophenyl ester (144 mg) were stirred in dry dimethylformamide (3 ml) at room temperature for two days. The mixture was loaded on an IBSIL-C8 5μ 250×20.2 mm column and was eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave N-Boc tryptophan daptomycin as a pale yellow solid (130 mg).

[0288] A preparation of deacylase enzyme was produced from recombinant Streptomyces lividans, which expresses the Actinoplanes utahensis deacylase enzyme. The enzyme in ethylene glycol (400 pμl) was added to the solution of N-Boc tryptophan daptomycin (100 mg) in HPLC grade water (20 ml). The solution was adjusted to pH 8.5 with sodium hydroxide (1 M). The mixture was stirred for 24 hours. The mixture was loaded on a C8 resin plug column, washed with water and eluted with methanol. Evaporation of the methanol gave a residue which was applied to an IBSIL-C8 5μ 250×20.2 mm column and was eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave deacylated N-Boc tryptophan daptomycin as a pale yellow solid (42 mg).

[0289] Deacylated N-Boc tryptophan daptomycin (20 mg) was stirred in dry dimethylformamide (2 ml) at room temperature. Undecyl isocyanate (2.25 mg) was added to the solution. After stirring at ambient temperature for 24 hours, the mixture was diluted with water (10 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave the undecyl urea of N-Boc tryptophan daptomycin as a pale yellow solid (21 mg).

[0290] N-Boc tryptophan daptomycin undecyl urea (21 mg) was stirred in trifluoroacetic acid (2 ml) and anisole (0.1 ml) at room temperature for 2 hours. Removal of the solvents under reduced pressure gave a residue which was loaded on an IBSIL-C8 5μ 250×20.2 mm column and eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave compound 181 as a pale yellow solid (0.8 mg).

[0291] In an analogous manner, compounds 86, 101-102, 206-207, 213-220, 246-251, 264 and 269 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those having ordinary skill in the art following the disclosure of the invention.

EXAMPLE 9a

Preparation of Compound 205

[0292] Deacylated N-Boc tryptophan daptomycin (50 mg) and nonaldehyde (4.1 mg) were stirred in dry dimethylformamide (2 ml) at room temperature. Sodium triacetoxy borohydride (3.6 mg) was added to the solution. The mixture was stirred for 24 hours, then loaded on an IBSIL-C8 5μ 250×20.2 mm column and eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave nonyl amino N-Boc tryptophan daptomycin as a pale yellow solid (14 mg).

[0293] Nonyl amino N-Boc tryptophan daptomycin (14 mg) was stirred in trifluoroacetic acid (2 ml) and anisole (0.1 ml) at room temperature for 2 hours. Removal of the solvents under reduced pressure gave a residue which was loaded on an IBSIL-C8 5μ 250×20.2 mm column and was eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol Evaporation of the methanol gave compound 7 as a pale yellow solid (5 mg).

EXAMPLE 10

Preparation of Compounds 356, 315-322, 332-337, 345-349 and 355

[0294] Daptomycin (5.0 g) was dissolved in water (25 ml) and adjusted to pH 9 with 5M sodium hydroxide. Di-tert-butyldicarbonate (1.5 g) was added and the mixture was adjusted to maintain pH 9 with 5 M sodium hydroxide until the reaction was complete (4 hours). The pH was adjusted to 7 and the mixture was loaded onto a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted from the column with methanol. Evaporation of the methanol gave Boc-protected daptomycin (5.08 g) as a yellow powder.

[0295] A preparation of deacylase enzyme was produced from recombinant Streptomyces lividans, which expresses the Actinoplanes utahensis deacylase enzyme. The enzyme in ethylene glycol (400 μl) was added to Boc-protected daptomycin (1 g) in water (100 ml) at pH 7-8. After incubation for 72 hours, the mixture was loaded on a Bondesil 40μ C8 resin column. The column was washed with water and the product was eluted from the column with 10% acetonitrile in water. The solvent was removed by evaporation to give deacylated Boc-protected daptomycin (440 mg) as a yellow powder.

[0296] Daptomycin undecyl urea synthesized from deacylated Boc protected daptomycin above using undecyl isocyanate instead of undecanoyl pentafluorophenol ester according to example 7 (100 mg) and 5-methoxyindole-3-carboxaldehyde (11 mg) in dry dimethylformamide (0.6 ml) was added sodium triacetoxyborohydride (76 mg). The mixture was stirred at room temperature for 24 hours before purification by preparative HPLC. The mixture was loaded on an IBSIL-C8 5μ 250×20.2 mm column and eluted at 25 ml/min with 30-60% acetonitrile in 5 mM ammonium phosphate gradient over 30 minutes. The desired fractions were collected at 21 minutes and freeze-dried. The freeze-dried residue was dissolved in water (2 ml) and applied to a plug of Bondesil 40μ C8 resin (500 mg). The Bondesil resin was washed with water (10 ml) and then the product was eluted with methanol (10 ml). Evaporation of the methanol gave compound 114 as a pale yellow solid (10 mg).

[0297] In an analogous manner, compounds 315-322, 332-337, 345-349 and 355 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those skilled in the art.

EXAMPLE 10a

Preparation of Compounds 307, 310, 295-306, 308-309, 311-312, 338-344 and 350-352

[0298] Daptomycin undecanoyl amide synthesized from deacylated Boc protected daptomycin by using undecanoyl pentafluorophenol ester according to examples 10 and 7 (60 mg) was stirred in dry dimethylformamide (2 ml) at room temperature. N-tBoc-L-tryptophan-p-nitrophenyl ester (31 mg) was added to the solution. The mixture was stirred for 24 hours. The mixture was loaded onto an IBSIL-C8 5μ 250×20.2 mm column and was eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave compound 307 as a pale yellow solid (25 mg).

[0299] Compound 307 (20 mg) was stirred in trifluoroacetic acid (2 ml) and anisole (0.1 ml) at room temperature for 2 hours. Removal of the solvents under reduced pressure gave a residue which was loaded on an IBSIL-C8 5μ 250×20.2 mm column and was eluted at 20 ml/min with 37% acetonitrile in 5 mM ammonium phosphate buffer. Fractions containing the desired compound were collected and freeze-dried. The freeze-dried residue was dissolved in water (5 ml) and applied to a Bondesil 40μ C8 resin column, washed with water and eluted with methanol. Evaporation of the methanol gave compound 310 as a pale yellow solid (4 mg).

[0300] In an analogous manner, compounds 295-306, 308-309, 311-312, 338-344 and 350-352 can be prepared as detailed in the above example by appropriate substitutions of reagents obvious to those skilled in the art.

EXAMPLE 11

[0301] Compounds according to Formula I were tested for antimicrobial activity against a panel of organisms according to standard procedures described by the National Committee for Clinical Laboratory Standards (NCCLS document M7-A5, Vol. 20, No. 2, 2000) except that all testing was performed at 37° C. Compounds were dissolved in 100% dimethyl sulfoxide and were diluted to the final reaction concentration (0.1 μg/mL-100 μg/mL) in microbial growth media. In all cases the final concentration of dimethyl sulfoxide incubated with cells is less than or equal to 1%. For minimum inhibitory concentration (MIC) calculations, 2-fold dilutions of compounds were added to wells of a microtiter plate containing 5×104 bacteria cells in a final volume of 100 μL of media (Mueller-Hinton Broth supplemented with 50 mg/L Ca2+). The optical densities (OD) of the bacterial cells, which measures bacterial cell growth and proliferation, were measured using a commercial plate reader. The MIC value is defined as the lowest compound concentration inhibiting growth of the test organism. The MIC (in μg/ml) values of representative compounds of the present invention are listed in Table III.

EXAMPLE 12

[0302] The mouse protection test is an industry standard for measuring the efficacy of a test compound in vivo [for examples of this model see J. J Clement, et al., Antimicrobial Agents and Chemotherapy, 38 (5), 1071-1078, (1994)]. As exemplified below, this test is used to demonstrate the in vivo efficacy of the compounds of the present invention against bacteria.

[0303] The in vivo antibacterial activity was established by infecting female CD-1 mice (Charles River Lab, Mass.) weighing 19-23 g intraperitoneally with from Methicillin Resistant S. aureus (MRSA) inoculum. The inoculum was prepared from Methicillin Resistant S. aureus (ATCC 43300). The MRSA inoculum was cultured in Mueller-Hinton (MH) broth at 37° C. for 18 hours. The optical density at 600 nm (OD600) was determined for a 1:10 dilution of the overnight culture. Bacteria (8×108 cfu) was added to 20 ml of phosphate buffered saline (Sigma P-0261) containing 5% hog gastric mucin (Sigma M-2378). All animals were injected with 0.5 ml of the inoculum, equivalent to 2×107 cfu/mouse, which is the dose causing ˜100% death of the animals without treatment.

[0304] The test compound was dissolved in 10.0 ml of 50 mM phosphate buffer to give a solution of 1 mg/ml (pH=7.0). This solution was serially diluted with vehicle by 4-fold (1.5 ml to 6.0 ml) to give 0.25, 0.063 and 0.016 mg/ml solutions. All the solutions were filtered with 0.2 m Nalgene syringe filter. Immediately after the bacterial inoculation, group 1 animals were subcutaneously (sc) injected with buffer (no test compound) and groups 2 to 5 were given test compound sc at 10.0, 2.5, 0.63, and 0.16 mg/kg, respectively. Group 6 animals received test compound sc at 10 mg/kg (or the highest therapeutic dose of a given compound) only for monitoring acute toxicity. These injections were repeated once at 4 hours after the inoculation for the respective groups. The injection volume at each time was 10 ml per kilogram of body weight. The results of the in vivo efficacy test are summarized in Table II, which provides a representative example of the results obtained for Compound 70. The 50% effective dose (ED50) is calculated on the basis of the number of mice surviving 7 days after inoculation. The ED50 was determined for other compounds of this invention in a similar manner. The ED50 in mg/kg of other representative compounds of the present invention are listed in Table III.

3TABLE II#Survival (7Groupof miceInoculated withTreatmentdays)15MRSA #43300Phosphate buffer0/52 × 107 cfu/mouse 10 ml/kg, s.c. × 225MRSA #43300Compound 705/52 × 107 cfu/mouse 10 mg/kg, s.c. × 235MRSA #43300Compound 703/52 × 107 cfu/mouse 2.5 mg/kg, s.c. × 245MRSA #43300Compound 701/52 × 107 cfu/mouse0.63 mg/kg, s.c. × 255MRSA #43300Compound 700/52 × 107 cfu/mouse0.16 mg/kg, s.c. × 265NoCompound 705/5 10 mg/kg, s.c. × 2The ED50 of compound 70 is calculated to be 1.51 mg/kg

[0305]

4

TABLE III

MIC
MIC
ED50

(μg/ml)
(μg/ml)
mg/kg

Compound #

S. aureus

E. faecalis

S. aureus

1
++
+
++

2
+++
+
+++

3
++
+

4
+
+

5
++
++

6
++
++

7
++
++

8
++
++

9
+++
++

10
+++
+
++

11
++
+

12
+++
++

13
+++
++

14
++
++

15
++
++

16
+++
++

17
++
++

18
++
+

19
++
++

20
+++
++

21
++
+

22
++
++

23
+++
++

24
+++
++
++

25
+++
++

26
+++
++

27
++
+

28
++
+

29
+

30
++
+

31
++
+

32
++
+

33
++
+

34
++
+

35
++
+

36
++
+

37
++
+

38
+++
+

39
+
+

40
++
+

41
+
+

42
++
+

43
++
+

44
++
++

45
+++
++
+++

46
++
++

47
++
++

48
+++
++

49
++
++

50
++
+

51
++
++

52
+++
+

53
++
+

54
++
++
++

55
+++
+

56
+++
++

57
++
+

58
+++
+

60
++
+

61
++
+

62
++
+

63
++
+

64
++
+

65
++
+

66
++
+

67
++
+

68
++
+

69
++
+

70
+++
+
++

71
++
+

72
++
+

73
++
+

74
++

75
++
+

76
+++
++
++

77
++
++

78
+
+

79
+++
++

80
+++
++

81
+++
++
+++

82
+++
++

83
+++
++

84
+++
++

85
+++
++
+++

86
+
+

87
+++
++

88
++
+

89
+++
++

90
++
++

91
++
+

92
++
+

93
++
++

94
+++
++

95
+++
++

96
+++
++

97
+++
++

98
+++
++

99
+++
++

100
+++
++

101
++
++

102
+++
+++

103
+++
+

104
++
++

105
+++
++

106
+++
++

107
++
++

108
++
++

109
++
++

110
++
++

111
+++
++

112
++
+

113
++
++

114
++
+

115
+++
+

116
+++
++

117
++
++

118
++
++

119
+++
++

120
++
++

121
+++
++

122
+++
+

123
++
+

124
++
+

125
++
++

126
++
++

127
+++
++

128
++
++

129
+++
+

130
+++
++

131
+++
+

132
++
++

133
+++
++

134
++
+

135
+++
+

136
+++
++

137
++
+

138
+++
+

139
+++
++

140
+++
++

141
++
+

142
+++
+

143
++
+

144
+++
++

145
++
++

146
+++
+

147
+++
++

148
++
++

149
++
+

150
+++
++

151
+++
++

152
++
++

153
++
+

154
++
++

155
++
++

156
+++
+

157
++
+

158
++
+

159
+++
+

160
++
+

161
+++
+

162
++
++

163
+++
++

164
+++
++

165
++
++

166
+++
++

167
+++
++

168
+++
++

169
+++
+

170
++
++

171
++
++

172
+++
++

173
+++
++

174
+++
++

175
++
++

176
+++
++

177
+
+

178
++
+

179
++
+

180
++
++

181
+++
+++
+++

182
++
+

183
+++
+

184
+++
+

185
++
+

186
++
+

187
+++
+

189

190

192
++
+

193
++
+

194
++
+

195
++
+

196
+++
+

197
++
+

198
++
+

199
+++
+

200
+

201
++
++

202

203
++
+

204
+++
++

205
++
+

206

207

208
++
++

209
+++
++

210
+++
++

211

++

212
+++
++
+++

213

214

215

216
++
+

217

218
+

219
+++
++

220
+++
+++

221
+
+

222
++
++

223
+++
++

224
++
+

225
++
+

226
++
+

227
+++
++

228
+++
++

229
+++
++

230
+++
+++

231
+++
++

232
+++
++

233
++
+

234
++
+

235
+++
++

236
++
+

237
+++
++

238
+++
++

239
+++
+

240
+++
++

241
++
++

242
++
+

243
++
+

244
+++
++

245

246
+

247
+

248
+

249
+

250
+++
+

251
++
+

252
++
++

253
+++
++

254
++
+

255
+++
++

256
+++
+++

257
++
+

258
+++
++

259
+++
+++

260
+++
++

261
++
++

262
++
++

263
+++
++

264
++
+

265
++
++

266
+++
+

267
++
+

268
++
++

269
+

270
+++
+

271
+++
+++

272
++
+

273
+++
++

274
+++
+++

275
+++
++

276
+++
+++

277
+++
+++

278
+++
+++

279
+++
++

280
+++
++

281
+++
++

282
+++
+++
+++

283
+++
++

284
+++
+++

285
+++
+++
+++

286
+++
+++

287
+++
+++

288
+++
+++

289
+++
++

290
++
++

291
+++
+++

292
+++
++

293
+++
++

294
++
+

295

++

296

297

++

298

299

++

300

++

301
+++
++

302
+++
++

303
+++
++

304

305
+++
++

306
+++
++

307
+++
++

308
+++
++

309
+++
++

310
+++
++

311
+++

312

313
+++

314
+++
++

315
++
+

316
+++

317

318
+++
+++

319
+++
++

320
+++
++

321
+++
++

322
+++
++

323
+++
++

324

++

325
+++
++

326
+++
++

327
+++
++

328

++

329

330
+++
++

331

++

332
+++
++

333

++

334
+++
+++

335
+++
++

336
+++
++

337
+++
+++

338
++
++

339
+++
++

340
+
+

341
++
++

342
+++
++

343
+++
++

344
+++
++

345
++
+++

346
+++
+++

347
++
+

348
++
+

349
++
+

350
++
+

351
++
++

352
++
++

355
++
++

356
+++
+++

358
++
++

359
+++

360
+++
++

361
+++

362
+++
++

363
+++

364
+++
++

365
+++

366
+++
++

367
+++
++

368
+++
++

369
+++

370
+++
++

371
+++
++

372
+++
++

373
+++
++

374
+++
++

375
+++

376
+++
++

377
+++
++

378
+++
++

379
+++
++

380
+++
++

381
+++
++

382
+++
++

383
+++
++

384
+++
++

385
+++
++

386
+++
++

387
+++
+++

388
++
++

389
+++
++

390
+++
++

391
+++
++

392
+++
++

393
+++
++

394
+++
++

395
+++
++

Wherein “+++” indicates that the compound has an MIC (μg/ml) of 1 μg/ml or less or an ED50 of 1 mg/kg or less;

“++” indicates that the compound has an MIC (μg/ml) or ED50 of greater than 1 μg/ml or 1 mg/kg, respectively but less than or equal to 10 μg/ml or ED50 of 10 mg/kg, respectively; and

“+” indicates that the compound has an MIC (μg/ml) of greater than 10 μg/ml or an ED50 of greater than 10 mg/kg.

[0306] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

	Number	Date	Country
	60170946	Dec 1999	US
	60208222	May 2000	US

Novel lipopeptides as antibacterial agents

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

Provisional Applications (2)