WO2007127135A2

WO2007127135A2 - Antibiotic compounds

Info

Publication number: WO2007127135A2
Application number: PCT/US2007/009640
Authority: WO
Inventors: Peter T. Meinke; Kun Liu; Libo Xu; Sheryl D. Debenham; Amy Hasler; Guo Q. Shi; Yong Zhang; Fengqi Zhang
Original assignee: Merck & Co., Inc.
Priority date: 2006-04-24
Filing date: 2007-04-20
Publication date: 2007-11-08
Also published as: WO2007127135A3

Abstract

The present invention relates to novel carboxylic acid derivatives of thiazolyl peptide antibiotics capable of treating serious bacterial infections in mammals, and particularly, in humans. These carboxylic acid analogs can also be versatile intermediates for the preparation of new derivatives with useful antibacterial activity.

Description

TITLE OF THE INVENTION ANTIBIOTIC COMPOUNDS

This application claims the benefit of US Provisional application 60/794,546, filed April 24, 2006. BACKGROUND OF THE INVENTION

Infections caused by bacteria are a growing medical concern as many of these bacteria are resistant to various antibiotics. Such microbes include Staphylococcus aureus, Staphylococcus hemolyticus, Pediococcus spp., and Streptococcus pyogenes, Streptococcus pneumoniae, P seudomonas aeruginosa, Vibrio cholerae, Vibrio parahemolyticus, Actinobacter calcoaeticus, Stenotrophomonas maltophilia.

Many thiazolyl peptide antibiotics exhibit potent antibacterial activity against a variety of Gram-pόsitive bacteria, including multiple drug-resistant strains. Their poor water solubility severely limits their usage as therapeutic agents. The present invention relates to novel carboxylic acid derivatives of thiazolyl peptide antibiotics capable of treating serious bacterial infections in mammals, and particularly, in humans. These carboxylic acid analogs can also be versatile intermediates for the preparation of new derivatives with useful antibacterial activity. Many of the novel thiazolyl peptide antibiotics of the present invention show much improved aqueous solubility over previously disclosed antibiotics (see WO2004/004646, WO2002/14354, WO2002/13834, WO2000/68413, WO200014100, WO2000/03722, WO2002/66046 and PCT US2005/33326, filed September 16, 2005). See also, P. Hrnciar et al., J. Org. Chem. 2002, 67(25), 8789-8793; B. Naidu, et al., Bioorganic & Med. Chem. Ltrs. (2004), 14(22), 5573-5577; M. Pucci, et al., Antimicrobial Agts. And Chemo., (2004), 48(10), 3697-3701; B. Naidu, et al, Tetrahedron Letters (2004), 45(17), 3531, Tetrahedron Letters (2004), 45(5), 1059-1063 ; M. D. Lee et al., J. Antibiotics Aug. 1994, Vol. 47 No. 8 pages 901-908; T. Otani et al., J. Antibiotics 1998, Vol. 51 No. 8, pages 715-721; and M. D. Lee et al., J. Antibiotics 199 »4, Vol. 47 No. 8 pages 894-900. The antibiotics of this invention thus comprise an important contribution to therapy for treating, inhibiting or preventing infections which are resistant to various known antibiotics. The carboxylic. acids of the claimed invention can be derived from thiazolyl antibiotics such as thiostrepton, GE2270A, A10255, S 54832, promothiocin, thioactin, siomycins, berninamycin, thiopeptin, nocathiacins, glycothiohexide, and nosiheptide. SUMMARY OF THE INVENTION

This invention is concerned with novel thiazolyl peptide antibiotics of the formula I:

or a pharmaceutically acceptable salt, ester, enantiomer, diasteriomer or mixture thereof.

wherein:

R independently represents hydrogen, and Ci_i2 alkyl;

Rl represents hydrogen, Cl -6 alkyl, and C3.6 cycloalkyl, OR, -(CH2)nNR2_» - (CH₂)n(O(CH2)2)l-6R9, -(CH2)_nC(O)NR₂, -(CH₂)_nC4-10 heterocyclyl, -P(O)(OH)₂, said heterocyclyl optionally substituted with 1 to 3 groups of R^; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R&;

R2 represents Rl, -OC(O)CF3₅ and ORi;

R3 represents hydrogen, -NR5R6, or -OR5, provided that when both R5 and R6 are attached to the same nitrogen they both are not hydrogen,

R4 represents hydrogen,

^or

R4a represents N(R)2;

R5 and R6 independently represent hydrogen, Cl -l 2 alkyl, -(CH2)nC4-iO heterocyclyl., - (CH2)_nNR7R8, -(CH₂)nCH(R₇)R8, -CH₂(R7)R8, -(CH₂)_nORi, -(CH₂)_nC(O)NR₇R8, - (CH2)_nC(=CH₂)CN, -(CH₂)_nC(=CH2)F, -(CH2)_nC6-10 aryl, -(CH₂)n(O(CH₂)2)l-6R9, - CH(R7)(CH2)_nNR7R8, -CH(R7)C(O)OR,-C3_6cylcoalkylC(O)OR, -CH(R7)CN, -(CHa)_nCN, - CH(R7)(CH2)nOR, -(CH₂)_nC(=CH2)CN, -(CH₂)_nC(O)NR7R8, -NHC(O)R7, NHR7, -CH2(C3- 6cylcoalkyl)CH₂OR, -C(CH3)₂C(O)OR, said aryl, and heterocyclyl optionally substituted with 1 to 3 groups of R^a; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R^a; or

R5 and R6 together with the nitrogen atom they are attached form a 4 to 10 membered heterocyclic ring optionally containing 1 to 2 additional heteroatoms selected from the group consisting of N₅ S and O and optionally substituted with 1 to 3 groups of R^a;

R7 and Rs independently represent hydrogen, hydroxyl, C\-6 alkoxy, Ci-I₂ alkyl, - (CH₂)_nNR5R6, -(CH₂)_nC4- 10 heterocyclyl, -(CH₂)_nC6-10 aryl, -(CH₂)_nNHNHC(O)C4-10 heterocyclyl, -(CH2)_nOR,

-C(O)Ci-6 alkyl, -C(O)Ci-6 alkylC(O)Rs, -C(O)C6-10 aryl, -C(O)(CH₂)_nC4-10 heterocyclyl, C(O)CHRsNR5R6, (CH₂)_nNHC(O)(CHR)_nNR5R6, -C(O)NH(CH₂)_nC4-10 heterocyclyl, - C(O)(CH₂)nNR5R6_> said aryl, and heterocyclyl optionally substituted with 1 to 3 groups of Ra; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of Ra or

R7 and R8 together with the nitrogen atom they are attached form a 4 to 10 membered heterocyclic ring optionally containing 1 to 2 additional heteroatoms selected from the group consisting of N, S and O and optionally substituted with 1 to 3 groups of Ra; or R-7 and Rg together with the carbon atom they are attached form a 3 to 10 membered carbocyclic ring optionally and optionally substituted with 1 to 3 groups of R^a;

R9 represents hydrogen, C\-β alkyl, (CH2)nC4-10 heterocyclyl, -(CHR)_nC(O)OR, - C(O)NR5R6, CN₃ OR, said alkyl and heterocyclyl optionally substituted with 1 to 3 groups of Ra

Ra represents hydrogen, halogen, -(CH₂)nOR, CF3, (CH2)_nC(O)OR, - (CH2)nC(O)(CH₂)nNR7R8,

-(CH2)_nC6-10 aiyl, -(CH₂)_nC4-10 heterocyclyl, =CH₂, S(O)₂R, C(O)R, (CH₂)_nC3-8 cycloalkyl, SO2NR5R6, (CH₂)C6-10 aiyl, N(R)₂, NO₂, CN, -O-, (Ci_₆ alkyl)O-, (aryl)O-, (Ci- <5 alkyl) S(O)0-2-_J Ci-I₂ alkyl, said alkyl, heterocyclyl, and aryl optionally substituted with 1 to 4 groups selected from the group consisting of Cl -6 alkyl, (CH₂)nOR₅ (CH₂)nN(R)₂, -O-; and

n represent 0-6, m represents 0-1, and p represents 0, 1 or 2, provided that the compound of formula I when Ri is hydrogen, R2 is OH, R3 is -NR5R6, R5 is hydrogen, R6 is (CH₂)nNR7R8,

n is 2, and R4 is

then R7 and Rg do not represent the compound where: R7 and Rg together are morpholino, R7 and Rs are both 2-hydroxyethyl or ethyl,

R7 is methyl or ethyl and R8 is 2-hydroxyethyl, or R7 is ethyl and R8 is 2-carboxymethyl.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described herein in detail using the terms defined below unless otherwise specified.

The compounds of the present invention may have asymmetric centers, chiral axes and chiral planes, and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. (See E.L. EHeI and S. H. Wilen Stereochemistry of Carbon Compounds (John Wiley and Sons, New York 1994), in particular pages 1119-1190).

When any variable (e.g. aryl, heterocycle, R₄, Ri etc.) occurs more than one time in any constituent, its definition on each occurrence is independent at every other occurrence. Also, combinations of substituents/or variables are permissible only if such combinations result in stable compounds.

When R^a is -O- and attached to a carbon it is referred to as a carbonyl group and when it is attached to a nitrogen (e.g., nitrogen atom on a pyridyl group) or sulfur atom it is referred to a N-oxide and sulfoxide group, respectively. The term "alkyl" refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 15 carbon atoms unless otherwise defined. It may be straight or branched. Preferred alkyl groups include lower alkyls which have from 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl and t-butyl. When substituted, alkyl groups may be substituted with up to 5 substituent groups, selected from the groups as herein defined, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with "branched alkyl group".

Cycloalkyl is a species of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings which are fused. Preferred cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. When substituted, cycloalkyl groups may be substituted with up to 3 substituents which are defined herein by the definition of alkyl.

The term "alkoxy" refers to those hydrocarbon groups having an oxygen bridge and being in either a straight or branched configuration and if two or more carbon atoms in length, they may include a double or a triple bond. Exemplary of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, isohexoxy allyloxy, propargyloxy, and the like.

"Halogen" or "halo" as used herein means fluoro, chloro, bromo and iodo.

The term "alkenyl" refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 10 carbon atoms and at least one carbon to carbon double bond. Preferred alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. Preferably, alkenyl is C2- C6 alkenyl.

Preferably, alkynyl is C2-C6 alkynyl. As used herein, "aryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. The term heterocyclyl, heterocycle or heterocyclic, as used herein, represents a stable 4- to 7-membered monocyclic or stable 8- to 12-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, S, SO2, and S(O), and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. The term heterocyclyl, heterocycle or heterocyclic includes heteroaryl moieties. Examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, 1,3-dioxolanyl, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidϊnyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2- oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl. quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl, thiamoφholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl. An embodiment of the examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzotbienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, 2- pyridinonyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl, thiamoφholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, thienyl and triazolyl. Preferably, heterocycle is selected from 2-azepinonyl, benzimidazolyl, 2- diazapinonyl, imidazolyl, 2-imidazolidinonyl, indolyl, isoquinolinyl, oxazolyl, morpholinyl, piperidyl, piperazinyl, pyridyl, pyrrolidinyl, 2-piperidinonyl, 2-pyrimidinonyl, 2-pyrollidinonyl, quinolinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, and thienyl. As used herein, "heteroaryl" is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic and wherein from one to four carbon atoms are replaced by heteroatoms selected from the group consisting of N, O, and S. Examples of such heterocyclic elements include, but are not limited to, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cϊnnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiazolyl, thienofuryl, thienothienyl, thienyl and triazolyl.

As used herein, unless otherwise specifically defined, substituted alkyl, substituted cycloalkyl, substituted aroyl, substituted aryl, substituted heteroaroyl, substituted heteroaryl, substituted arylsulfonyl, substituted heteroaryl-sulfonyl and substituted heterocycle include moieties containing from 1 to 3 substituents, substituents in addition to the point of attachment to the rest of the compound. Preferably, such substituents are selected from the group which includes but is not limited to F, Cl, Br, CF₃, NH₂, N(C₁-C₆ alkyl)₂, NO₂, CN, (C₁-C₆ alkyl)O-, (aryl)O-, (C₁-C₆ alkyl)S(O)_m-, (C₁-C₆ alkyl)C(O)NH-, H₂N-C(NH)-, (C₁-C₆ alkyl)C(O)-, (C₁-C₆ alkyl)OC(O)-, (C₁-C₆ alkyl)OC(O)NH-, phenyl, pyridyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thienyl, furyl, isothiazolyl and C₁-C_2Q alkyl, (CH2)nOH, CF3, (CH2)_nC(O)OH, (CH2)_nC(O)OC 1 -6 alkyl, (CH₂)_nC(O)NR7R8, (CH2)_nC5-l 0 heterocyclyl,

SO₂NR₅Ro₅ (CH₂)C6-10 aryl, N(R)₂, NO₂, CN, (Cl-6 alkyl)O-, (aryl)O-, (Ci_6 alkyl)S(O)0-2-, Cl _12 alkyl, said heterocyclyl, and aryl optionally substituted with 1 to 3 groups selected from the group consisting of (CH2)nOR, (CH₂)_nN(R)2, -Os-

When a functional group is termed "protected", this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T. W. et al. Protective Groups in Organic Synthesis Wiley, New York (1991). Examples of suitable protecting groups are contained throughout the specification. The compounds of the present invention are basic therefore salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric. gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic. methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- toluenesulfonic acid and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.

The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described by Berg et ah, "Pharmaceutical Salts," J. Pharm. ScI, 1977:66:1-19.

An embodiment of this invention is realized when Rl represents Ci-6 alkyl, preferably methyl,; and all other variables are as described herein.

An embodiment of this invention is realized when Ri represents -(CH2)nC4-10 heterocyclyl, and all other variables are as described herein. A sub-embodiment of this invention is realized when the heterocyclyl is selected from the group consisting of phenyl, pyrimidinyl, morpholinyl, piperazinyl, pridinyl, pyrazolyl, indolyl, furanyl, isoindazolyl, oxazolyl, pyrazinyl, pyrrolyl, imidazolyl, triazolyl or teterazolyl said X groups optionally substituted with 1 to 3 groups of Ra).

Another embodiment of this invention is realized when Ri represents hydrogen, and all other variables are as described herein.

Another embodiment of this invention is realized when R2 represents Ri, and all other variables are as described herein.

Another embodiment of this invention is realized when R2 represents ORi, and all other variables are as described herein. An embodiment of this invention is realized when R2 represents Cl _6 alkyl, preferably methyl,; and all other variables are as described herein.

An embodiment of this invention is realized when R2 represents -(CH2)nC4-10 heterocyclyl, and all other variables are as described herein. A sub-embodiment of this invention is realized when the heterocyclyl is selected from the group consisting of phenyl, pyrimidinyl, morpholinyl. piperazinyl, pridinyl, pyrazolyl, indolyl, furanyl, isoindazolyl, oxazolyl, pyrazinyl, pyrrolyl, imidazolyl, triazolyl or teterazolyl said X groups optionally substituted with 1 to 3 groups of R^a).

Another embodiment of this invention is realized when R3 represents -NR5R6 provided that when both R5 and Kβ are attached to a nitrogen they are not both hydrogen and all other variables are as described herein.

Another embodiment of this invention is realized when R3 represents — OR₅, and all other variables are as described herein.

Another embodiment of this invention is realized when R4 represents hydrogen, and all other variables are as described herein.

Another, embodiment of this invention is realized when R4 represents

all other variables are as described herein. A subembodiment of this invention is realized when R4_a is -N(CH3)2, -NH2, -NHCH3, -N+(CH3)2θ-.

Another embodiment of this invention is realized when R4 represents

and all other variables are as described herein. A subembodiment of this invention is realized when R₄₃ is hydrogen and R is hydrogen. Another embodiment of this invention is realized when R₄ represents

and all other variables are as described herein. A subembodiment of this invention is realized when R4_a is -N(CH3)2, -NH2, -NHCH3, -N+(CH3)2θ-.

Another embodiment of this invention is realized when R4 represents

and all other variables are as described herein. A subembodiment of this invention is realized when R₄₉ is hydrogen and R is hydrogen.

Another embodiment of this invention is realized when one of R5 and Rβ is hydrogen and the other is hydrogen, Cl -l 2 alkyl, -(CH2)nC4-lO heterocyclyl, - (CH2)n(O(CH₂)2)l-6R9, -(CH₂)_nNR7R8, -(CH₂^CH(R₇)Rg₅ -CH₂(R₇)Rs, -(CH₂)_nORi, - (CH2)_nC(O)NR₇R8, -(CH₂)_nC(=CH₂)CN, -(CH₂)_nC(=CH₂)F, -(CH₂)_nC6-10 aryl, -

(CH2)_nC(=CH2)CN, -(CH2)nC(O)NR7R8 said heterocyclyl optionally substituted with 1 to 3 groups of R^a; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R^a. A sub-embodiment of this invention is realized when one of R5 and R6 is hydrogen and the other is -(CH2)nC4-lO heterocyclyl or -(CH2)nNR₇R8- Still another sub-embodiment of this invention is realized when said heterocyclyl selected from the group consisting of pyrimidinyl, morpholinyl, piperazinyl, pridinyl, pyrazolyl, indolyl, furanyl, isoindazolyl, pyrazinyl, pyrrolyl, imidazolyl, triazolyl or teterazolyl.

Another embodiment of this invention is realized when R₇ and R8 independently are selected from hydrogen, hydroxyl, Ci_6 alkoxy, C1-12 alkyl, -(CH₂)nNR5R6₅ -(CH2)_nC4-10 heterocyclyl, -(CH2)_nC6-l 0 aryl, -C(O)(CH2)_nC4-l 0 heterocyclyl, -C(O)(CH₂)nNR5R6, said aryl, and heterocyclyl optionally substituted with 1 to 3 groups of Ra; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R^a Still another embodiment of this invention is realized when R₇ and Rs are independently selected from the group consisting of hydrogen, Ci_6 alkyl (said alkyl group optionally substituted with 1 to 6 groups of Ci -4 alkoxy or OH), -(CH₂)nN(R)2₅ -(CH2)_nX (wherein X represents phenyl, pyriniidinyl, morpholinyl, piperazinyl, pridinyl, pyrazolyl, indolyl, furanyl, isoindazolyl, oxazolyl, pyrazinyl, pyrrolyl, imidazolyl. triazolyl or teterazolyl said X groups optionally substituted with 1 to 3 groups of R^a).

Still another embodiment of this invention is realized by structural formula II:

wherein R3 is -NHCR7R8C(O)NRsR6,» provided that when R5 and R6 are both attached to a nitrogen they both are not hydrogen, and all other variables are as described herein.

Another-embodiment of this invention is realized when R3 is — NH(CH2)nX_* wherein X is selected from the group consisting of phenyl, pyrimidinyl, morpholinyl, piperazinyl, pridinyl, pyrazolyl, indolyl, furanyl, isoindazolyl, oxazolyl, pyrazinyl, pyrrolyl, imidazolyl, triazolyl and teterazolyl said X groups optionally substituted with 1 to 3 groups of Ra, provided that when X is morpholinyl, n is not 2, Ri is hydrogen or methyl and R2 is Ri or OR. As sub- embodiment of this invention is realized when X is morpholinyl. Another sub-embodiment of this invention is realized when X is oxazolyl. Preferred compounds of this invention are found in Table 1 below:

The compounds of this invention are broad spectrum antibiotics useful in the treatment of bacterial infections. They demonstrate antibacterial activity primarily against S. aureus, E. faecalis, E. faecium, S. pneumonieae, B. subtilus including species that are resistant to many known antibiotics. The minimum inhibitory concentration (MIC) values range from 0.0001 to less than 200 μg/mL for test strains such as Staphylococuus aureus, Staphylococuus hemolyticus, Streptococcus pyogenes, Streptococcus pneumoniae, and E. feacalis. The compounds of the invention can be formulated in pharmaceutical compositions by combining the compounds with a pharmaceutically acceptable carrier. Examples of such carriers are set forth below. The compounds may be employed in powder or crystalline form, in liquid solution, or in suspension. They may be administered by a variety of means; those of principal interest include: topically, orally and parenterally by injection (intravenously or intramuscularly) . Compositions for injection, one route of delivery, may be prepared in unit dosage form in ampules, or in multidose containers. The injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder (lyophillized or non-lyophillized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. In injectable compositions, the carrier is typically comprised of sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections. Also, various buffering agents, preservatives and the like can be included.

Topical applications may be formulated in carriers such as hydrophobic or hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.

Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral compositions may utilize carriers such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.

The dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, the sensitivity of the pathogen to the Compound, the virulence of the infection and other factors. Such matters, however, are left to the routine discretion of the physician according to principles of treatment well known in the antibacterial arts.

The compositions for administration to humans per unit dosage, whether liquid or solid, may contain from about 0.01% to as high as about 99% of Compound I, one embodiment of the range being from about 10-60%. The composition will generally contain from about 15 mg to about 2.5 g of Compound I, one embodiment of this range being from about 250 mg to 1000 mg. In parenteral administration, the unit dosage will typically include pure Compound I in sterile water solution or in the form of a soluble powder intended for solution, which can be adjusted to neutral pH and isotonicity. The invention described herein also includes a method of treating a bacterial infection in a mammal in need of such treatment comprising the administration of the compound of formula I to the mammal in an amount effective to treat the infection.

One embodiment of the methods of administration of a compound of formula I includes oral and parenteral methods, e.g., i.v. infusion, i.v. bolus and Lm. injection.

For adults, about 5-50 mg of a compound of formula I per kg of body weight given one to four times daily is preferred. The preferred dosage is 250 mg to 1000 mg of the antibacterial given one to four times per day. More specifically, for mild infections a dose of about 250 mg two or three times daily is recommended. For moderate infections against highly susceptible gram positive organisms a dose of about 500 mg three or four times daily is recommended. For severe, life-threatening infections against organisms at the upper limits of sensitivity to the antibiotic, a dose of about 1000-2000 mg three to four times daily may be recommended.

For children, a dose of about 5-25 mg/kg of body weight given 2, 3, or 4 times per day is preferred; a dose of 10 mg/kg is typically recommended.

Throughout the instant application, the following abbreviations are used with the following meanings:

DCM dichloromethane

DIEA diisopropylethylamine

D DMMFF N,N-dimethylformamide

DMAP 4-Dimethylaminopyridine

DMSO Dimethyl sulfoxide

DSC N,N'-disuccinimidyl carbonate

EDC 1 -(3 -dimethylaminopropyl)-3 -ethylcarbodi-imide hydrochloride

EI-MS Electron ion-mass spectroscopy

Et ethyl

EtOAc ethyl acetate

EtOH ethanol eq. equivalent(s)

F FAABB--MMSS Fast atom bombardment-mass spectroscopy

HOAc acetic acid

HOBT, HOBt Hydroxybenztriazole

HPLC High pressure liquid chromatography

Me methyl MeOH methanol

MF Molecular formula

MHz Megahertz

MPLC Medium pressure liquid chromatography

NMM N-Methylmorpholine

NMR Nuclear Magnetic Resonance

Ph phenyl

Pr propyl prep. Prepared

PyBOP (benzotriazole- 1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate

TEA Triethylamine

TFA Trifluoroacetic acid

TFAA trifloroacetic anhydride

THF Tetrahydrofuran

TLC Thin layer chromatography

TMS Trimethylsilane

The compounds of the present invention can be prepared according to Schemes 1- 2, using appropriate materials, and are further exemplified by the following non-limiting examples. Nocathiacin-I and the process for making can be found in US6,218,398, 6,287,827 and US2004/0018963 all incorporated herein by reference in their entirety. The structure of Nocathiacin I is:

In Scheme 1, the carboxylic acid intermediate can be formed first, then coupled to a variety of neucleophiles, including alcohols, and primary and secondary amines, to generate esters and amides. In Scheme 2, the product can be obtained in a one-pot fashion. The compounds illustrated in the examples are not, however, to be construed as forming the only genus that is considered as the invention. The following examples further illustrate details for the preparation of compounds of the present invention. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare the compounds of the present invention. All temperatures are in degrees Celsius unless otherwise noted. Processes for making compounds with substituents YR' are exemplified in the non- limiting examples below (See also US6,218,398, 6,287,827 and US2004/0018963 all incorporated herein by reference in their entirety). Coupling of HYR' can be performed, for example, with HOBT/EDC in DMF, PyBOP in DIEA. Alternatively, activated esters or mix anhydrides can be formed first, then coupled to HYR' to generate the desired products.

wherein YR' represents R3 and Rl, R2₅ and R4 are as described herein.

Scheme 2

Example 1

To a suspension of nocathiacin-I (see B. Naidu, et al., Bioorganic & Medicinal Chemistry Letters (2004), 14(22), 5573-5577 and US6.218,398, both incorporated herein in their entirety for how to make nocathiacin I.) (100 mg, 0.07 mmol) in tetrahydrofuran (4 mL) at 0 ⁰C were added pyridine (0.1 mL, 1.2 mmol) and trifluoroacetic anhydride (0.1 mL, 0.7 mmol) slowly. After addition, the reaction mixture was allowed to warm to room temperature and stirring was continued for 6 h. A second portion of pyridine (0.05 mL) and TFAA (0.05 mL) were added, and after 4 h the reaction was quenched by addition of a small amount of water while being kept in ice-bath and stirred for 30 minutes at room temperature. It was then concentrated until solid started to precipitate out. More water was added and the solid was collected by filtration. After being washed with water, the solid was dissolved in methanol/chloroform, evaporated repeatedly until dry. The crude acid product was obtained as an orange crystalline solid (94 mg, 80% pure). LC-MS analysis indicated that this material consisted of approximately 1 :1 acid and trifluoroacetylated acid, which can be used in the subsequent coupling reactions without further purification. An analytical sample was obtained by adding a drop of saturated sodium bicarbonate solution to 10 mg the mixed acid in DMSO, followed by reversed-phase HPLC purification. ¹H NMR (500 MHz₃ DMSO-cft) 11.94 (s. IH). 10.77 (s, IH), 9.10 (s, IH), 8.70 (s, IH), 8.66 (s, IH), 8.57 (m, 2H), 8.54 (s, IH), 8.22 (s, IH), 8.03 (s, IH) (d, IH, J = 9.5 Hz), 7.90 (s, IH), 7.86 (d, IH, J = 11.5 Hz), 7.75 (d, IH, J = 8.5 Hz), 7.38 (m, 2H), 7.20 (d, IH, J = 7 Hz), 6.38 (d, IH, J = 12.0 Hz), 5.76 (dd, IH, J = 11.0 and 4.5 Hz), 5.72 (d, IH, J = 9.5 Hz), 5.24 (dd, IH, J = 11.0 and 4.5 Hz), 5.06 (m, 2H)₅ 5.00 (d, IH, J = 8.0 Hz), 4.80 (d, IH, J = 10.5 Hz), 4.54 (d, IH, J = 11.0 Hz), 4.30 (d, IH, J = 9.5 Hz), 4.26 (m, IH), 4.16 (d, IH, J = 10.5 Hz), 4.05 (d, IH, J = 8.0 Hz), 3.92 (s, 3H), 3.13 (s, br, IH), 2.89 (s, 3H), 2.87 (s, 3H), 2.13 (m, IH), 2.01 (s, 3H), 1.95 (d, IH, J = 14.5 Hz)₅ 1.61 (s, 3H), 1.57 (s, br, 3H), 0.81 (d, 3H, J = 6.5 Hz). MS: 1369.71 (M+H)⁺, 685.42 ([M+2H]/2)⁺.

Example 2

To a solution of Nocathiacin-I (6 g, 4.18 mmol) in benzene (150 mL) and methanol (24 mL) at 0 ⁰C was added a solution of trimethylsilyl diazomethane in hexanes (2.0 M, 6.26 mL, 12.52 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 4 h. Volatiles were evaporated, and the solid residue was dried under vacuum overnight. Methyl- nocathiacin-I was obtained as a yellow powder (5.86 g, 80% pure by LC-MS), and was used in the next step without further purification.

To a solution of methyl-nocathiacin-I (5.86 g, 80% pure) in tetrahydrofuran (360 mL) at O⁰C, was added pyridine (6.53 mL, 81 mmol) and trifluoroacetic anhydride (5.7 mL, 40 mmol) slowly. After addition, the reaction mixture was allowed to warm to room temperature and stirring was continued for 8 h. The reaction was quenched by adding small amount of water to it while being kept in ice-bath and stirred for 30 minutes at room temperature. It was then concentrated until solid started to precipitate out. More water was added and the solid was collected by filtration. After being washed with water, the solid was dissolved in dimethylsulfoxide and purified by reversed-phase HPLC (10-60% acetonitrile with 0.1% TFA). Product was obtained as a mixture of acid and trifluoroacetylated acid after lyophilization (2.56 g, 44% yield). For most of the coupling reactions, this intermediate gave satisfactory results because the trifluoroacetyl group can be easily removed with either sodium bicarbonate solution or N,N-diisopropylethylamine. An analytical sample was obtained similarly as described for example 1. ¹H NMR (500 MHz, CD₃OD) 8.94 (s₅ IH), 8.62 (d, IH₅ J = 9.5 Hz), 8.60 (s, IH), 8.53 (s, IH), 8.43 (s, IH), 8.17 (s, IH), 8.15 (d, IH, J = 11.0 Hz)₃ 8.00 (s, IH), 7.95 (s, IH), 7.91 (d, IH₃ J = 7.0 Hz)₅ 7.84 (d₃ IH₅ J = 8.5 Hz), 7.43 (t, IH, J = 7.8 Hz)₃ 7.23 (d₅ IH₅ J = 7.0 Hz), 6.14 (d, IH, J = 12.5 Hz)₅ 5.91 (dd₅ IH, J = 9.3 and 1.8 Hz)₅ 5.78 (dd, IH, J = 10.5 and 5.0 Hz), 5.38 (dd, IH, J = 11.5 and 5.0 Hz), 5.21 (m, IH)₅ 5.06 (d, IH, J = 12.5 Hz), 4.96 (d, IH, J = 10.5 Hz), 4.60 (d, IH₃ J = 11.0 Hz), 4.41 (d, IH, J = 9.5 Hz)₅ 4.36 (dd, IH, J = 10.5 Hz)₅ 4.23 (s, 3H)₃ 4.15 (m, IH)₅ 4.06 (dd, IH₅ J = 9.8 and 1.8 Hz)₅ 3.95 (s, 3H), 3.21 (m, IH)₅ 3.02 (s, 6H), 2.84 (m, IH)₅ 2.18 (m, 2H)₅ 2.05 (s, 3H)₅ 1.75 (S, 3H), 1.41 (d, 3H₅ J = 6.0 Hz), 1.00 (d₅ 3H₅ J = 7.0 Hz). MS: 1383.87 (M+H)⁺, 692.41 ([M+2H]/2)⁺.

The product of example 2 (1.04 g, 0.65 mmol) was mixed with iV-(3-aminopropyl)morpholine (0.11 mL, 0.78 mmol), 1-hydroxybenzotriazole (0.088 g, 0.65 mmol) and EDC (0.18 g, 0.97 mmol) in anhydrous DMF (8 mL). After Ih, 2 mL of saturated sodium bicarbonate solution was added and the mixture was stirred at room temperature for 30 minutes to remove the trifluoroacetyl group. The reaction mixture was directly purified by reversed-phase HPLC (10- 45% acetonitrile with 1% TFA). The desired compound was obtained as a light yellow solid after lyophilization (bis TFA salt, 0.64 g, 57% yield).

To convert to the HCl salt, the above obtained bis-TFA salt (0.64 g, 0.37 mmol) was dissolved in deionized water (130 mL) and passed through a column of AG1-X2 resin (Cl form, 40 g). Eluant and washings were combined and lyophilized to give the HCl salt of the desired compound as a light yellow fluffy solid (0.57 g, 98% yield). ¹H NMR (500 MHz, CD₃OD) 8.61 (d, IH, J = 9.5 Hz)₅ 8.60 (s, IH), 8.45 (s, IH), 8.42 (s, IH), 8.18 (s, IH), 8.13 (d, IH, J = 11 Hz), 8.10 (s, IH), 7.88 (s, IH), 7.83 (d, IH, J = 6.5 Hz), 7.82 (d, IH, J = 8.5 Hz), 7.43 (t, IH, J = 7 Hz)₃ 7.23 (d, IH, J = 7 Hz), 6.15 (d, IH₃ J = 12.5 Hz)₃ 5.91 (dd, IH, J = 9.4 and 1.5 Hz), 5.77 (dd, IH, J = 11.5 and 4.5 Hz), 5.42 (dd, IH, J - 11.5 and 4.5 Hz)₃ 5.21 (m, IH), 5.07 (d, IH₅ J = 13.0 Hz), 5.00 (d, IH, J = 10.5 Hz), 4.61 (d, IH, J = 11.0 Hz), 4.44 (d, IH₃ J = 10.0 Hz)₃ 4.38 (m, IH)₃4.32 (s, 3H), 4.29 (s, IH), 4.15 (m, IH), 4.07 (dd, IH, J = 9.5 Hz), 3.96 (s, 3H), 3.87 (m, 2H)₃ 3.54 (m, 2H), 3.19 (m, 4H), 3.02 (s, 6H), 2.78 (m, IH), 2.17 (m, 3H), 2.07 (s, 3H), 2.04 (m, IH), 1.75 (s, 3H)₃ 1.41 (d, 3H, J = 6.0 Hz), 1.00 (d, 3H, J = 7.0 Hz). MS: 1509.44 (M+H)⁺, 755.67 ([M+2H]/2)⁺.

The product of example 2 (30 mg, 0.020 mmol) was mixed with iV-(2-hydroxylethyl)piperazine (3.2 mg, 0.024 mmol), 1-hydroxybenzotriazole (3.1 mg, 0.020 mmol) and EDC (5.8 mg, 0.03 mmol) in anhydrous DMF (0.4 mL). After Ih, the reaction mixture was deacylated with saturated sodium bicarbonate and purified by reversed-phase HPLC with a step gradient of 10- 30% acetonitrile/water containing 0.1% TFA over 20 minutes. The product was obtained as a light yellow solid after lyophilization (bis TFA salt, 10.3 mg, 30% yield). ¹H NMR (500 MHz, CD₃OD) 8.62 (d, IH, J = 9.0 Hz), 8.61 (s, IH)₃ 8.43 (s, IH)₅ 8.34 (s, br, IH)₅ 8.18 (s, IH), 8.16 (d, IH, J = 11.0 Hz), 8.02 (s, IH), 7.91 (d, IH, J = 7.0 Hz)₃ 7.88 (s₃ IH), 7.84 (d, IH, J = 8.5 Hz), 7.44 (t₃ IH, J = 7.8 Hz), 7.24 (d, IH₃ J = 6.5 Hz), 6.15 (d, IH, J = 12.5 Hz), 5.91 (dd, IH₃ J = 9.5 and 2.3 hz), 5.77 (dd, IH, J = 10.3 and 5.0 Hz), 5.38 (dd, IH, J = 11.3 and 5.0 Hz)₃ 5.21 (m, IH), 5.07 (d, IH, J = 12.5 Hz)₃ 4.97 (d, IH, J = 11.0 Hz), 4.60 (d₃ IH, J = 11.0 Hz), 4.41 (d, IH, J = 9.5 Hz), 4.37 (m, IH)₃ 4.32 (d, IH, J = 11.0 Hz)₃ 4.25 (s, 3H), 4.15 (m, IH), 4.07 (dd, IH, J = 9.8 Hz)₃ 3.96 (S₃ 3H)₃ 3.87 (m, 2H)₃ 3.20 (m, 2H), 3.02 (s, 6H)₃ 2.84 (m, IH), 2.17 (m, 2H), 2.05 (s, 3H)₃ 1.75 (s, 3H), 1.42 (d, 3H, J = 6.0 Hz)₃ 1.00 (d, 3H₃ J = 7.5 Hz). MS: 1495.8 (M+H)⁺, 748.8 ([M+2H]/2)⁺.

The product of example 1 (630 mg, 0.43 mmol) was mixed with N-(3-aminopropyl)morpholine (75 mg, 0,52 mmol), 1-hydroxybenzotriazole (80 mg, 0.52 mmol) and EDC (125 mg, 0.65 mmol) in anhydrous DMF (3 mL). After stirring at room temperature for 1 h, the reaction mixture was deacylated with saturated sodium bicarbonate, and purified in multiple runs by reversed-phase HPLC with a step gradient of 5-45% acetonitrile/water modified with 0.1% TFA over 20 minutes. The product was obtained as a light yellow solid after lyophilization (bis TFA salt, 270 mg, 41% yield). ¹H NMR (500 MHz, CD₃OD-rf4) 8.62 (d, IH, J = 9.5 Hz)₅ 8.56 (s, IH), 8.43 (s, IH), 8.41 (s, IH), 8.19 (s, IH), 8.14 (d, IH₅ J = 11.0 Hz), 7.88 (s, IH), 7.86 (d, IH, J = 7.0 Hz), 7.84 (m, 2H), 7.43 (t, IH, J = 8.0 Hz), 7.22 (d, IH, J = 7.0 Hz), 6.13 (d, IH, J - 13.0 Hz)₅ 5.91 (dd, IH₅ J = 9.5 and 2.0 Hz)₅ 5.78 (dd₅ IH, J = 11.0 and 5.0 Hz), 5.39 (dd, IH₅ J = 11.3 and 5.3 Hz), 5.21 (m, IH), 5.06 (d, IH, J = 12.5 Hz), 4.96 (d, IH, J = 10.5 Hz), 4.59 (d, IH, J = 11.5 Hz), 4.42 (d, IH₅ J = 10.0 Hz)₅ 4.37 (m, IH)₅ 4.29 (d, IH₅ J = 10.5 Hz)₅ 4.05-4.18 (m, 4H), 3.96 (s, 3H), 3.80 (m, 2H), 3.53-3.60 (m, 4H), 3.29 (t, 2H, J = 7.8 Hz), 3.12-3.22 (m, 3H), 3.03 (S₅ 6H)₅ 2.80 (m, IH)₅ 2.18 (s, 3H)₅ 2.07 (s₅ 3H), 1.75 (s, 3H)₅ 1.42 (d, 3H₅ J = 6.0 Hz)₅ 1.00 (d, 3H, J = 7.0 Hz). MS: 1495.27 (M+H)⁺, 748.61 ([M+2H]/2)⁺.

"One-pot" procedure: To a suspension of nocathiacin-I (75 mg₅ 0.052 mmol) in THF(I mL) was added anhydrous pyridine (84 DL, 1.04 mmol) to give a clear solution. Trifuoroacetic anhydride (72 DL, 0.72 mmol) was added. The reaction solution was stirred at room temperature for 1 hour, then transferred to a solution of 3-morpholinopropyl-amine (225 DL) THF (0.5 mL). After 10 minutes, the volatiles were removed in vacuo. The residue was purified by preparative reversed phase HPLC to generate the desired compound (37 mg, 48 % yield).

Example 6

To a solution of the acid of example 1 (50 mg, 0.03 mmol) in anhydrous DMF were added N- (N,N-dimethylglycyl)-ethylenediamine (TFA salt, 12 mg, 0.045 mmol) (prepared from ethylenediamine and N,N-dimethylglycine) and PYBOP (19 mg, 0.036 mmol), followed by N₅N- diisopropylethylamine (6.3 DL₃ 0.036 mmol). After stirring at room temperature for 1 h, the reaction mixture was treated with saturated sodium bicarbonate, and purified by reversed-phase HPLC with a step gradient of 10-90% methanol/water modified with 0.1% TFA over 20 minutes. The product was obtained as a light yellow solid after lyophilization (bis TFA salt, 22 mg, 43% yield). ¹H NMR (600 MHz, CD₃OD) 8.86 (s, IH), 8.60 (d, IH, J = 9.6 Hz), 8.55 (s, IH), 8.40 9s_s IH), 8.36 (s, IH), 8.16 (s, IH), 8.12 (d, IH, J = 10.8 Hz), 7.88 (s, IH), 7.84 (d, IH, J - 6.6 Hz)₅ 7.81 (d, IH, J = 9.0 Hz), 7.80 (s, IH), 7.41 (t, IH, J = 7.8 Hz), 7.21 (d, IH, J = 6.6 Hz), 6.12 (d, IH, J = 12.6 Hz), 5.88 (d, IH, J = 9.6 Hz), 5.75 (dd, IH, J = 11.1 and 5.1 Hz)₅ 5.37 (dd, IH, J = 11.7 and 5.1 Hz), 5.19 (m, IH), 5.04 (d, IH, J = 12.6 Hz), 4.95 (d, IH, J - 10.2 Hz), 4.57 (d, IH, J = 11.4 Hz), 4.40 9d, IH, J = 9.6 Hz), 4.35 (m, IH), 4.28 (d, IH, J = 10.2 Hz), 4.12 (m, IH), 4.05 (dd, IH, J = 9.6 and 1.8 Hz), 3.93 (s, 3H), 3.89 (s, 3H), 3.60 (m, 2H), 3.18 (m, IH), 3.00 (s, 6H), 2.90 (s, 6H)₅ 2.77 (m₅ IH), 2.15 (s, 2H), 2.04 (s, 3H)₅ 1.73 (s, 3H), 1.39 (d, 3H, J = 6.0 Hz), 0.98 (d, 3H, J = 7.2 Hz). MS: 1497.66 (M+H)⁺, 749.21 ([M+2H]/2)⁺.

Example 7

Following the procedure described for example 3 except using dimethylamine as the amine component to afford the product as a yellow lyophilized solid. ¹H NMR (500 MHz, CD3OD): δ 8.84 (S₃ 1 H); 8.64-8.60 (m, 2 H); 8.44 (s, 1 H); 8.19-8.13 (m, 3 H); 7.97 (s, 1 H); 8.15 (d, J = 5.0 Hz, 2 H); 7.84 (d, J = 8.2 Hz, 1 H); 7.44 (t, J = 7.3 Hz, 1 H); 7.24 (d, J = 6.9 Hz₃ 1 H); 6.14 (d, J = 12.3 Hz, 1 H); 5.91 (d, J = 8.7 Hz₅ 1 H); 5.77 (dd, J = 6.2 Hz, 1 H); 5.38 (dd, J = 4.3 Hz, 1 H); 5.22 (s, 1 H); 5.07 (d, J = 12.6 Hz, 1 H); 4.59 (d, J = 11.2 Hz₃ 1 H); 4.41-4.29 (m, 3 H); 4.21 (S₃ 3 H); 4.16-4.14 (m₃ 1 H); 4.07 (d, J *= 9.6 Hz₃ 1 H); 3.95 (s, 3 H); 3.22 (s, 1 H); 3.16 (s₅ 2 H); 3.03 (s, 5 H); 2.85 (s, 1 H); 2.19 (s, 2 H); 2.05 (s₃ 3 H); 1.76 (s₃ 3 H); 1.41 (d, J = 5.7 Hz₃ 3 H); 1.00 (d₃ J = 7.1 Hz₃ 3 H).

Example 8

Following the procedure described for example 5 except using 3-(2-aminoethyl)pyridine as the amine component to afford the product as a yellow lyophilized solid. ¹H NMR (500 MHz₅ CD₃OD): δ 8.86 (s, IH); 8.79 (s, IH); 8.69 (d, J=3.2 Hz, IH); 8.60 (d, J=9.4 Hz, IH); 8.55 (s, IH); 8.48 (d, J=8 Hz, IH); 8.41 (s, IH); 8.30 (s, IH); 8.17 (s, IH); 8.12 (d, J=10.9 Hz₅ IH); 7.94 (dd, J=5.9, 7.7 Hz₃ IH); 7.83 (m, 4H); 7.41 (t, J=7.1 Hz₅ IH); 7.21 (d, J=7.1 Hz₅ IH); 6.12 (d, J=12.4 Hz, IH); 5.89 (dd, J=I.2, 9.6 Hz₅ IH); 5.76 (dd₅ J=4.8, 10.3, IH); 5.37 (dd, J=4.8,l 1.4 Hz, IH); 5.20 (bs, IH); 5.04 (d, J=12.5 Hz, IH); 4.94 (d, J=10.5 Hz, IH); 4.57 (d, J=I 1.4 Hz, IH); 4.39 (d, J=9.9 Hz, IH); 4.35 (m, IH); 4.28 (d, J=10.7 Hz, IH); 4.13 (m, IH); 4.05 (dd, J=1.6, 9.7 Hz₅ IH); 3.94 (s, 3H); 3.79 (bs, 2H); 3.44 (bs, IH); 3.20 (m, 3H); 3.01 (s, 6H); 2.98 (m, IH); 2.16 (s, 2H); 2.05 (s, 3H); 1.94 (m, IH); 1.74 (s, 3H); 1.40 (d, J=6Hz, 3H); 0.98 (d, J=7Hz, 3H).

Example 9

Following the procedure described for example 5 except using histamine as the amine component to afford the product as a yellow lyophilized solid. ¹H NMR (500 MHz, CD3OD): δ 8.86 (s, 1 H); 8.82 (s, 1 H); 8.61 (d, J = 9.5 Hz₃ 1 H); 8.56 (s, 1 H); 8.41 (s, 1 H); 8.35 (s, 1 H); 8.17 (s, 1 H); 8.13 (d, J = 11.2 Hz, 1 H); 7.88-7.80 (m, 4 H); 7.41 (m, 2 H); 7.21 (d, J = 6.9 Hz₃ 1 H); 6.13 (d, J = 12.4 Hz, 1 H); 5.89 (d, J = 9.3 Hz₅ 1 H); 5.75 (dd, J=5, 11.3Hz, IH); 5.37 (dd, J=4.8, 11.5 Hz, IH); 5.20 (s, 1 H); 5.04 (d, J=12.4 Hz, IH); 4.95 (d, J=10.5 Hz, IH); 4.58 (d, J = 11.5 Hz₃ 1 H); 4.40 (d, J = 9.7 Hz, 1 H); 4.35 (t, J = 5.5 Hz, 1 H); 4.29 (d, J = 10.7 Hz, 1 H); 4.06 (t, J = 4.8 Hz, 1 H); 4.05 (d, J=I.9, 9.6 Hz, IH); 3.94 (s₃ 3 H); 3.76 (s, 2 H); 3.19 (s, 1 H); 3.08 (t, J = 6.6 Hz, 2 H); 3.00 (d, J = 12.2 Hz, 6 H); 2.80 (m, IH); 2.15 (s, 2 H); 2.04 (s, 3 H); 1.74 (s, 3 H); 1.40 (d, J = 6.5 Hz, 3 H); 0.98 (d, J = 7.1 Hz, 3 H).

To a solution of the acid of example 1 (20 mg, 0.015 mmol), triethylamine (6.3 DL, 0.045 mmol), and 4-dimethylaminopyridine (catalytic amount) in dichloromethane (1 mL) was isopropenylchloroformate (5DL, 0.045 mmol) at room temperature. After 5 minutes, l-(2- hydroxyethyl)-4-methylpiperazine (43 mg, 0.15 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The solvent was evaporated, and the residue was purified on preparative reversed-phase HPLC. The product was obtained as a light yellow solid after lyophilization. ¹H NMR (CD₃OD₅ 500MHz): d 8.61 (d, J=9.5, 1 H), 8.57 (m, 2 H)₃ 8.43 (s, 1 H), 8.18 (S₅ 1 H)₅ 8.14 (d₅ J=Il. OHz, 1 H), 7.87 (m, 1 H)₅ 7.82 (m, 1 H), 7.42 (t, J=7.8Hz, 1 H)₅ 7.22 (d, J=7.0 Hz, 1 H), 6.19 (d, J=12.5 Hz, 1 H), 5.90 (m, 1 H)₅ 5.78 (dd, J=10.5 Hz, 4.5 Hz₅ 1 H), 5.39 (m₅ 1 H)₅ 5.21 (s, 1 H), 5.06 (d, J=12.5 Hz, IH), 4.96 (d, J=10.5Hz, 1 H), 4.57 (m, 3 H), 4.41 (d, J=9.5 Hz, 1 H), 4.37 (m₅ 1 H), 4.30 (d, 10.5 Hz, 1 H), 4.06 (m, 1 H), 3.95 (s, 3 H), 3.20 (s, 2 H)₅ 3.02 (s, 6 H), 2.96 (m₅ 2 H)₅ 2.89 (s, 3 H), 2.80 (m, 1 H)₅ 2.17 (m, 2 H), 2.05 (s, 3 H), 1.75 (s, 3 H), 1.40 (d, J=6 Hz₅ 3 H)₅ 0.99 (d, J=7.5 Hz₅ 3 H).

Following the procedure described for example 10 except using l-(2-hydroxyethyl)-morpholine as the neucleophile to afford the product as a yellow lyophilized solid. ¹H NMR (500 MHz₅ CD₃OD): δ 8.66 (s, 1 H), 8.61 (d, J=9.5, 1 H)₅ 8.55 (s, 1 H)₅ 8.43 (s, 1 H), 8.18 (s, 1 H), 8.13 (d, J=I LOHz₃ 1 H), 7.87-7.83 (m, 4 H), 7.42 (t, J=7.8Hz, 1 H), 7.21 (d, J=7.0 Hz, 1 H), 6.12 (d, J=12.5 Hz, 1 H), 5.90 (dd, J=9.5 Hz, 1.5 Hz, 1 H), 5.78 (dd, J=10.5 Hz, A.5 Hz, 1 H)₃ 5.39 (m, 1 H), 5.21 (m, 1 H), 5.06 (d, J=12.5 Hz, IH), 4.79 (m, 1 H), 4.58 (d, J=I l Hz, 1 H)₅ 4.40 (d₅ J=9.5 Hz₃ 1 H), 4.36 (m, 1 H), 4.27 (d, 10.5 Hz₅ 1 H), 4.13 (m, 1 H)₅ 4.05 (m₃ 1 H)₃ 3.95 (s, 3 H)₅ 3.34 (m₃ 3 H), 3.20 (s, 2 H)₃ 3.02 (s₅ 6 H), 2.80 (m, 1 H), 2-17 (d, J=1.5 Hz, 2 H), 2.06 (s, 3 H), 1.75 (s, 3 H), 1.41 (d, J=6 Hz, 3 H), 0.99 (d, J=7.5 Hz, 3 H).

Following the procedure described for example 10 except using l-(3-hydroxypropyl)-morpholine as the neucleophile to afford the product as a yellow lyophilized solid. ¹H NMR (500 MHz, CD₃OD): δ 8.61 (d, J=9.5, 1 H), 8.60 (s, 1 H), 8.55 (s, 1 H), 8.43 (s, 1 H), 8.18 (s, 1 H)₅ 8.14 (d, J=ILOHz₃ 1 H), 7.87-7.82 (m, 4 H)₃ 7.42 (t, J=7.8Hz, 1 H)₅ 7.21 (d, J=7.0 Hz, 1 H), 6.12 (d, J=12.5 Hz₅ 1 H), 5.90 (dd, J=9.5 Hz, 1.5 Hz₃ 1 H), 5.78 (dd, J=10.5 Hz₃ 4.5 Hz₃ 1 H)₃ 5.39 (m, 1 H), 5.21 (s, 1 H)₃ 5.06 (d, J=12.5 Hz₃ IH), 4.94 (d, J=10.5Hz, 1 H), 4.58 (d, J=Il Hz₃ 1 H), 4.52 (t, J=6.0 Hz, 2 H), 4.41 (d, J=9.5 Hz, 1 H), 4.36 (m, 1 H)₃ 4.28 (d, 10.5 Hz₃ 1 H), 4.13 (m, 1 H), 4.06 (m, 1 H), 3.95 (s, 3 H)₃ 3.34 (m, 3 H)₃ 3.20 (s, 2 H)₃ 3.02 (s, 6 H), 2.81 (m, 1 H), 2.28 (m, 2 H), 2.17 (d, J=I .5 Hz, 2 H), 2.06 (s, 3 H), 1.75 (s₃ 3 H)₃ 1.41 (d, J=6 Hz₃ 3 H), 0.99 (d, J=7.5 Hz, 3 H).

To a suspension of nocathiacin-I (75 mg, 0.052 mmol) in THF (1 mL) was added anhydrous pyridine (84 DL, 1.04 mmol) to give a clear solution. Trifuoroacetic anhydride (72 DL, 0.72 mmol) was then introduced. The reaction mixture was stirred at room temperature for 1 hour before it was transferred to a solution of anhydrous methanol (200 DL) in THF (0.5 mL). After 10 minutes, the volatiles were removed in vacuo. The residue was treated with DIEA (0.1 mL) in a mixture of 1 :1 mixture of water and acetonitrile (2 mL) for 10 minutes. Purification on preparative reversd phase HPLG generated the desired product (36 mg, 51 % yield) ¹H NMR (CD₃OD, 600 MHz) D 8.82(s, 1 H)₃ 8.59 (d, 9.6 Hz, 1 H), 8.51(s, 1 H)₅ 8.48 (d, 6.6 Hz, 1 H) ,8.41 (s, 1 H), 8.15 (s, 1 H), 8.11 (d, 10.8 Hz, 1 H), 7.83(d, 6.6 Hz, 1 H), 7.80 (m, 3 H), 7.39 (t, 7.8 Hz, 1 H)₃ 7.18 (d, 7.2 Hz, 1 H), 6.08 (d, 12.6 Hz, 1 H), 5.87 (d, 9.6Hz, 1 H), 5.76 (m, 1 H), 5.35 (m, 1 H), 5.19 (m, 1 H), 5.02 (d, 12.6 Hz, 1 H), 4.91 (d, 10.2 Hz, 1 H), 4.54 (d, 9.6 Hz, 1 H), 4.38 (d, 9.6 Hz, 1 H), 4.33 (m, 1 H), 4.24 (d, 10.2 Hz, 1 H), 4.11 (m, 1 H), 4.02 (d, 9.6 Hz, 1 H), 3.97 (s, 3 H), 3.92 (s, 3 H), 3.18 (s,l H), 3.00 (s, 6 H), 2.78 (m, 1 H), 2.16 (m, 2 H), 2.04 (s, 3 H), 2.02 (s, 1 H), 1.73 (s, 3 H), 1.39 (d, 6.0 Hz, 3 H), 0.97 (d, 7.2 Hz, 3 H). LCMS: 1383.3 (M+H)⁺.

The product of example 2 (200 mg, 0.15 mmol) was mixed with EDC (60 mg₅ 0.29 mmol) and pentafluorophenol (134 mg, 0.073 mmol) in DMF (5 mL). After 30 min at room temperature, 4- fluorophenethylamine (38.3 DL, 0.29 mmol) and DIEA (25 DL, 0.14 mmol ) were added. The reaction mixture was stirred at room temperature for 10 hours, and directly purified by preparative reverse phase HPLC (preparative C- 18 column using CH3CN/water/l%TFA as mobile phase, gradient elution 20-60%) to give the desired product (110 mg, 51% yield). ¹H NMR (CD₃OD, 600 MHz) D 8.59 (d, 9.6 Hz, 1 H), 8.50 (s, 1 H), 8.41 (s, 1 H), 8.26 (s, 1 H), 8.15 (s, 1 H), 8.12 (d, 10.8 Hz, 1 H), 7.85 (d, 6.0 Hz₅ 1 H)₅ 7.80-7.79 (m, 2 H)₅ 7.39 (t, 7.8 Hz/1 H), 7.27 (m, 2 H), 7.19 (d, 6.6 Hz₅ 1 H)₅ 7.00 (d, 8.4 Hz₅ 2 H)₅ 6.09 (d₅ 12 Hz₅ 1 H)₅ 5.88(d₅ 10.2 Hz, 1 H)₅ 5.77-5.75 (m₅ 1 H), 5.35 (m, 1 H)₅ 5.19 (m, 1 H)₅ 5.02 (d, 12.6 Hz₅ 1 H), 4.92 (d, 10.2 Hz₅ 1 H), 4.54 (d₅ 11.4 Hz₅ 1 H), 4.38 (d, 9.6 Hz₅ 1 H), 4.33 (m, 1 H), 4.25 (d, 10.8Hz, 1 H),

4.12 (m, 1 H), 4.02 (d, 9.6 Hz, 1 H), 3.92 (s, 3 H), 3.62 (t, 7.2 Hz, 2 H), 3.18 (m, 1 H), 3.00 (s, 6 H), 2.93 (t, 7.2 Hz₅ 2 H), 2.79 (m, 1 H)₅ 2.16 (s, 2 H)₅ 2.04 (s, 3 H)₅ 1.73 (s, 3 H), 1.40 (d, 5.4 Hz, 3 H)₅ 0.97 (d, 7.2 Hz₅ 3 H). LCMS: 1491.0 (M+H)⁺. Example 15

To a solution of the product of example 14 (20 mg, 0.0134 mmol) in DMF (1 mL) was added cesium carbonate (7 mg, 0.02 mmol). After 20 min at room temperature, ethyl bromopyruvate (13 mg, 0.067 mmol) was added. The reaction solution was stirred at room temperature for 6 hours, and was directly purified by preparative reverse phase HPLC (preparative C-18 column using CH₃CN/water/l%TFA as mobile phase, gradient elution 20-60%) to give the desired product after lyophilization (11.6 mg, 57% yield). LCMS: 1475.0 (M+H)⁺.

To a solution of the product of example 15 (8 mg, 0.0054 mmol) and DIEA (19 DL, 0.108 mmol) in a mixture of water and DMF (4 mL, 1:1) was added N-(2-chloroethyl)morpholine hydrochloride (15 mg, 0.082 mmol). The reaction mixture was stirred at 40 ⁰C for 30 hours, and directly purified by preparative reverse phase HPLC (preparative C- 18 column using CH₃CN/water/l%TFA as mobile phase, gradient elution 20-60%) to give the desired product (lmg, 12% yield). ¹H NMR (CD₃OD₅ 600 MHz) D 8.59 (d, 9.6 Hz, 1 H), 8.50 (s, 1 H), 8.41 (s, 1 H), 8.26 (s, 1 H), 8.15 (s, 1 H)₅ 8.12 (d, 10.8 Hz, 1 H), 7.85 (d, 6.0 Hz, 1 H), 7.80-7.79 (m, 2 H), 7.30 (t, 7.8 Hz, 1 H), 7.12 (m, 2 H), 7.01 (d, 6.6 Hz, 1 H), 6.84 (d₅ 8.4 Hz, 2 H)₅ 6.09 (d, 12 Hz, 1 H)₅ 5.88(d, 10.2 Hz₅ 1 H), 5.77-5.75 (m, 1 H), 5.35 (m, 1 H), 5.19 (m, 1 H), 5.02 (d, 12.6 Hz, 1 H), 4.92 (d, 10.2 Hz, 1 H), 4.54 (d, 11.4 Hz, 1 H), 4.38 (d, 9.6 Hz, 1 H), 4.33 (m, 1 H), 4.25 ( d, 10.8Hz₅ 1 H), 4.12 (m, 1 H), 4.02 (d_s 9.6 Hz, 1 H), 3.92 (s, 3 H), 3.62 (t, 7.2 Hz, 2 H), 3.18 (m, 1 H), 3.00 (S₅ 6 H), 2.93 (t, 7.2 Hz, 2 H)₅ 2.79 (m, 1 H), 2.75 (m, 4 H), 2.64 ( m, 2 H)₅ 2,49 (M, 6 H), 2.16 (S₅ 2 H), 2.04 (s, 3 H), 1.73 (s, 3 H)₅ 1.40 (d₅ 5.4 Hz₅ 3 H), 0.97 (d, 7.2 Hz, 3 H). LCMS: 1589.0 (M+H)⁺.

The product of example 2 (500 mg, 0.365 mmol) was mixed with EDC (105 mg, 0.548 mmol)₅ HOBt (50 mg, 0.365 mmol) in DMF (5 mL). To this solution was added 3-hydroxyazetidine hydrochloride (80 mg, 0.73 mmol). The reaction mixture was stirred at room temperature for 8 hours, and directly purified by preparative reverse phase HPLC (preparative C- 18 column using CH₃CN/water/l%TFA as mobile phase, gradient elution 20-60%) to give the desired product as its TFA salt (280 mg, 50 % yield). ¹H NMR (DMSO-^₆, 500 MHz) Q9.08 (s, 1 H)₅ 8.64 (s, 1 H), 8.56 (m, 2 H), 8.52 (s, 1 H)₅ 8.49 (s, 1 H)₅ 8.20 (s, 1 H)₅ 7.98 (s, 1 H), 7.87 (s, 1 H), 7.85 (d, 1 IHz₅ 1 H)₅ 7.73 (d₅ 8.5 Hz₅ 1 H)₅ 7.36 (m, 2 H)₅ 7.18 (d₅ 7.5 Hz₅ 1 H)₅ 6.36 (s, 1 H)₅ 6.01 (d₅ 6.6 Hz₅ 1 H)₅ 5.75 (dd₅ 10.5 Hz₅ 5.0 Hz₅ 1 H)₅ 5.70 (d, 10.5 Hz₅ 1 H)₅ 5.22 (m, 1 H), 5.04 (m₅ 2 H)₅ 4.98 (m, 1 H), 4.80 (m, 2 H)₅ 4.56 (m₅ 1 H)₅ 4.52 (d, 11 Hz₅ 1 H), 4.32 (m, 2 H)₅ 4.28 (d, 9.0 Hz₅ 1 H)₅ 4.24 (m, 1 H)₅ 4.14 (d₅ 10.5 Hz₅ 1 H)₅ 4.03 (m, 1 H)₅ 3.90 (s, 3 H)₅ 3.82 (dd₅ 10.5 Hz₅ 4.5 Hz₅ 1 H)₅ 3.12 (s, 1 H)₅ 2.88 (s, 3 H)₅ 2.86 (s, 3 H), 2.11 (m, 1 H)₅ 1.99 (s, 3 H)₅ 1.92 (m, 1 H)₅ 1.59 (s, 3 H), 1.15 (d, 5.5 Hz₅ 3 H)₅ 0.80 (d, 7.0 Hz₅ 3 H). LCMS: 1424.6 (M+H)⁺.

To a suspension of the product of example 17 (TFA salt, 50 mg 0.035 mmol) in dry THF (3 mL) were added carbon tetrachloride (50 DL₅ 0.525 mmol), DIEA (61 DL, 0.35 mmol), DMAP (catalytic amount), and di-^-butylphosphite (34 mg, 0.176 mmol). The reaction mixture was stirred at 60 ⁰C for 36 hours and purified directly on preparative reverse phase HPLC. The appropriate fractions were combined and lyophilized to give the di-t-butylphosphate product as a white powder ( 25 mg, 44 % yield).

The di-t-butylphosphate thus obtained was dissolved in 0.2 mL of formic acid. After 10 minutes at room temperature, the reaction mixture was directly applied to a preparative reverse phase HPLC. The appropriate fractions were combined and lyophilized to get the desired phosphonic acid product as a white solid (16 mg, 65 % yield). ¹H NMR (DMSO-J₆, 600 MHz) D9.07 (s, 1 H), 8.61 (s, 1 H), 8.55 (m, 2 H), 8.51 (s, 1 H), 8.43 (s, 1 H), 8.37 (s, 1 H)₅ 8.18 (s, 1 H), 7.95 (s, 1 H), 7.82 (d, 10.8 Hz, 1 H), 7.71 (d, 8.4 Hz, 1 H), 7.33 (m, 2 H), 7.16 (d, 6.6 Hz, 1 H), 6.34 (m, 1 H), 5.99 ( d, 12 Hz, 1 H), 5.73 (m, 1 H), 5.68 (d, 9.6 Hz, 1 H)₅ 5.19 (m, 1 H)₅ 5.00 (m, 3 H)4.76 (d, 10.8 Hz₅ 1 H)₅ 4.52 (d, 10.8 Hz, 1 H)₅ 4.47 (m, 1 H)₅ 4.36 (m, 1 H), 4.28 (m, 1 H), 4.22 (m, 2 H), 4,12 (d₅ 10.2 Hz, 1 H)₅ 4.01 (d, 9.0 Hz₅ 1 H), 3.88 (s, 3 H)3.77 (m, 1 H)₅ 3.10 (s. 1 H), 2.86 (s, 3 H), 2.83 (s, 3 H), 2.50 (m, 1 H), 1.97 (s, 3 H), 1.89 (m, 1 H), 1.57 (s, 3 H), 1.12 (s, 3 H), 0.77 (d, 6.5 Hz, 3 H). LCMS: 1704.7 (M+H)⁺.

To a solution of nocathiacin I (1.43 g, 1 mmol) in DMF (20 mL) were added N- iodoethylmorpholine hydroiodide (5 mmol) and ethyldiisopropylamiπe (10 mmol). After 18 h at

25 ⁰C₅ diethyl ether (60 mL) was added and the solid precipitate was collected by filtration.

Purification by preparative HPLC on a C-18 column afforded the alkylated product (800 mg) as a white powder. ¹H NMR (CD₃COD) D (selected signals) 8.82 (s, IH), 8.65 (s, IH) 8.63 (d, J =

9.5 hz, IH)₅ 8.43 (s, IH), 8.41 (s, IH), 8.18 (s, IH), 8.15 (d, J = 12.0 Hz₅ IH), 7.98 (s, IH), 7.89 (s, IH), 7.88 (d, J = 6.5 Hz, IH), 7.84 (d, J = 8.5 Hz, IH), 7.43 (t, J = 7.5 Hz₃ IH), 7.24 (d, J =

7.0 Hz, IH), 6.68 (s, IH)₅ 6.15 (d, J = 8.4 Hz, IH), 5.59 (d, J = 8.5 hz, IH). LCMS: 1551.6

(M+H)⁺. ^•

The product thus obtained was transformed to the carboxylic acid product following the procedure as described for the preparation of example 1.

LCMSi MSS-O (M⁺+!)

The carboxylic acid product of example 19 (15mg, O.Olmmol) was dissolved in anhydrous DMF (0.5mL). To this solution were added pentaflurophenol (9.2mg) and EDC (3.8mg). After 30 min at room temperature, propargylamine (2.8mg) was added. The reaction mixture was stirred for another 15 min, acidified by adding TFA, and directly purified by preparative reverse phase HPLC to give the desired product as its TFA salt. ¹HNMR (500MHz, CD₃OD) P 8.62 (d, J = 9.5 Hz, IH), 8.58 (s, IH), 8.45 (s, IH), 8.47 (s, IH), 8.44 (s, IH)₅ 8.19 (s, IH), 8.16 (s, IH), 8.15 (d, J = 11.0 Hz, IH)₅ 7.91 (s, IH), 7.83 (d, J = 8.5 Hz,lH), 7.43 (t, J = 8.0 Hz, IH), 7.23 (d, J = 7.0 Hz, IH), 6.12 (d, J - 12.0 Hz, IH), 5.91 (d, J = 6.5 Hz, IH), 5.78 (dd, J = 11.0, 5.0 Hz, IH), 5.40 (dd, J = 11.0, 5.0 Hz, IH), 5.19 (s, IH), 5.06 (d, J = 12.5 Hz, IH), 4.97 (d_s J = 11.0 Hz, IH), 4.66 (s, IH), 4.57 (d, J = 11.0 Hz, IH), 4.44 (d, J= 10 Hz, IH), 4.40 (s, IH), 4.37 (d, J = 4.0 Hz, IH), 4.30 (d, J = 10.5, IH), 4.12 (d, J = 6.5 Hz₅ IH), 4.07 (d₅ J = 9.5 Hz, IH), 3.96 (s, 3H), 3.69 (d, J = 4.0 Hz, 2H), 3.05 (m, 2H), 2.94 (s, 6H), 2.79 (m, IH), 2.66 (s, 4H), 2.14 (s, 2H), 2.09 (s, 3H), 1.99 (s, IH), 1.72 (s, 3H), 1.43 (d, J = 5.5 Hz, 3H), 0.96 (d, J = 5.0 Hz, 3H). LCMS (ESI, m/z): 761.26 (1/2M+1).

The antibacterial activity of the compounds of Formula I can be determined using the assay methods described below. Materials:

Cation-Adjusted Mueller Hinton Broth (MH; BBL)

50% Lysed Horse Blood (LHB; BBL) (stored frozen)

RPMI 1640 (BioWhittaker) Human Serum (Pel-Freez)

RPMI 1640 (BioWhittaker)

Haemophilus Test Medium (HTM, Remel)

Trypticase Soy Broth (TSB, 5 mL/tube; BBL)

0.9% Sodium Chloride (Saline; Baxter) Trypticase Soy + 5% Sheep Blood Agar Plates (TSA; BBL)

Sabouraud Dextrose Agar Plates (BBL)

Chocolate Agar Plates (BBL)

2X Skim Milk (Remel)

Microbank Beads (Kramer Scientific) MIC 2000 Microtiter plate inoculator.

2X Trypticase Soy Broth (TSB, BBL) + 15% glycerol/50% horse serum.

96- Well Microtiter plates, lids, inoculum trays (Dynex Laboratories)

8-Channel Finn Multichannel pipettor, 0.5-10 DL volume

METHODS:

MEDIA PREPARATION

Cation-Adjusted Mueller Hinton Broth (BBL): Prepared according to manufacturer's instructions (22 gms dissolved in 1000 mL water; autoclaved 22 minutes). Stored refrigerated.

Filter-sterilized before use using a Corning 0.45 Tm cellulose acetate filter.

50% Lysed Horse Blood: Defibrinated horse blood is diluted 1:1 with sterile distilled water; frozen, thawed and re-frozen (at least 7 times), then centrifuged. Stored frozen at -20⁰C.

Cation-Adjusted Mueller Hinton + 2.5% Lysed Horse Blood: Aseptically add 5 mL 50% lysed horse blood to 100 mL Cation- Adjusted Mueller Hinton Broth. Filter-sterilize before use using a Corning 0.45 Tm cellulose acetate filter. Cation- Adjusted Mueller Hinton + 50% Human Serum: Aseptically add 50 mL Human Serum to 50 mL 2X Cation-Adjusted Mueller Hinton Broth. Filter-sterilize before use using a Corning 0.45 Tm cellulose acetate filter.

Haemophilus Test Medium (Remel): Received prepared from manufacturer. Filter-sterilized before use using a Corning 0.45 Tm cellulose acetate filter.

0.9% Sodium Chloride (Saline; Abbott Labs): Received prepared from manufacturer.

2X Skim Milk (Remel): Received prepared from manufacturer.

AU agar plates are received prepared from manufacturer.

CONDITIONS AND FOR REPRESENTATIVE STRAINS

INOCULUM

BACILLUS, INCUBATION CONDITIONS, 35°C; MICS READ AT

STAPHYLOCOCCUS, 18-22 HOURS;

ENTEROCOCCUS:

ESCHERICHL; CATION-ADJUSTED MUELLER HINTON (CAMHB;

BBL); INOCULUM = 10⁵ CFU/ML

STREP. PNEUMONIAE: INCUBATION CONDITIONS, 35°C; MICS

READ AT 22-24 HOURS;

CATION-ADJUSTED MUELLER HINTON+ 2.5% LYSED

HORSE BLOOD (LHB); INOCULUM = 10⁵ CFU/ML

HAEMOPHILUS INCUBATION CONDITIONS, 35°C; MICS

INFLUENZAE: READ AT 18-22 HOURS;

HAEMOPHILUS TEST MEDIUM (HTM; REMEL);

INOCULUM = 10⁵ CFU/ML

CANDIDA: INCUBATION CONDITIONS, 35°C; MICS READ AT 24

HOURS; RPMI 1640 MEDRJM (BIO WHITTAKER)

INOCULUM = 10³ CFU/ML

HIGHEST CONCENTRATION OF ANTIBIOTIC TESTED = 64 DG/ML (WHEN STARTING FROM A 1 MG/ML SOLTST IN 50% DMSO) FINAL CONCENTRATION OF DMSO PER WELL = 3.2%

SELECTION AND MAINTENANCE OF ISOLATES

The type of strains listed above can be obtained from publicly available sources.

The strain of Haemophilus influenzae used in to assay the compound of this invention is a mouse pathogen used for in vivo testing at Merck. The Escherichia coli strain used in to assay the compound of this invention is a cell wall permeable strain. The Candida albicans strain is used as a control. These culture are maintained as frozen stocks at —80⁰C in a) Microbank beads; b)

2X Skim Milk; or c) in 2X Trypticase Soy Broth + 15% glycerol/50% horse serum

{Haemophilus and Streptococcus pneumoniae).

INOCULUM PREPARATION

Selected isolates are sub-cultured onto either Chocolate Agar Plates {Haemophilus influenzae), onto Trypticase Soy + 5% Sheep Blood Agar Plates {Streptococcus pneumoniae,

Staphylococcus aureus, Escherichia coli, Enterococcus, Bacillus) or onto Sabouraud Dextrose Agar {Candida) and incubated at 35⁰C. Haemophilus and Streptococcus pneumoniae are incubated in 5% CO₂; all other isolates are incubated in ambient air. Isolates are sub-cultured 2X before assay.

Colonies are selected from plates and used to prepare an inoculum equivalent to a

0.5 McFarland standard in Trypticase Soy Broth. An inoculum with a density equivalent to a 1.0 McFarland standard is prepared for Streptococcus pneumoniae. The inoculum density for all cultures is ~10⁸ CFU/mL in TSB. This TSB inoculum is diluted 1:10 in sterile saline (4 mL inoculum + 36 mL saline; equivalent to ~10⁷ CFU/mL) and kept on ice until used to inoculate microtiter plates.

Colony counts are performed on randomly-selected isolates to confirm CFU/well (TSB inoculum plated out 10^"5, 10^'6 onto either TSA II + 5% SB or onto chocolate agar plates, incubated overnight, 35°C, CO₂)

PLATE FILLING

AU wells of 96- well microtiter plates (Dynex) are filled with 100 TL media. Haemophilus test media plates are prepared to test Haemophilus influenzae; Cation- Adjusted Mueller Hinton + 5% Lysed Horse Blood plates are prepared to test Streptococcus pneumoniae; Cation-Adjusted Mueller Hinton Broth plates are prepared to test Enterococcus, Staphylococcus aureus, Escherichia coli and Bacillus subtilis. RPMI 1640 is used to test Candida. The MICs against S. aureus Smith are determined in Cation-adjusted Mueller Hinton and in Cation- Adjusted Mueller Hinton + 50% Human Serum, to determine if the compound is inactivated by some component in serum (indicated by an increase in the MIC). Filled plates are wrapped in plastic bags (to minimize evaporation), stored frozen and thawed before use.

PREPARATION OF COMPOUNDS

The compounds are prepared on a weight basis. Compounds are prepared to 2-10 mg/mL in 100% DMSO₃ then diluted to lmg/rnL in a 1:1 dilution of DMSO/2x CAMHB (final concentration=50%DMSO/50% CAMHB). Compounds are serially diluted 1:1 in 50% DMSO/50% CAMHB in BD Biosciences Deep Well Polypropylene 96 well plates (starting concentration 1-5 mg/mL).

MICROBROTH DILUTION ASSAY

Using a Finn Automated Multichannel Pipette, (0.5-10 GL volume) 6.4 TLs of antimicrobial working solutions are added to wells of filled microtiter plates (concentration of antimicrobial in first well = 512-64 microg/mL; concentration of DMSO = 3.2%).

Antimicrobials are added in this manner to keep constant the amount of DMSO in each well (to keep compounds solubilized and to account for the possibility of non-specific killing by the

DMSO. The last row contains a growth control of 3.2% DMSO. With each assay, controls are run. They are Penicillin G and chloramphenicol, prepared in the same manner as the compounds. Ertapenem is included as a control for the serum protein binding assay.

PLATE INOCULATION All wells of microtiter plates are inoculated with (saline-diluted) culture using the

MIC 2000 System, an automated plate inoculating device which delivers an inoculum of 1.5 TL per well. Plates are incubated at 35⁰C in ambient air. An uninoculated plate is also incubated as a sterility check. Results are recorded after 22-24-hours' incubation. Plates were read to no growth. The MIC is defined as the lowest antimicrobial level which resulted in no growth after 22-24-hours' incubation.

The Compounds of formula I demonstrate antibacterial activity against various strains of 5. aureus, E.faecalis, E.faecium, B. subtilis and S. pneumoniae. Compounds of formula I also demonstrate antibacterial activity against various species that are resistant to many known antibiotics such as methicillin-resistant S. aureus (MRSA)₅ vancomycin-resistant Enterococcus sp. (VRE), multidrug-resistant E. faecium, macrolide-resistant S. aureus and S. epidermidis, and linezolid-resistant S. aureus and E. faecium. The minimum inhibitory concentration (MIC) values for these test strains range from 0.0001 to 200 Og/mL. MICs are obtained in accordance to the NCCLS guidelines. Select compounds of this invention have been found to have minimum inhibitory concentration (MIC) values that are at least a 10 fold improvement over the compounds disclosed in P. Hrnciar, et. al., J. Org. Chem. 2002, 67, 8789- 8793 against certain tested strains. See Table 2 where compounds A and B (Examples 5 and 3 of claimed invention) were compared with compound C (example 7 of J. Org. Chem. 2002, 67, 8789-8793).

TABLE 2

Compound A Organism Strain Serum % MIC ue/mL

Enterococcus Faecalia CLB 21560 0 0.01595 Staphylococcus Aureus CL 5814 0 0.00565 Staphylococcus Aureus CL 8260 0 0.0075 Staphylococcus Aureus MB 2865 50 0.03

Compound B Organism Strain Serum % MIC ue/mL

Enterococcus Faecalia CLB 21560 0 0.0325 Staphylococcus Aureus CL 5814 0 0.0075 Staphylococcus Aureus CL 8260 0 0.015 Staphylococcus Aureus MB 2865 50 0.06

Compound C Organism Strain Serum % MIC uε/mL

Enterococcus Faecalia CLB 21560 0 0.25375 Staphylococcus Aureus CL 5814 0 0.030475 Staphylococcus Aureus CL 8260 0 0.125475 Staphylococcus Aureus MB 2865 50 0.14

Claims

WHAT IS CLAIMED IS:

1. A compound of structural formula I:

wherein:

R independently represents hydrogen, and Ci_i2 alkyl;

Ri represents hydrogen, C\.β alkyl, and C3-6 cycloalkyl, OR, -(CH2)nNR2, -

(CH2)n(O(CH₂)2)l-6R9, -(CH2)_nC(O)NR₂, -(CH₂)_nC4-10 heterocyclyl, -P(O)(OH)₂, said heterocyclyl optionally substituted with 1 to 3 groups of R^a; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R^a;

R2 represents Ri , -OC(O)CF3 _f and ORi ;

R3 represents hydrogen, -NR5R6, or -OR5, provided that when both R5 and Rg are attached to the same nitrogen they both are not hydrogen,

R4 represents hydrogen R4a represents N(R)2;

R5 and R6 independently represent hydrogen, C_1-12 alkyl, -(CH2)nC4_io heterocyclyl. - (CH₂)nNR7R8, -(CH₂)_nCH(R₇)R₈, -CH₂(R₇)R₈, -(CH₂)_nORl, -(CH₂)_nC(O)NR₇R₈, - (CH₂)nC(=CH₂)CN, -(CH₂)nC(=CH₂)F₅ -(CH₂)_nC6-10 aiyl, -(CH₂)_n(O(CH₂)2)l-6R9, - CH(R7)(CH2)_nNR7R8, -CH(R7)C(O)OR,-C3_6cylcoalkylC(O)OR, -CH(R₇)CN, -(CH2)_nCN, - CH(R7)(CH2)_nOR, -(CH2)_nC(=CH2)CN, -(CH2)nC(O)NR₇R8, -NHC(O)R7, NHR7, -CH2(C3- 6cylcoalkyl)CH2θR, -C(CH3)2C(O)OR, said aryl, and heterocyclyl optionally substituted with 1 to 3 groups of R^a; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R^a; or

R5 and R₆ together with the nitrogen atom they are attached form a 4 to 10 membered heterocyclic ring optionally containing 1 to 2 additional heteroatoms selected from the group consisting of N, S and O and optionally substituted with 1 to 3 groups of R^a;

R7 and R₈ independently represent hydrogen, hydroxy!, C_{1 -6} alkoxy, C_1-12 alkyl, -

(CH2)_nNR5R6, -(CH2)_nC4-10 heterocyclyl, -(CH2)_nC6-10 aryl, -(CH2)_nNHNHC(O)C4-10 heterocyclyl, -(CH2)_nOR, -C(O)Ci_6 alkyl, -C(O)Ci_6 alkylC(O)R5, -C(O)C6-10 aryl, -C(O)(CH2)_nC4-10 heterocyclyl,

C(O)CHR5NR5R6, (CH₂)_nNHC(O)(CHR)_nNR₅R₆, -C(O)NH(CH2)_nC4-10 heterocyclyl, -

C(O)(CH2)nNR5R6, said aryl, and heterocyclyl optionally substituted with 1 to 3 groups of R^a; said alkyl optionally substituted with 1 to 6 hydroxy and/or optionally substituted by one to three groups of R^a or

R₇ and R₈ together with the nitrogen atom they are attached form a 4 to 10 membered heterocyclic ring optionally containing 1 to 2 additional heteroatoms selected from the group consisting of N, S and O and optionally substituted with 1 to 3 groups of R^a; or

R7 and R₈ together with the carbon atom they are attached form a 3 to 10 membered carbocyclic ring optionally and optionally substituted with 1 to 3 groups of R^a;

R9 represents hydrogen, C_{1 -6} alkyl, (CH2)nC4-10 heterocyclyl, -(CHR)_nC(O)OR, - C(O)NR5R6, CN, OR, said alkyl and heterocyclyl optionally substituted with 1 to 3 groups of R^a Ra represents hydrogen, halogen, -(CH2)nOR, CF3, (CH2)_nC(O)OR, - (CH2)nC(O)(CH2)nNR-7R8,

-(CH2)nC6-10 aryl, -(CH₂)_nC4-10 heterocyclyl, =CH2, S(O)2R, C(O)R, (CH2)_nC3-8 cycloalkyl, SO2NR5R6, (CH2)C6-10 aryl, N(R)2, NO2, CN₅ -O-, (Ci-6 alkyl)O, (aiyl)O-, (Ci- 6 alkyl)S(O)0-2-₃ Ci_i2 alkyl, said alkyl, heterocyclyl, and aryl optionally substituted with 1 to 4 groups selected from the group consisting of C\.β alkyl, (CH2)nOR, (CH2)nN(R)2_> -O-; and

n represent 0-6, m represents 0-1, and p represents 0, 1 or 2, provided that the compound of formula I when Ri is hydrogen, R2 is OH, R3 is -NR5R6, R5 is hydrogen, R6 is (CH2)nNR7R8,

then R7 and Rs do not represent the compound where: R7 and R8 together are morpholino, R7 and R8 are both 2~hydroxyethyl or ethyl,

R7 is methyl or ethyl and Rg is 2-hydroxyethyl, or R7 is ethyl and Rs is 2-carboxymethyl.

2. The compound according to claim 1 wherein R3 represents -C(O)NRsRs provided that when both R5 and Rg are attached to a nitrogen they are not both hydrogen.

3. The compound according to claim 2 wherein R3 is — (C(O)NH(CH2)nC5~ 10 heterocyclyl, Rl is hydrogen or C 1-6 alkyl, R2 is Rl or -OR, or Ci_6 alkyl, R4 represents

4. The compound according to claim 1 wherein R3 represents OR5.

5. The coipound according to claim 1 represented by structural formula II:

wherein Ri is hydrogen or methyl, R₂ is R₁ or OR, and R₃ is -NH(CH2)_nX or - NHCR₇R₈C(O)NR₅R₆, wherein X is selected from the group consisting of phenyl, pyrimidinyl, morpholinyl, piperazinyl, pridinyl, pyrazolyl, indolyl, furanyl, isoindazolyl, oxazolyl₅ pyrazinyl, pyrrolyl, imidazolyl, triazolyl and teterazolyl said X groups optionally substituted with 1 to 3 groups of R^a, provided that when X is morpholinyl, n is not 2,.

6. A compound according to claim 6 wherein R3 is -NH(CH2)nX , n is 3 and X is morpholinyl.

7. A compound according to claim 6 wherein R3 is -NH(CH2)nX , n is 3 and

X is oxazolyl.

8. A compound according to claim 1 which is: TABLEl