EP1471923A1 - Amido-macrolides - Google Patents

Amido-macrolides

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Publication number
EP1471923A1
EP1471923A1 EP03713257A EP03713257A EP1471923A1 EP 1471923 A1 EP1471923 A1 EP 1471923A1 EP 03713257 A EP03713257 A EP 03713257A EP 03713257 A EP03713257 A EP 03713257A EP 1471923 A1 EP1471923 A1 EP 1471923A1
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EP
European Patent Office
Prior art keywords
substituted
unsubstituted
compound
formula
compounds
Prior art date
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EP03713257A
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German (de)
English (en)
Other versions
EP1471923A4 (fr
Inventor
Gary Ashley
Simon James Shaw
Yandong C/o Kosan Biosciences Inc. LI
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Kosan Biosciences Inc
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Kosan Biosciences Inc
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Publication of EP1471923A1 publication Critical patent/EP1471923A1/fr
Publication of EP1471923A4 publication Critical patent/EP1471923A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • Erythromycins are macrolide antibiotics, glycosylated polyketides originally discovered in 1952 in the metabolic products of a strain of Streptomyces erythreus, now classified as Saccharopolyspora erythraea.
  • the antibiotic occurs in various forms, designated A, B, C, and D. Since their discovery, many have worked to prepare derivatives of the molecule to improve or modify its properties. The focus of much of this work involved chemical modification of the naturally produced erythromycin molecule. For example, clarithromycin is a semi-synthetic antibiotic that is made by chemically methylating the hydroxyl group at C-6.
  • Azalides such as azithromycin
  • erythromycin derivatives where the C-9 ketone has been replaced with a N-CH 2 unit through a Beckmann rearrangement. See, for example, O'Connell et al., "Azalides and methods of making same," U.S. Patent No. 6,270,768, incorporated herein by reference.
  • Ketolides are erythromycin derivatives where the C-3 cladinose sugar is chemically removed and the resulting free hydroxyl group converted into a keto group.
  • Phan et ah "2-Halo-6-O-substituted ketolide derivatives ”
  • U.S. Patent No. 6,124,269 describes ketolides with a cyclic carbamate group at C-ll and C-12 and an O-alkylaryl group at C-6.
  • Agouridas et al., "Erythromycin compounds” U.S. Patent No.
  • 5,635,485 also describes ketolides with a cyclic carbamate group at C-ll and C-12 but which have a -OMe group at C-6 and an alkylaryl group at the carbamate nitrogen.
  • the complexity of the macrolide molecule has limited medicinal chemistry efforts to produce derivatives of the naturally occurring erythromycins and their precursors.
  • PKS's modular polyketide synthases
  • PKS's are multifunctional enzymes that catalyze the formation of the polyketide chains through repeated reactions between acylthioesters.
  • the Sac. erythraea PKS known as 6-deoxyerythronolide B synthase (DEBS)
  • DEBS 6-deoxyerythronolide B synthase
  • DEBS produces the polyketide macrolactone 6-deoxyerythronolide B, which is processed by additional tailoring enzymes present in Sac. erythraea to make erythromycins A-D.
  • the collective assembly of the PKS gene and the genes for the tailoring enzymes are referred to as the biosynthetic gene cluster.
  • the organization of the gene cluster is described in Summers et al., "Polyketide-associated sugar biosynthesis genes," U.S. Patent No. 5,998,194, incorporated herein by reference. Recombinant methods using vectors encoding a variety of PKS's, including the PKS from Sac.
  • Khosla et al. "Recombinant production of novel polyketides," U.S. Patent Nos. 5,672,491, 5,830,750, 5,962,290, 6,022,731, and 6,077,696; Khosla et al, "Recombinant combinatorial genetic library for the production of novel polyketides," U.S. Patent No. 5,712,146; Khosla et al., “Method to produce novel polyketides," U.S. Patent Nos. 6,066,721, 6,221,641, and 6,261,816; Barr et al., "Production of polyketides in bacteria and yeast," U.S. Patent Nos.
  • the present invention fulfills this need by providing novel erythromycins, ketolides, and azalides.
  • the invention provides various compounds and pharmaceutically acceptable salts thereof.
  • the invention provides compounds and pharmaceutically acceptable salts thereof of the formula I
  • R 1 is C Cg substituted or unsubstituted alkyl, C 2 -C 8 substituted or unsubstituted alkenyl, C 2 -C 8 substituted or unsubstituted alkynyl, C -C 15 substituted or unsubstituted aryl, C 5 -C 2 o substituted or unsubstituted arylalkyl, C 5 -C 20 substituted or unsubstituted biarylalkyl, C 5 -C 2 o substituted or unsubstituted arylalkenyl, or C 5 -C o substituted or unsubstituted arylalkynyl;
  • R 2 is H, C ⁇ -C substituted or unsubstituted alkyl, C 2 -C 4 substituted or unsubstituted alkenyl, or C 2 -C substituted or unsubstituted alkynyl;
  • R 3 is H, C ⁇ -C alkanoyl, or benzoyl;
  • R 4 is H or OH
  • R 5 is H, OH, C ⁇ -C 4 alkoxy, C 2 -C 4 alkenyloxy, or C 2 -C 4 alkynyloxy;
  • the invention provides compounds of formula I where
  • R 2 is H and m is 0.
  • the invention provides compounds of formula I where R 2 is H and m is 1.
  • the invention provides compounds of formula I where R 2 is H and m is 2.
  • the invention provides compounds of formula I which have any of the following structures:
  • the invention provides compounds of formula II and pharmaceutically acceptable salts thereof
  • R is H, C ⁇ -C 4 alkanoyl, or benzoyl
  • R 4 is H or OH
  • R 5 is H, -C 4 alkoxy, C 2 -C 4 alkenyloxy, or C 2 -C 4 alkynyloxy
  • the invention provides compounds of formula II where m is 0. In other embodiments, the invention provides compounds of formula II where m is 1. In yet other embodiments, the invention provides compounds of formula II where m is 2.
  • the invention provides compounds of formula II which have any of the following structures:
  • the invention provides compounds of formula III and pharmaceutically acceptable salts thereof
  • R 1 is C 1 -C 8 substituted or unsubstituted alkyl, C 2 -C 8 substituted or unsubstituted alkenyl, C 2 -C 8 substituted or unsubstituted alkynyl, C 4 -C 15 substituted or unsubstituted aryl, C 5 -C 20 substituted or unsubstituted arylalkyl, C 5 -C 20 substituted or unsubstituted biarylalkyl, C 5 -C 20 substituted or unsubstituted arylalkenyl, or C 5 -C o substituted or unsubstituted arylalkynyl;
  • R 2 is H, C C substituted or unsubstituted alkyl, C 2 -C 4 substituted or unsubstituted alkenyl, or C 2 -C 4 substituted or unsubstituted alkynyl;
  • R 3 is H, -C 4 alkanoyl, or benzoyl;
  • R 4 is H or OH
  • R 5 is H, C 1 -C 4 alkoxy, C 2 -C alkenyloxy, or C 2 -C 4 alkynyloxy;
  • the invention provides compounds of formula III in which m is 0.
  • the invention provides compounds of formula III in which m is 1.
  • the invention provides compounds of formula II in which m is 2.
  • the invention provides compounds of formula III where R 2 is H and m is 0.
  • the invention provides compounds of formula IH where R 2 is H and m is 1.
  • the invention provides compounds of formula III where R 2 is H and m is 2.
  • the invention provides compounds of formula III in which R 1 is a C -C 15 substituted or unsubstituted aryl, C 5 -C 2 o substituted or unsubstituted arylalkyl, C 5 -C 0 substituted or unsubstituted biarylalkyl, C 5 -C 20 substituted or unsubstituted arylalkenyl, or C 5 -C o substituted or unsubstituted arylalkynyl; and R 2 is H.
  • the invention provides compounds of formula IN and pharmaceutically acceptable salts thereof
  • R 1 is C C 8 substituted or unsubstituted alkyl, C 2 -C 8 substituted or unsubstituted alkenyl, C 2 -C 8 substituted or unsubstituted alkynyl, C 4 -C ⁇ 5 substituted or unsubstituted aryl, C 5 -C 0 substituted or unsubstituted arylalkyl, C 5 -C o substituted or unsubstituted biarylalkyl, C 5 -C 20 substituted or unsubstituted arylalkenyl, or C 5 -C 2 o substituted or unsubstituted arylalkynyl;
  • R 2 is H, C ⁇ -C 4 substituted or unsubstituted alkyl, C 2 -C 4 substituted or unsubstituted alkenyl, or C 2 -C substituted or unsubstituted alkynyl;
  • R 3 is H, C ⁇ -C 4 alkanoyl, or benzoyl
  • R 5 is H, C ⁇ -C 4 alkoxy, C 2 -C alkenyloxy, or C -C 4 alkynyloxy;
  • R 8 is H or F;
  • the invention provides compounds of formula IV in which R 2 is H.
  • the invention provides compounds of formula IV in which R 1 is C 6 -Ci 5 substituted or unsubstituted aryl, Cs-C 20 substituted or unsubstituted arylalkyl, C 5 -C 20 substituted or unsubstituted biarylalkyl, C 5 -C 2 o substituted or unsubstituted arylalkenyl, or C 5 -C 2 o substituted or unsubstituted arylalkynyl; and R 2 is H.
  • the invention provides compounds of formula IV in which R 1 is C 6 -C 15 substituted or unsubstituted aryl, C 5 -C 2 o substituted or unsubstituted arylalkyl, C 5 -C 2 o substituted or unsubstituted biarylalkyl, C 5 -C 20 substituted or unsubstituted arylalkenyl, or C 5 -C 2 o substituted or unsubstituted arylalkynyl; R 2 is H; and X is NH.
  • the invention provides compounds of formula IV which have any of the following structures:
  • the invention provides compounds of formulas I, III, or IV in which R 1 is a group having any of the following formulas:
  • the invention provides compounds of the formula V and pharmaceutically acceptable salts thereof
  • R 1 is -C 8 substituted or unsubstituted alkyl, C 2 -C 8 substituted or unsubstituted alkenyl, C 2 -C 8 substituted or unsubstituted alkynyl, C 4 -C 15 substituted or unsubstituted aryl, C 5 -C 20 substituted or unsubstituted arylalkyl, C 5 - 0 substituted or unsubstituted biarylalkyl, C 5 -C 2 o substituted or unsubstituted arylalkenyl, or C 5 -C o substituted or unsubstituted arylalkynyl;
  • R 2 is H, -C 4 substituted or unsubstituted alkyl, C 2 -C substituted or unsubstituted alkenyl, or C 2 -C substituted or unsubstituted alkynyl;
  • R 4 is H or OH;
  • R 6 is OH or OMe;
  • the invention provides pharmaceutical formulations and medicaments.
  • Such pharmaceutical formulations and medicaments include any of the compounds of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation or medicament includes any compound of formulae I - V and a pharmaceutically acceptable carrier.
  • the invention provides a method of treating a bacterial infection with compounds of the formulae I - V.
  • a method of treating a bacterial infection includes administering a compound of formulae I-V to a patient in need thereof.
  • the bacterial infection results from Gram positive bacteria, Gram negative bacteria or anaerobic bacteria.
  • the bacteria are Staphylococcus aureus, Streptococcus epidermidis, Strep, pneumoniae, Strep, pyogenes, enterococci, Moraxella catarrhalis or Haemophilus influenzae.
  • the bacterial infection is community-acquired pneumonia, acute exacerbated chronic bronchitis, acute sinusitis, tonsillitis/pharyngitis, upper respiratory tract infection, lower respiratory tract infection, skin infection, soft tissue infection, meningitis, hospital-acquired infection, bone infection or joint infection.
  • the invention provides a method of treating a gastric motility disease.
  • the method includes treating a patient with gastric motility disease with a compound of formula I.
  • the gastric motility disease is gastro-esophageal reflux disease (GERD), post-operative ileus, diabetes, or gastroparesis.
  • Figure 1 shows a variety of 15-amidoerythromycins prepared according to the methods of the invention.
  • Figure 2 shows some embodiments of additional 15-amidoerythromycins that can be prepared according to the methods of the invention.
  • Figure 3 shows a synthetic procedure that may be used to prepare certain macrolides according to the invention.
  • R 1 is as defined in the specification and can include any of the groups shown in Figures 1 and 2.
  • the present invention relates to erythromycin derivatives, intermediates thereto, and methods for their use in the treatment of disease.
  • inventive compounds possess antibacterial activity against Gram positive, Gram negative, and anaerobic bacteria, and are useful as broad-spectrum antibacterial agents for the treatment of bacterial infections in humans and animals. These compounds are effective against diverse strains including but not limited to Staphylococcus aureus, Streptococcus epiderniidis, Strep, pneumoniae, Strep, pyogenes, enterococci, Moraxella catarrhalis and Haemophilus influenzae.
  • Exemplary infections that may be treated include community-acquired pneumonia, acute exacerbated chronic bronchitis, acute sinusitis, tonsillitis/pharyngitis, upper and lower respiratory tract infections, skin and soft tissue infections, meningitis, hospital-acquired infections, and bone and joint infections.
  • Certain of the inventive compounds also possess prokinetic activity and are useful in the treatment of diseases of gastric motility, including but not limited to gastro-esophageal reflux disease (GERD), post-operative ileus, diabetes, and gastroparesis.
  • GSD gastro-esophageal reflux disease
  • crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention.
  • some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also encompassed within the scope of this invention.
  • the salts of the compounds of this invention refer to nontoxic "pharmaceutically acceptable salts.”
  • Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts.
  • Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, glucoheptonic acid, lactobionic acid, and dodecylsulfonic acid.
  • a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, glucoheptonic acid, lactobionic acid, and dode
  • the present invention includes within its scope prodrugs of the compounds of this invention.
  • prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound.
  • examples of prodrugs of the inventive compounds include but are not limited to 2 '-O-esters such as acetates, propionates, hemisuccinates, stearates, and the like.
  • the term "administering" shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.
  • substituents and substitution patterns on the compounds of this invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.
  • substituents include alkyl, alkenyl, alkynyl, aryl, halo, trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, alkanoyl, alkanoyloxy, amino, alkylamino, aralkylamino, cycloalkylamino, heterocycloamino, dialkylamino, alkanoylamino, thio, alkylthio, cycloalkylthio, heterocyclothio, ureido, nitro, cyano, carboxy, caroboxylalkyl, carbamyl, alkoxycarbonyl, alkylthiono, arylthiono, alkylsulfonyl, sulfonamindo, aryloxy, and the like, in addition to those otherwise specified herein.
  • the substituent may be further substituted, for example, by halo, hydroxy, al
  • arylalkylcarboxamido refers to a group of the formula
  • terapéuticaally effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • alkyl refers to straight or branched chain hydrocarbons, optionally substituted.
  • alkenyl refers to a straight or branched chain hydrocarbon with at least one carbon-carbon double bond.
  • Alkynyl refers to a straight or branched chain hydrocarbon with at least one carbon-carbon triple bound.
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • cycloalkyl refers to optionally substituted, saturated cyclic hydrocarbon ring systems, preferably containing 1 to 3 rings and 3 to 7 carbons per ring which may be further fused with an unsaturated C 3 -C carbocyclic ring.
  • Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cydohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl.
  • Exemplary substituents include one or more alkyl groups or one or more groups described above as alkyl substituents.
  • aryl refers to aromatic monocyclic, fused bicyclic, or fused polycyclic hydrocarbon or heterocyclic groups having 1 to 20 carbon atoms in the ring portions, such as phenyl, naphthyl, pyrrolyl, indolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadazolyl, isothiazolyl, furyl, thienyl, oxadiazolyl, pyridinyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrazinyl, triazinyl, triazolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, quinolinyl-
  • N-oxide isoquinolinyl, benzimidazolyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, benzisothiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzpyrazolyl, indolinyl, isochromanyl, isoindolinyl, naphthyridinyl, phthalazinyl, purinyl, quinazolinyl, and the like.
  • Aryl groups may be substituted.
  • biasing refers to a combination of two bonded, nonfused aryl groups as defined above.
  • exemplary biaryl groups include biphenyl, furylphenyl, phenylfuryl, thienylphenyl, phenylthienyl, pyridylphenyl, phenylpyridyl, furylpyridyl, pyridylfuryl, pyrrolylpyridyl, pyridylthienyl, isoxazolylthienyl, isoxazolylphenyl, and the like.
  • the above defined groups may be substituted by one or more substituents.
  • substituents include but are not limited to alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, hydroxy, alkoxy, haloalkoxy, cycloalkoxy, oxo, aryloxy, alkanoyl, alkanoyloxy, amino, alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, sulfonamido, carbonyl, carboxyl, alkoxycarbonyl, amidocarbonyl, oxoalkyl, cyano, nitro, carbamyl, guanidine, amidino, sulfonyl, and the like.
  • R 1 is Q-Q substituted or unsubstituted alkyl, C 2 -C 8 substituted or unsubstituted alkenyl, C 2 -C 8 substituted or unsubstituted alkynyl, C4-Q5 substituted or unsubstituted aryl, C 5 -C o substituted or unsubstituted arylalkyl, C 5 -C 20 substituted or unsubstituted biarylalkyl, C 5 -C 2 o substituted or unsubstituted arylalkenyl, or C 5 -C 20 substituted or unsubstituted arylalkynyl;
  • R 2 is H, C 1 -C 4 substituted or unsubstituted alkyl, C 2 -C 4 substituted or unsubstituted alkenyl, or Q-Q substituted or unsubstituted alkynyl;
  • R 3 is H, Q-Q alkanoyl,
  • R 1 is Q-Q 5 substituted or unsubstituted arylalkyl, C 6 -Q 5 substituted or unsubstituted biarylalkyl, Q-Q 5 substituted or unsubstituted arylalkenyl, or C 6 -C 15 substituted or unsubstituted arylalkynyl.
  • compounds of formula (I) are provided wherein R 1 is Q-Q 5 substituted or unsubstituted aryl, Q-Qo substituted or unsubstituted arylalkyl, or Q-Qosubstituted or unsubstituted biarylalkyl; and R 2 is H.
  • the azidoerythromycin is treated with an acylating reagent, for example acetic anhydride, propionic anhydride, benzoic anhydride, and the like, at ambient temperature in an inert solvent such as ethyl acetate, dichloromethane, or acetonitrile, to produce the 2'-O-acyl azidoerythromycin.
  • an acylating reagent for example acetic anhydride, propionic anhydride, benzoic anhydride, and the like
  • an inert solvent such as ethyl acetate, dichloromethane, or acetonitrile
  • the azide is then reduced using a phosphine such as trimethylphosphine or triphenylphosphine in a solvent such as tetrahydrofuran (THF) or a mixture of THF and dichloromethane, and the intermediate phosphinimine is reacted with a carboxylic acid, a carbodiimide such as l-[3-(dimethylamino)propyl]-2-ethylcarbodiimide hydrochloride (EDCI) or dicyclohexyl-carbodiimide, and a coupling adjuvant such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), or l-hydroxy-7- azabenzotriazole (HABt), to produce the 2'-O-acyl-amidoerythromycin.
  • a phosphine such as trimethylphosphine or triphenylphosphine in a solvent such as tetrahydr
  • the azidoerythromycin is treated with trimethylphosphine, and the resulting phosphinimine is reacted with a carboxylic acid and a coupling agent such as hexafluorophosphate (HBTU), O-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU), or a similar coupling agent which incorporates the coupling agent and adjuvant into one molecule.
  • HBTU hexafluorophosphate
  • HATU O-(7-azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate
  • the carboxylic acids used in the preparation of the inventive compounds include alkanoic acids, alkenoic acids, alkynoic acids, N-protected amino acids and/or peptides such as N-BOC, N-Cbz, and N-FMOC amino acids and/or peptides, benzoic acids, heterocyclic carboxylic acids, biaryl carboxylic acids, arylacetic acids, biarylacetic acids, and the like, each of which may be substituted by a variety of groups.
  • the carboxylic acids used in the preparation of the inventive compounds are biarylacetic acids.
  • One means of preparing these biarylacetic acids is via a Suzuki coupling as illustrated in Scheme 2.
  • a haloarylacetic ester is coupled with an arylboronic acid in the presence of a palladium catalyst and base to provide the biarylacetic ester.
  • the ester is subsequently saponified to provide the biarylacetic acid.
  • Suitable palladium catalysts include tetrakis(triphenylphosphine)palladium, palladium on carbon with triphenylphosphine, and similar sources of palladium(O).
  • the 2'-O-acyl azidoerythromycin is reacted with trimethylphosphine to generate the phosphinimine, which is then alkylated by reaction with an alkyl halide or alkyl sulfonate, R 2 X.
  • Suitable alkylating agents include but are not limited to methyl iodide, methyl bromide, methyl triflate, ethyl tosylate, ethyl triflate, ethyl iodide, allyl bromide, and propargyl bromide.
  • the resulting phosphonium salt is hydrolyzed, and the product amine is acylated with the carboxylic acid, R !
  • the method comprises the steps of (1) chemically synthesizing a racemic chlorinated diketide thioester; (2) growing a culture of a first organism in the presence of the racemic chlorinated diketide thioester so as to produce a chlorinated erythronolide; (3) partially purifying the chlorinated erythronolide; (4) chemically converting the chlorinated erythronolide into the azido erythronolide; (5) growing a culture of a second organism in the presence of the azido erythronolide, so as to produce an azidoerythromycin; and (6) purifying the azidoerythromycin.
  • a method for preparing 15- azidoerythromycin comprises the steps of (1) chemically synthesizing racemic (2S* 5R*)-5-chloro-3-hydroxy-2-methylpentanoyl N- propionylcysteamine thioester; (2) growing a culture of a first organism in the presence of racemic (2S* 3R*)-5-chloro-3-hydroxy-2-methylpentanoyl N- propionylcysteamine thioester so as to produce 15-chloro-6-deoxyerythronolide B; (3) partially purifying the 15-chloro-6-deoxy erythronolide B so produced; (4) chemically converting the 15-chloro-6-deoxyerythronolide B into 15-azido-6-deoxyerythronolide B; (5) growing a culture of a second organism in the presence of the 15-azido-6- deoxyerythronolide B, so as to produce
  • a method for preparing 14-azido-14- desmethylerythromycin comprises the steps of (1) chemically synthesizing racemic (2S* JR*)-4-chloro-3-hydroxy-2-methylbutanoyl N- propionylcysteamine thioester; (2) growing a culture of a first organism in the presence of racemic (2S* 3R*)-4-chloro-3-hydroxy-2-methylbutanoyl N- propionylcysteamine thioester so as to produce 15 -chloro-6-deoxy erythronolide B; (3) partially purifying the 14-chloro-14-desmethyl-6-deoxyerythronolide B so produced; (4) chemically converting the 14-chloro-14-desmethyl-6-deoxyerythronolide B into 14-azido-14-desmethyl-6-deoxy erythronolide B; (5) growing a culture of a second organism in the presence of the 14-azi
  • a method for preparing 15- (azidomethyl)-erythromycin comprises the steps of (1) chemically synthesizing racemic (2S* 3R*)-4-chloro-3-hydroxy-2-methylhexanoyl N- propionylcysteamine thioester; (2) growing a culture of a first organism in the presence of racemic (2S*, 5R*)-4-chloro-3-hydroxy-2-methylhexanoyl N- propionylcysteamine thioester so as to produce 15-(chloromethyl)-6- deoxyerythronolide B; (3) partially purifying the 15-(chloromethyl)-6- deoxyerythronolide B so produced; (4) chemically converting the 15-(chloromethyl)- 6-deoxyerythronolide B into 15-(azidomethyl)-6-deoxyerythronolide B; (5) growing a culture of a second organism in the presence of the 15-(a
  • the thioester so produced is (2S* 3R*)-5-chloro-3-hydroxy-2-methylpentanoate N- propionylcysteamine thioester.
  • the aldehyde used is chloroacetaldehyde
  • the thioester so produced is (2S* 3R*)-4-chloro-3-hydroxy-2-methylbutanoate N- propionylcysteamine thioester.
  • the aldehyde used is 4-chlorobutyraldehyde
  • the thioester so produced is (2S* 3R*)-6-chloro-3-hydroxy-2-methylhexanoate N- propionylcysteamine thioester.
  • the preparation of 13-substituted 6-deoxyerythronolide B compounds is described in Khosla et al, U.S. Patent Nos. 6,080,555, 6,274,560, 6,066,721, and 6,261,816; and Ashley et al, U.S. Patent 6,492,562; each of which is incorporated herein by reference.
  • 15-chloro-6-deoxyerythronolide B is prepared by a method in which racemic (2S* 3R*)-5-chloro-3-hydroxy-2- methylpentanoyl N-propionylcysteamine thioester is provided to a 6- deoxyerythronolide B synthase ("DEBS") that is unable to act on its natural substrate, propionyl CoA, due to a mutation in the ketosynthase domain of module 1 of DEBS.
  • DEBS 6- deoxyerythronolide B synthase
  • This recombinant DEBS can be expressed in the natural host that normally produces erythromycin, Saccharopolyspora erythraea, or the entire PKS gene cluster can be inserted by plasmid in a suitable host such as S. coelicolor (see e.g., Jacobsen et al, Science 277: 367-369 (1997), incorporated herein by reference) or S. lividans which has been modified to delete its endogenous actinorhodin polyketide synthesis mechanism.
  • the host is S. coelicolor CH999/pJRJ2, which expresses a mutant 6-DEB synthase having an inactivated module 1 ketosynthase.
  • a cell free system as described in Khosla et al, "Synthesis of polyketides from diketides," U.S. Patent No. 6,080,555; Khosla et al, “Cell-free synthesis of polyketides,” U.S. Patent 6,274,560; and PCT Publication No. WO 97/02358, each of which is incorporated herein by reference, may also be employed by producing the relevant PKS proteins recombinantly and effecting their secretion or lysing the cells containing them.
  • a typical cell-free system would include the appropriate PKS, NADPH and an appropriate buffer and substrates required for the catalytic synthesis of polyketides.
  • the appropriate thioester diketide substrates are provided to PKS enzymes other than the 6-dEB synthase of Saccharopolyspora erythraea.
  • PKS enzymes include the 6-dEB synthase of Micromonospora megalomicea and its KS1° derivative described in McDaniel &
  • the appropriate thioester diketide substrates are provided to any of the above PKS enzymes in a host cell that is capable of performing one or more of the post-PKS hydroxylation and/or glycosylation steps leading to the erythromycins.
  • a chlorinated erythronolide B is prepared by providing a chlorinated thioester diketide to a strain comprising both DEBS and a 6- hydroxylase, for example the eryF hydroxylase from Sac. erythraea.
  • the production of 14-chloro-14-desmethylerythronolide B is described in Example 72 below.
  • 15-chloro-6-deoxyerythronolide B The preparation of 15-chloro-6-deoxyerythronolide B is detailed in Example 2 below. Using the same methods but starting with the appropriate diketide thioesters described above, 14-chloro-14-desmethyl-6-deoxy erythronolide B and 15- (chloromethyl)-6-deoxyerythronolide B are prepared as described below in Examples 71 and 73.
  • the chlorinated erythronolide so produced is reacted with sodium azide and sodium iodide in DMSO so as to produce the azido erythronolide by displacement of the chloride with azide. This is detailed in the Examples below.
  • 15-azido-6-deoxyerythronolide B is prepared from 15- chloro-6-deoxyerythronolide B (Example 3)
  • 14-azido-14-desmethyl-6- deoxyerythronolide B is prepared from 14-chloro-14-desmethyl-6-deoxyerythronolide B (Example 74)
  • 15-(azidomethyl)- 6-deoxyerythronolide B is prepared from 15- (chloromethyl)- 6-deoxyerythronolide B (Example 75)
  • 15-azido-erythronolide B is prepared from 15-chloro-erythronolide B
  • 14-azido-14-desmethyl-erythronolide B is prepared from 14-chloro-14-desmethyl-erythronolide B
  • 15-(azidomethyl)- erythronolide B is prepared from 15-(chloromethyl)-erythronolide B.
  • the azidoerythronolide prepared according to one of the above methods is then added to a culture of a bioconversion organism capable of glycosylating at the C-3 and C-5 positions, and optionally hydroxylating at C-6, hydroxylating at the C-12 position and/or methylating a 3-O- mycarosyl group, depending on the strain and fermentation conditions employed.
  • the bioconversion organism is Saccharopolyspora erythraea K39-14V, a strain in which the module 1 ketosynthase of the native DEBS genes has been inactivated as described in Santi et al., "Hosts for the biosynthesis of polyketides," PCT publication WO 01/83803, incorporated herein by reference.
  • K39- 14V contains the enzyme activities needed to convert an erythronolide into the erythromycin A derivative, i.e., hydroxylation at C-6, addition of mycarose to the 3- OH, addition of desosamine to the 5-OH, hydroxylation at C-12, and methylation of the mycarose.
  • K39-14V is supplied with 15- azido-6-deoxyerythronolide B under fermentation conditions wherein 15- azidoerythromycin A is the predominant product. This is detailed below in Example 4.
  • K39-14V is supplied with 15-azido-6- deoxyerythronolide B under fermentation conditions wherein 15-azidoerythromycin B is the predominant product, for example under conditions of limited oxygen.
  • K39-14V is supplied with 14-azido-14- desmethyl-6-deoxyerythronolide B under fermentation conditions wherem 14-azido- 14-desmethylerythromycin A is produced.
  • K39-14V is supplied with 14-azido-14-desmethyl-6-deoxyerythronolide B under fermentation conditions wherein 14-azido-14-desmethylerythromycin B is produced (Example 76).
  • K39-14V is supplied with 15- (azidomethyl)-6-deoxyerythronolide B under fermentation conditions wherein 15- (azidomethyl)-erythromycin A is produced.
  • K39-14V is supplied with 15 -(azidomethyl)-6-deoxy erythronolide B under fermentation conditions wherein 15-(azidomethyl)-erythromycin B is produced (Example 77).
  • a bioconversion strain that lacks an active 12-hydroxylase, encoded by the eryK gene or a homologue, is used to convert the azido-6-deoxyerythromycin into the azidoerythromycin B.
  • the azidoerythromycin B compounds are produced using a mutant of K39-14V wherein the eryK gene encoding the 12-hydroxylase has been inactivated or deleted.
  • the resulting azidoerythromycins are isolated as described for 15- azidoerythromycin A in Example 4 below.
  • the cells are removed from the fermentation by centrifugation, and the broth is extracted to remove the azidoerythromycins.
  • the extraction is performed using absorption onto a solid resin, such as XAD or HP20.
  • the azidoerythromycins are subsequently eluted from the resin with an organic solvent such as methanol, ethanol, acetone, acetonitrile, or ethyl acetate.
  • the extraction is performed by mixing the broth with an immiscible organic solvent in which the azidoerythromycin is soluble, such as ethyl acetate, dichloromethane, or chloroform. After concentration of the extract, the residue is subjected to chromatography to provide purified azidoerythromycins.
  • an immiscible organic solvent in which the azidoerythromycin is soluble such as ethyl acetate, dichloromethane, or chloroform.
  • these compounds are prepared from azidoerythromycins as illustrated in Scheme 4 and exemplified in Example 80.
  • the azidoerythromycin is first treated with hydroxylamine, typically in the presence of an acid catalyst such as acetic acid and in a mixture of water and isopropanol as solvent, to produce the 9-oxime.
  • This procedure is exemplified in detail with 15- azidoerythromycin A in Example 48 below.
  • the 9-oxime is protected, for example using an acetal such as 1,1-diisopropoxycyclohexane (DIPCH) or 2,2- dimethoxypropane in the presence of a mild acid catalyst such as pyridinium p- toluenesulfonate (PPTS).
  • DIPCH 1,1-diisopropoxycyclohexane
  • PPTS pyridinium p- toluenesulfonate
  • Other protecting groups such as trialkylsilyl ethers, can also be used to protect the 9-oxime.
  • the 2'- and 4" -OH groups are next protected, for example as trimethylsilyl ethers by treatment with a mixture of trimethylsilylimidazole and chlorotrimethylsilane, as described below in Example 50.
  • This process is exemplified below in Example 51.
  • a palladium catalyst for example palladium acetate and triphenylphosphine
  • the 9-oxime and 2'- and 4"-OH groups are deprotected by treatment with acetic acid in acetonitrile.
  • R 3 , R 4 , and m are as defined above;
  • R 5 is H, Q-Q alkoxy, Q-Q alkenyloxy, or Q-Q alkynyloxy; and
  • R 8 is H or F.
  • the cladinose is removed from the compound of formula (I) by treatment with a mild acid in the presence of water, for example using PPTS in a mixture of acetone and water as exemplified below in Example 54, or using aqueous HCl.
  • a mild acid for example using PPTS in a mixture of acetone and water as exemplified below in Example 54, or using aqueous HCl.
  • the 3-OH is oxidized to the ketone (Example 56).
  • Suitable oxidation methods include but are not limited to the Corey-Kim (N- chlorosuccinimide, dimethylsulfide, triethylamine), Swern (oxalyl chloride, DMSO, triethylamine), Moffat (dicyclohexylcarbodiimide, DMSO), and modified Pfizer- Moffat (l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide, pyridinium trifluoroacetate, DMSO) conditions.
  • NFBS N- fluorobenzenesulfonimide
  • R 1 , R 2 , R 3 , R 4 , R 8 , and m are as defined above and R 5 is H, Q-Q alkoxy, Q- Q alkenyloxy, or Q-Q alkynyloxy.
  • compounds of formula (III) are provided wherein R 1 is C 6 -C 15 substituted or unsubstituted arylalkyl, Q-Q5 substituted or unsubstituted biarylalkyl, Q-Q 5 substituted or unsubstituted arylalkenyl, or Q-Q 5 substituted or unsubstituted arylalkynyl.
  • R 1 is C 6 -C 15 substituted or unsubstituted arylalkyl, Q-Q5 substituted or unsubstituted biarylalkyl, Q-Q 5 substituted or unsubstituted arylalkenyl, or Q-Q 5 substituted or unsubstituted arylalkynyl.
  • R 2 is
  • R 5 is alkoxy are provided.
  • R 1 is Q-Q 5 substituted or unsubstituted aryl, Q-Qosubstituted or unsubstituted arylalkyl, or Q-Qosubstituted or unsubstituted biarylalkyl;
  • R 2 is H; and
  • R 5 is alkoxy.
  • the compound of formula (II) is treated with trimethylphosphine, and the resulting phosphinimine is reacted with a carboxylic acid, a coupling agent such as a carbodiimide, and a coupling adjuvant such as HOBt, HABt, or HOSu as discussed above and exemplified below in Example 65.
  • a coupling agent such as a carbodiimide
  • a coupling adjuvant such as HOBt, HABt, or HOSu as discussed above and exemplified below in Example 65.
  • the compound of formula (II) is treated with trimethylphosphine, and the resulting phosphinimine is reacted with a carboxylic acid and a coupling agent such as O-benzotriazol-l-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-l-yl)- N,N,N',N' -tetramethyluronium hexafluorophosphate (HATU), or a similar coupling agent which incorporates the coupling agent and adjuvant into one molecule.
  • HBTU O-benzotriazol-l-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate
  • HATU O-(7-azabenzotriazol-l-yl)- N,N,N',N' -tetramethyluronium hexaflu
  • R benzoyl
  • Suitable alkylating agents include but are not limited to methyl iodide, methyl bromide, methyl triflate, ethyl tosylate, ethyl triflate, ethyl iodide, allyl bromide, and propargyl bromide.
  • compounds of formula (IV) wherein R 1 , R 2 , R 3 , R 8 , and m are as defined above and R 5 is H, Q-Q alkoxy, Q-Q alkenyloxy, or Q-Q alkynyloxy, and X is O or NR 7 , wherein R 7 is H, Q-Q alkyl, or C 6 -C 20 arylalkyl.
  • R 1 is C 6 -Q 5 substituted or unsubstituted arylalkyl, Q-Q 5 substituted or unsubstituted biarylalkyl, C 6 -C 15 substituted or unsubstituted arylalkenyl, or Q-Q 5 substituted or unsubstituted arylalkynyl.
  • R 1 is Q-Q 5 substituted or unsubstituted aryl, Q-C 20 substituted or unsubstituted arylalkyl, Q-Qo substituted or unsubstituted biarylalkyl, Q-C 20 substituted or unsubstituted arylalkenyl, or Q-Qo substituted or unsubstituted arylalkynyl; and R 2 is H.
  • R 1 is Q-Q 5 substituted or unsubstituted aryl, Q-C 20 substituted or unsubstituted arylalkyl, Q-Qn substituted or unsubstituted biarylalkyl, Q-C 20 substituted or unsubstituted arylalkenyl, or Q-Qo substituted or unsubstituted arylalkynyl;
  • R 2 is H; and
  • X is NH.
  • Example 57 A representative procedure for mesylation is given in Example 57.
  • the 11-O-mesylate is treated with 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) to produce the 10,11-anhydrocompound, as detailed in Example 58.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
  • a representative procedure for fluorination is given in Example 60.
  • 1,1- carbonyldiimidazole and a strong base for example sodium bis(trimethylsilyl)amide (NaHMDS) in an inert solvent such as tetrahydrofuran at a temperature between - 10°C and 30 °C
  • an amine for example ammonia
  • potassium tert-butoxide provides the 11,12-cyclic carbamate.
  • the cladinose is removed, for example by treatment with aqueous acid, and the resulting 3-OH is subjected to hydrogenolysis to reduce the azide to the amine.
  • Hydrogenolysis is performed in a solvent such as methanol in the presence of a metal catalyst, such as palladium or palladium supported on an inert material such as charcoal, and an acid, such as HCl, acetic acid, or similar, under a moderate pressure of hydrogen gas, such as that provided by a hydrogen balloon.
  • the acid may be generated in situ, for example by addition of chlorotrimethylsilane to methanol.
  • the resulting ammonium salt is condensed with a carboxylic acid R ⁇ OOH as described above.
  • the 3-OH is oxidized to the 3-oxo group, for example using the Dess-Martin periodinane, Swern oxidation, Corey-Kim oxidation, or similar.
  • the 2' -protecting group is removed by methanolysis. This procedure is exemplified in detail in Example 81 below.
  • a representative procedure for debenzoylation is given in Example 62.
  • R 1 , R 2 , R 4 , R 6 and m are as defined above; and R 9 is H or Q-Q alkyl.
  • compounds of formula (V) are provided wherein R 1 is Q-Q 5 substituted or unsubstituted arylalkyl, C 6 -C 15 substituted or unsubstituted biarylalkyl, Q-Q 5 substituted or unsubstituted arylalkenyl, or Q-Q 5 substituted or unsubstituted arylalkynyl.
  • R 1 is Q-Q 5 substituted or unsubstituted aryl, Q-Qo substituted or unsubstituted arylalkyl, or Q-Qo substituted or unsubstituted biarylalkyl.
  • the oximinoether is then reduced, for example using sodium borohydride, under conditions wherein the azide group remains intact.
  • the resulting azalide is then alkylated, for example methylated using formaldehyde and formic acid as described for the production of azithromycin in Kobrehel & Djokic, U.S. Patent No. 4,517,359, incorporated herein by reference, to produce the azidoazalide.
  • the azidoazalide is converted into an embodiment of the compounds of formula (V) as illustrated in Scheme 12.
  • the 2'-OH is protected, for example as the acetate or benzoate, and the azide is reduced by treatment with a phosphine, for example trimethylphosphine or triphenylphosphine, and the amide is formed by condensation with carboxylic acid R ! COOH as described above.
  • the 2' -protecting group is removed by methanolysis.
  • the azidoazalide is converted into an embodiment of the compounds of formula (V) by protecting the 2'-OH as the acetate or benzoate, reducing the azide to the amine by hydrogenolysis and forming the amide by condensation of the aminoazalide with R ! COOH as described above.
  • This invention further provides a method of treating bacterial infections, or enhancing the activity of other anti-bacterial agents, in warm-blooded animals, which comprises administering to the animals a compound of the invention alone or in admixture with a diluent or in the form of a medicament according to the invention.
  • the compounds When the compounds are employed for the above utility, they may be combined with one or more pharmaceutically acceptable carriers, e.g., solvents, diluents, and the like, and may be administered orally in such forms as tablets, capsules, dispersible powders, granules, or suspensions containing for example, from about 0.5% to 5% of suspending agent, syrups containing, for example, from about 10% to 50% of sugar, and elixers containing, for example, from about 20% to 50% ethanol, and the like, or parenterally in the form of sterile injectable solutions or suspensions containing from about 0.5% to 5% suspending agent in an isotonic medium.
  • These pharmaceutical preparations may contain, for example, from about 0.5% up to about 90% of the active ingredient in combination with the carrier, more usually between 5% and 60% by weight.
  • compositions for topical application may take the form of liquids, creams or gels, containing a therapeutically effective concentration of a compound of the invention admixed with a dermatologically acceptable carrier.
  • any of the usual pharmaceutical media may be employed.
  • Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include sterile water, polyethylene glycols, non-ionic surfactants and edible oils such as corn, peanut and sesame oils, as are appropriate to the nature of the active ingredient and the particular form of administration.
  • Adjuvants customarily employed in the preparation of pharmaceutical compositions may be advantageously included, such as flavoring agents, coloring agents, preserving agents, and antioxidants, for example, vitamin E, ascorbic acid, BHT and BHA.
  • the active compounds may also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropyl-cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
  • the effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration and the severity of the condition being treated. However, in general, satisfactory results are obtained when the compounds of the invention are administered at a daily dosage of from about 0.1 mg/kg to about 400 mg/kg of animal body weight, preferably given once a day, or in divided doses two to four times a day, or in sustained release form. For most large mammals, including humans, the total daily dosage is from about 0.07 g to 7.0 g, preferably from about 100 mg to 1000 mg.
  • Dosage forms suitable for internal use comprise from about 100 mg to 500 mg of the active compound in intimate admixture with a solid or liquid pharmaceutically acceptable carrier. This dosage regiment may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • compositions and medicaments are carried out by any method known in the art, for example, by mixing the active ingredient(s) with the diluent(s) to form a pharmaceutical composition (e.g., a granulate) and then forming the composition into the medicament (e.g., tablets).
  • a pharmaceutical composition e.g., a granulate
  • the medicament e.g., tablets
  • a seed culture of Streptomyces coelicolor CH999/pJRJ2 was made by inoculating 1 mL of frozen mycelium into a 250 mL baffled flask containing 50 mL of FKA medium (corn starch, 45 g L; corn steep liquor, 10 g/L; dried, inactivated brewers yeast, 10 g/L; and CaCO 3 , 1 g/L), 0.050 mL of 50 mg/mL thiostrepton in DMSO (filter sterilized), and 0.500 mL of 50% Antifoam B.
  • the flask was incubated at 30 °C with shaking at 175 rpm for 48 hours (Innova floor shaker).
  • the culture was transferred into a 2.8-L baffled flask containing 500 mL of FKA medium, 0.500 mL of 50 mg/mL thiostrepton in DMSO, and 5 mL of 50% Antifoam B, and the flask was incubated at 30 °C with shaking at 175 rpm for 48 hours.
  • a 10-L stirred tank bioreactor (B. Braun) was autoclaved, filled with 5 L of sterile FKA medium and 5 mL of 50 mg/mL thiostrepton in DMSO, and then inoculated with 500 mL (10%) of seed culture.
  • the bioreactor was run for 24 hours at 30 °C with stirring at 600 rpm, sparged with air at 1.33 LPM, and the pH was maintained at 6.50 by automated addition of 2.5 N NaOH and 2.5 N H 2 SO 4 .
  • Three liters of the above culture was used to inoculate a 100-L bioreactor containing 55 L of sterile FKA medium with 2 g/L Tastone 310 added prior to sterilization.
  • the fermentor agitation rate was set at a tip speed of 2.5 m s, the temperature was maintained at 30°Q the pH was controlled at pH 6.5 by automated addition of 2.5 N NaOH and 2.5 N H 2 SO 4 , and the airflow was set at 0.4 vvm.
  • Foaming was controlled by automated addition of 50% Antifoam B.
  • the dissolved oxygen was maintained at > 50% air saturation by cascade control using the agitation rate (tip speed of 2.5-3.0 m/s) and back pressure (0.1-0.4 bar) in that order.
  • the 15-chloro-6-deoxyerythronolide B was isolated by solid phase extraction.
  • the broth was clarified by centrifugation and loaded onto a column containing HP-20 resin (Rohm and Haas) at a concentration of 1 L resin/20 g 15-chloro-6- deoxyerythronolide B.
  • the column was then equilibrated with 5 column volumes of water at a flow rate of 2-4 mL/cm 2 -min.
  • the loaded resin was washed with 2 column volumes of water followed by 2 column volumes of 30% methanol in water.
  • the 15- chloro-6-deoxyerythronolide B was eluted from the resin with methanol.
  • the fractions containing 15-chloro-6-deoxy-erythronolide B were identified by HPLC with ELSD detection.
  • the methanol fractions containing 15-chloro-6-deoxyerythronolide B were pooled, and the volatiles were removed under reduced pressure.
  • the dried solids were extracted with 3-5 L of methanol and filtered to yield a solution containing 6-10 mg/mL 15-chloro-6-deoxyerythronolide B, which was diluted with an equal volume of water.
  • This solution was loaded onto a column of HP20SS (1 L resin/20 g of 15- chloro-6-deoxyerythronolide B), which was then washed with 2 column volumes of 50% aqueous methanol.
  • the 15-chloro-6-deoxyerythronolide B was then eluted with 70% methanol in water, and the fractions were analyzed by HPLC.
  • a seed culture of Saccharopolyspora erythraea K39-14V was made by inoculating a 1 mL aliquot of frozen mycelium into each of three 250 mL baffled flasks containing 50 mL of VI medium (com starch, 16 g/L; com dextrin, 10 g/L; soya meal flour, 15 g/L; com steep liquor, 5 g/L; soy bean oil, 6 g/L; sodium chloride, 2.5 g/L; ammonium sulfate, 1 g/L; and CaCO , 4 g/L) and 0.100 mL of Antifoam B.
  • VI medium com starch, 16 g/L; com dextrin, 10 g/L; soya meal flour, 15 g/L; com steep liquor, 5 g/L; soy bean oil, 6 g/L; sodium chloride, 2.5 g/L; ammonium sulfate
  • the flasks were incubated at 34 °C with shaking at 175 rpm for 48 hours (Innova floor shaker). Each culture was transferred into a 2.8-L baffled flask containing 500 mL of VI medium and 1 mL of Antifoam B, and the flasks were incubated at 34 °C with shaking at 175 rpm for 48 hours.
  • the fermentor agitation rate was set at a tip speed of 2-4 m/s, the pH was controlled at pH 7.0 by automated addition of 2.5 N NaOH and 2.5 N H 2 SO 4 , the temperature was maintained at 34°C, and the airflow was set at 0.15 vvm. Foaming was controlled by automated addition of 50% Antifoam B.
  • Each fermentor was inoculated with a 500- mL seed culture prepared above. During the fermentation, the dissolved oxygen was maintained at > 80% air saturation by cascade control using agitation rate (tip speed of 2-4 m/s)airflow (0.15-0.5 vvm), and oxygen enrichment in that order.
  • the 15-azido-erythromycin A was isolated by solid phase extraction.
  • the broth was adjusted to pH 9 using 2.5 N NaOH, clarified by centrifugation, and loaded onto a column containing HP-20 resin (Rohm and Haas) at a concentration of 1 L resin/20 g 15-azidoerythromycin A.
  • the column was then equilibrated with 5 column volumes of water at a flow rate of 2-4 mL/cm 2 -min.
  • the loaded resin was washed with 2 column volumes of water.
  • the 15-azido-erythromycin A was eluted from the resin with 5 column volumes of methanol.
  • the fractions containing 15-azidoerythromycin A were identified by HPLC, pooled, and the volatiles were removed under reduced pressure.
  • the dried solids were mixed with 800 mL of acetone and 3.2-L of hexane for 20 minutes. The mixture was then filtered using a #4 Whatman filter paper. The solids were extracted twice in this manner, and the filtrates were combined and evaporated.
  • the crude product was dissolved in methanol and diluted with an equal volume of water. This solution was loaded onto a column of HP20SS (1 L resin 20 g of 15-azido-erythromycin A), which was then washed successively with 1 column volume of 50% aqueous methanol, 3 column volumes of 3:2 methanol/water, 3 column volumes of 7:3 methanol/water, 10 column volumes of 4:1 methanol/water, and finally 5 column volumes of 100% methanol. The fractions were analyzed by HPLC. Product-containing fractions were pooled and evaporated to dryness to yield 13 g of 15-azidoerythromycin A of 95% purity.
  • the solution is stirred at room temperature for 45 minutes before transferring to a solution of the carboxylic acid (0.092 mmol, 1.5 eq), l-[(3-(dimethylamino)propyl]-l-ethylcarbodiimide hydrochloride (0.019 g, 0.099 mmol, 1.6 eq) and 1-hydroxybenzotriazole (0.017 g, 0.124 mmol, 2.0 eq) in dichloromethane or tetrahydrofuran (1.0 ml) also stirred at room temperature for 45 minutes. The resulting solution is stirred at room temperature for 3 to 14 hours before partitioning between ethyl acetate (10 ml) and NaHCO3 (10 ml).
  • aqueous phase is extracted with ethyl acetate (3 x 10 ml) and the combined organics further washed with brine (25 ml) before drying (Na2SO4), filtering, and concentrating under reduced pressure.
  • the residue is dissolved in methanol (2 ml) and stirred at 50 °C for 14 hours before concentrating under reduced pressure.
  • This compound was prepared according to the method of Example 6 using 4- phenylbutyric acid.
  • This compound was prepared according to the method of Example 6 using benzyloxyacetic acid.
  • This compound was prepared according to the method of Example 6 using (3,4,5-trimethoxybenzoyl)formic acid.
  • This compound was prepared according to the method of Example 6 using (4- methyl-5-phenyloxazol-2-yl)acetic acid.
  • This compound was prepared according to the method of Example 6 using quinoline-3-carboxylic acid.
  • This compound was prepared according to the method of Example 6 using pyridine-3-carboxylic acid.
  • This compound was prepared according to the method of Example 6 using indole-3 -carboxylic acid.
  • This compound was prepared according to the method of Example 6 using 4- biphenylacetic acid.
  • This compound was prepared according to the method of Example 6 using 3- (3-furyl)phenylacetic acid.
  • This compound was prepared according to the method of Example 6 using 3- (3-thienyl)phenylacetic acid.
  • This compound was prepared according to the method of Example 6 using 2- (2-thienyl)phenylacetic acid.
  • This compound is prepared according to the method of Example 6 using 3-(4- pyridyl)phenylacetic acid.
  • This compound is prepared according to the method of Example 6 using 3-(2- furyl)pyridyl-5-acetic acid.
  • This compound is prepared according to the method of Example 6 using 3-(2- thienyl)-pyridyl-5-acetic acid.
  • This compound is prepared according to the method of Example 6 using 3-(2- pyrrolyl)-pyridyl-5 -acetic acid.
  • This compound is prepared according to the method of Example 6 using 5- phenylthienyl-2-acetic acid.
  • This compound is prepared according to the method of Example 6 using 5-(2- pyridyl)-thienyl-2-acetic acid.
  • This compound is prepared according to the method of Example 6 using 5-(3- isoxazolyl)-thienyl-2-acetic acid.
  • This compound is prepared according to the method of Example 6 using 3-(5- (2-pyridyl)thien-2-yl)propionic acid.
  • IPCH ketal (4.2 g), 2 M methyl bromide in ether (3.9 mL), 7 mL of THF, and 7 mL of DMSO was cooled on ice.
  • N-Chlorosuccinimide (0.191 g, 1.427 mmol, 1.5 eq) was added to a solution of dimethyl sulfide (0.085 ml, 1.712 mmol, 1.8 eq) and dichloromethane (5 ml) at -15 °C.
  • the solution was stirred at -15 °C for 15 minutes before adding a solution of 1,1-carbonyldiimidazole (0.167 g, 1.032 mmol, 3.0 eq) in tetrahydrofuran (3.0 ml) dropwise.
  • the solution was stirred at -15 °C for 15 minutes before warming to room temperature over a period of 40 minutes. Sat. aq. NaHCO3 (25 ml) was added followed by ethyl acetate (50 ml), and the solution was partitioned. The organics were further washed with sat. aq.
  • the solution is stirred at room temperature for 45 minutes before transferring to a solution of the carboxylic acid (0.092 mmol, 1.5 eq), l-[(3-(dimethylamino)propyl]-l- ethylcarbodiimide hydrochloride (0.019 g, 0.099 mmol, 1.6 eq) and 1- hydroxybenzotriazole (0.017 g, 0.124 mmol, 2.0 eq) in dichloromethane or tetrahydrofuran (1.0 ml) also stirred at room temperature for 45 minutes. The resulting solution is stirred at room temperature for 14 hours before partitioning between ethyl acetate (10 ml) and NaHCO 3 (10 ml).
  • the aqueous phase is extracted with ethyl acetate (3 10 ml) and the combined organics further washed with brine (25 ml) before drying (Na 2 SO 4 ), filtering, and concentrating under reduced pressure.
  • the product is purified by silica gel chromatography using acetone/hexane + 1% Et 3 N.
  • the solution is stirred at room temperature for 45 minutes before transferring to a solution of the carboxylic acid (0.092 mmol, 1.5 eq), l-[(3- (dimethylamino)propyl]-l-ethylcarbodiimide hydrochloride (0.019 g, 0.099 mmol, 1.6 eq) and 1-hydroxybenzotriazole (0.017 g, 0.124 mmol, 2.0 eq) in dichloromethane or tetrahydrofuran (1.0 ml) also stirred at room temperature for 45 minutes. The resulting solution is stirred at room temperature for 14 hours before partitioning between ethyl acetate (10 ml) and NaHCO3 (10 ml).
  • the aqueous phase is extracted with ethyl acetate (3 x 10 ml) and the combined organics further washed with brine (25 ml) before drying (Na2SO4), filtering, and concentrating under reduced pressure.
  • the product is purified by silica gel chromatography using acetone/hexane + 1% Et 3 N.
  • the solution is stirred at room temperature for 45 minutes before transferring to a solution of the carboxylic acid (0.092 mmol, 1.5 eq), l-[(3-(dimethylamino)propyl]-l-ethylcarbodiimide hydrochloride (0.019 g, 0.099 mmol, 1.6 eq) and 1-hydroxybenzotriazole (0.017 g, 0.124 mmol, 2.0 eq) in dichloromethane or tetrahydrofuran (1.0 ml) also stirred at room temperature for 45 minutes. The resulting solution is stirred at room temperature for 14 hours before partitioning between ethyl acetate (10 ml) and NaHCO3 (10 ml).
  • the aqueous phase is extracted with ethyl acetate (3 x 10 ml) and the combined organics further washed with brine (25 ml) before drying (Na2SO4), filtering, and concentrating under reduced pressure.
  • the product is purified by silica gel chromatography using acetone/hexane + 1% Et 3 N.
  • step (b) Oxime protection.
  • the crude oxime from step (a) above (34.0 g, 42.5 mmol) was suspended in 120 ml of CH 2 C1 2 and treated with 1,1 -diisopropoxycyclohexane (51.0 ml, 246.8 mmol, 6 eq.) and pyridinium p-toluenesulfonate (24.8 g, 98.8 mmol, 2 eq.) for 15 hours at ambient temperature.
  • the mixture was diluted with 600 ml of CH C1 2 , and then washed sequentially with saturated NaHCO , water, and brine.
  • the organic phase was dried with MgSO 4 , filtered, and evaporated to yield brown syrup.
  • the temperature of the mixture was lowered to -15 C, and liquid NH3 was added in by condensation of anhydrous ammonia gas using a dry ice condenser.
  • the mixture was capped and stirred for 2h at -15 C.
  • Liquid NH3 was dripped in above o mixture again before the mixture was capped, and stirred for another 2h at 0 C.
  • the mixture was capped and stirred at 0 C for another lh after liquid NH3 was dripped in the mixture for the third time.
  • 15 ml of a 1 N solution of potassium tert-butoxide in THF 15 mmol, 1.2 eq.
  • step (c) Cladinose removal.
  • the crude product from step (b) was dissolved in 120 ml of EtOH and 120 ml of 2N aqueous HCl, and the resulting solution was heated at
  • EXAMPLE 82 2'-O-benzoyl-6-O-methyl-3-descladinosyl-ll-amino-ll-deoxy-3-15- aminoerythromycin A 11,12-cyclic carbamate hydrochloride 400 mg of 10% activated palladium on carbon was weighed out into a round bottom flask under nitrogen, and 50 ml of methanol was added in slowly followed by sequential addition of 400 mg of 2'-O-benzoyl-6-O-methyl-3-descladinosyl-ll- amino-ll-deoxy-3-15-azidoerythromycin A 11,12-cyclic carbamate (0.53 mmol) (Example 81) and 0.13 mL (2 eq.) of trimethylsilyl chloride.
  • step (b) The compound from step (b) above was dissolved in 2 ml of methanol and heated at 70 C under stirring overnight. Evaporation of the methanol under vacuum gave a yellow solid, which was purified by flash column to provide final 15- amidoketolide.
  • step (e) The compound from step (b) above was dissolved in 2 ml of methanol and heated at 70 C under stirring overnight. Evaporation of the methanol under vacuum gave a yellow solid, which was purified by flash column to provide final 15- amidoketolide.
  • This compound was prepared according to the methods of Example 83, using
  • This compound was prepared according to the methods of Example 83, using 2-([l ,2,4]-tetrazol-l-yl)pyrid-5-ylacetic acid.
  • step (c) 3-(2-furanyl)phenylacetic acid.
  • a solution of the methyl 3-(2- furanyl)phenylacetate from step (b) in 5 ml of methanol was added 10 ml of IN NaOH aqueous solution, the resulting solution was stirred overnight before diluting with 40 of ml IN NaOH aqueous solution and washing with 2 X 30 ml CH 2 C1 2 .
  • the aqueous solution was then acidified with 2N HCl, and extracted with 3 X 30 ml of CH2CI 2 .
  • the organic extracts were combined and were dried with MgSO 4 , filtered, and evaporated to give 0.32 g of the product as a golden-colored solid.
  • Compounds prepared according to this general procedure include: (a) 3-quinolylacetic acid, using 3-bromoquinoline; and (b) 2-([l,2,4]-triazol-l-yl) ⁇ yrid-5-yl-acetic acid, using 2-([l,2,4]-tetrazol-l- yl)- 5-bromo-pyridine.
  • the resulting solution was heated at 70 C for 30 min before it was diluted with 20 ml of water, and extracted with 3 X 30 ml of ethyl acetate. The organic extracts were combined and washed sequentially with water, and brine. The organic phase was dried with MgSO 4 , filtered, and evaporated to give 60 mg of a slight yellow solid.
  • MICs Minimum inhibitory concentrations
  • NCCLS broth microdilution procedure for susceptibility testing for bacteria that grow aerobically (National Committee for Clinical Laboratory Standards, 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4 th ed. Approved standard.
  • Stock solutions were prepared on the day of the test and appropriate aliquots were added to cation adjusted Mueller-Hinton broth (CAMHB) or Haemophilus test media. Two-fold serial dilutions were prepared and added to wells in microtiter plates. Final test concentrations ranged from 16 to 0.015 ug/ml. Broth cultures of bacteria inoculated from growth on overnight plates for all test bacteria except Streptococcus pneumoniae and Haemophilus influenzae were incubated at 35°C and then adjusted to the Kirby Bauer standard and diluted in
  • CAMHB to achieve a final inoculum concentration of approximately 5xl0 5 CFU/ml.
  • Inocula for S. pneumoniae and H. influenzae were prepared by directly suspending colonies from an overnight plate, adjusting the turbidity and diluting as above.
  • S. pneumoniae media was supplemented with 2.5% lysed horse blood. All plates were incubated in ambient air at 35°C for 20-24 h for S. pneumoniae and Haemophilus influenzae and 16-20 h for all other bacteria. The MIC endpoints were determined by reading the lowest concentration of test compound that completely inhibited the growth of the test bacteria. Results for compounds described in the above Examples are listed in Table 1.

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Abstract

L'invention concerne divers composés de macrolides comme ceux ayant les formules suivantes, dans lesquelles les variables ont les valeurs indiquées dans la description et les revendications.
EP03713257A 2002-01-17 2003-01-17 Amido-macrolides Withdrawn EP1471923A4 (fr)

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US8278281B2 (en) 2005-08-24 2012-10-02 Rib-X Pharmaceuticals, Inc. Triazole compounds and methods of making and using the same
CN101973979B (zh) * 2010-10-27 2013-04-17 浙江大学 一种抗菌化合物及其应用
CN102707059A (zh) * 2012-05-31 2012-10-03 华中农业大学 一种检测泰乐菌素和替米考星残留的胶体金试纸条及其使用方法和应用

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