Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
  • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
  • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
  • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
  • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
  • C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/04—Systems containing only non-condensed rings with a four-membered ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/06—Systems containing only non-condensed rings with a five-membered ring
  • C07C2601/08—Systems containing only non-condensed rings with a five-membered ring the ring being saturated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2601/00—Systems containing only non-condensed rings
  • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
  • C07C2601/14—The ring being saturated

Definitions

• This invention pertains generally to treating infections caused by gram- negative bacteria. More specifically, the invention described herein pertains to treating gram-negative infections by inhibiting activity of UDP-3-0-(R-3-hydroxydecanoyl)-N- acetylglucosamine deacetylase (LpxC).
• the present invention provides small molecule inhibitors of LpxC, pharmaceutical formulations containing such inhibitors, methods of treating patients with such pharmaceutical formulations, and methods of preparing such pharmaceutical formulations and inhibitors.
• the invention described herein pertains to treating gram-negative infections by administering compounds capable of inhibiting activity of UDP-3-0-(R-3-hydroxydecanoyl)-N-acetylglucosamine deacetylase (LpxC), either alone or in combination with a second antibacterial agent.
• LpxC UDP-3-0-(R-3-hydroxydecanoyl)-N-acetylglucosamine deacetylase
• LpxC the enzyme uridyldiphospho-3-0-(R- hydroxydecanoyl)-N-acetylglucosamine deacetylase
• LpxC is essential for survival and presents an ideal target for antibiotic activity in Gram-negative bacterial species.
• WO 2008/154642 (published 18 December 2008) all disclose compounds having antibacterial anti-LpxC activity.
• the commercial development of these LpxC inhibitors has been complicated by toxicity of these compounds in mammalian animals at concentrations at or near those required for antibacterial activity.
• the compounds presented herein are significantly better tolerated than other closely related compounds having anti-LpxC activity.
• an object of this invention to provide compounds and combinations of such compounds for use in the preparation of non-toxic antibacterials and other pharmaceuticals capable of inhibiting gram-negative bacterial infections.
• An additional object of the present invention is to provide synergistic combinations of antibacterial agents with LpxC inhibitors, which have intrinsic antibacterial properties as well the ability to improve permeability of the outer membrane of gram-negative bacteria to other antibacterial agents.
• synergistic combinations of drugs could have many advantages over conventional single compound chemotherapy, including lowered side-effects of drugs due to lower doses used or shorter time of treatment, more rapid cure of infection shortening hospital stays, increasing spectrum of pathogens controlled, and decreasing incidence of development of resistance to antibiotics.
• the present invention provides novel compounds, pharmaceutical formulations including the compounds, methods of inhibiting UDP-3-0-(R-3- hydroxydecanoyl)-N-acetylglucosamine deacetylase (LpxC), and methods of treating gram-negative bacterial infections.
• the invention provides compounds of Formula I:
• A is a substituted C 3 -C 6 cycloalkyl, wherein at least one substituent is a C C 3 primary alcohol;
• R 1 , R 2 and R 3 independently are selected from hydrogen and substituted or unsubstituted C C 3 alkyl, or R 1 and R 2 , together with the carbon atom to which they are attached, form an unsubstituted C 3 -C 6 cycloalkyl group, or R 2 and R 3 , together with the carbon atom and Q to which they are attached, form a substituted or unsubstituted heterocyclic ring, having from 5 to 8 ring atoms
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising an effective amount of an antibacterial compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
• the present invention provides a method of inhibiting a deacetylase enzyme in gram-negative bacteria, thereby affecting bacterial growth, comprising administering to a patient in need of such inhibition an LpxC-inhibitory compound of Formula I or a stereoisomer or pharmaceutically acceptable salt thereof.
• the present invention provides a method of inhibiting LpxC in gram-negative bacteria, thereby modulating the virulence of a bacterial infection, comprising administering to a patient in need of such inhibition an LpxC- inhibitory compound of Formula I or a stereoisomer or pharmaceutically acceptable salt thereof.
• the present invention provides a method for treating a subject having a bacterial infection comprising administering to the subject in need thereof an antibacterially effective amount of a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof.
• the bacterial infection is a gram-negative bacterial infection.
• the bacteria is Pseudomonas aeruginosa, Burkholderia (e.g., Burkholderia cepacia), Enterobacteriaceae, Franciscellaceae (e.g., Franciscella tularensis), Serratia, Proteus, Klebsiella, Enterobacter, Citrobacter, Salmonella, Providencia, Yersinia (e.g., Yersinia pestis), Morganella or Escherichia coli.
• the bacteria is Pseudomonas aeruginosa, Burkholderia, Franciscellaceae, Enterobacter, Yersinia or Escherichia coli.
• the bacteria is Pseudomonas aeruginosa. In another such embodiment the bacteria is Escherichia coli. In another embodiment the bacteria is Stenotrophomonas maltophila, Alcaligenes xylosoxidans, Haemophilus, Neisseria species, Cedecea or Edwardsiella species. In a further specific embodiment the subject is a mammal and in certain embodiments, a human.
• One aspect of the invention provides pharmaceutical compositions comprising an inhibitor of LpxC and a second antibacterial agent.
• the second antibacterial agent is selected from the group consisting of vancomycin, linezolid, azithromycin, imipenem, teicoplanin, daptomycin, clindamycin, rifampin, cefotaxime, gentamicin, novobiocin, and telavancin.
• the second antibacterial agent is vancomycin or rifampin.
• the LpxC inhibitor is a compound of Formula I:
• A is a substituted C 3 -C 6 cycloalkyl, wherein at least one substituent is a C C 3 primary alcohol;
• R 1 , R 2 and R 3 independently are selected from hydrogen and substituted or unsubstituted C C 3 alkyl, or R 1 and R 2 , together with the carbon atom to which they are attached, form an unsubstituted C 3 -C 6 cycloalkyl group, or R 2 and R 3 , together with the carbon atom and Q to which they are attached, form a substituted or unsubstituted heterocyclic ring, having from 5 to 8 ring
• Another aspect of the invention provides methods for treating a patient with a gram-negative bacterial infection, comprising co-administering a synergistic amount, for example an in vivo synergistic amount, of an antibacterial agent and an LpxC inhibitor of Formula I.
• the antibacterial agent is selected from the group consisting of vancomycin, linezolid, azithromycin, imipenem, teicoplanin, daptomycin, clindamycin, rifampin, cefotaxime, gentamicin, novobiocin, and telavancin.
• the antibacterial agent is vancomycin or rifampin.
• Figure 1 illustrates in vivo synergy of compound 1-1 and vancomycin in bacterial strain ATCC 43816.
• the present invention provides novel compounds, methods for inhibiting LpxC in gram-negative bacteria, and novel methods for treating bacterial infections.
• the compounds provided herein can be formulated into pharmaceutical formulations and medicaments that are useful in the methods of the invention.
• the invention also provides for the use of the compounds in preparing medicaments and pharmaceutical formulations, for use of the compounds in inhibiting LpxC, and for use of the compounds in treating bacterial infections in a subject.
• the invention further provides compositions and methods for treating gram-negative infections by administering compounds capable of inhibiting activity of UDP-3-0-(R-3-hydroxydecanoyl)-N- acetylglucosamine deacetylase (LpxC), either alone or in combination with administering a second antibacterial compound.
• UDP-3-0-(R-3-hydroxydecanoyl)-N- acetylglucosamine deacetylase LpxC
• LpxC is an abbreviation that stands for UDP-3-0-(R-3- hydroxydecanoyl)-N-acetylglucosamine deacetylase.
• Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), i-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 ) 3 CCH 2 -).
• Alkoxy refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, f-butoxy, sec-butoxy, n-pentoxy, and the like.
• Amino refers to the group -NH 2 .
• Primary alcohol refers to the group -alkyl-OH, wherein the hydroxyl radical is connected to a primary carbon. Examples include -CH 2 OH (hydroxymethyl), -CH 2 CH 2 OH (hydroxymethyl) and -CH(CH 3 )CH 2 OH (1 -hydroxypropan-2-yl).
• Alkynyl refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of acetylenic -C ⁇ C- unsaturation. Examples of such alkynyl groups include acetylenyl (-C ⁇ CH), and propargyl (-CH 2 C ⁇ CH).
• Carboxyl or “carboxy” refers to -COOH or salts thereof.
• Cycloalkyl refers to cyclic alkyl groups of from 3 to 13 carbon atoms having single. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like.
• Halo or halogen refers to fluoro, chloro, bromo, and iodo and is typically fluoro or chloro.
• Heterocycle refers to a saturated or unsaturated group having a single ring, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen. In one implementation, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -S0 2 - moieties.
• Niro refers to the group -N0 2 .
• Niroso refers to the group -NO.
• Substituted refers to a group having one or more hydrogens replaced with substituents selected from the group consisting of alkoxy, acyl, acylamino, acyloxy, amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, guanidino, halo, hydroxy, nitro, S0 3 H, sulfonyl, sulfonyloxy, thioacyl, thiol, and alkylthio, wherein said substituents are as defined herein.
• substituted also refers to a group having two hydrogens replaced with a single double bonded oxygen atom (an oxo group) or a single double bonded sulfur atom (thioxo).
• the substituted group has 1 to 3 of the aforementioned substituents. In other implementations, the substituted group has 1 to 2 of the aforementioned substituents
• Sulfonyl refers to the group -S0 2 -alkyl, -S0 2 -substituted alkyi, -S0 2 - alkenyl, -S0 2 -substituted alkenyl, wherein alkyi, substituted alkyi, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl are as defined herein.
• Sulfonyl includes groups such as methyl-S0 2 -.
• “Sulfonyloxy” refers to the group -OS0 2 -alkyl, -OS0 2 -substituted alkyi, -OS0 2 -alkenyl, -OS0 2 -substituted alkenyl, -OS0 2 -alkynyl, -OS0 2 -substituted alkynyl, wherein alkyi, substituted alkyi, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl are as defined herein.
• Thioacyl refers to the groups H-C(S)-, alkyl-C(S)-, substituted alkyl-C(S)-, alkenyl-C(S)-, substituted alkenyl-C(S)-, alkynyl-C(S)-, and substituted alkynyl-C(S)-, wherein alkyi, substituted alkyi, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl are as defined herein.
• Thiol refers to the group -SH.
• Alkylthio refers to the group -S-alkyl, wherein alkyl is as defined herein.
• sulfur may be oxidized to -S(O)-.
• the sulfoxide may exist as one or more stereoisomers.
• arylalkyloxycarbonyl refers to the group (aryl)-(alkyl)-0- C(O)-.
• references to a certain element such as hydrogen or H is meant to include all isotopes of that element.
• a substituent group is defined to include hydrogen or H, it also includes deuterium and tritium.
• the subject invention also includes isotopically-labeled compounds of the present invention, that are structurally identical to those disclosed herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 8 0, 17 0, 31 P, 32 P, 35 S, 18 F and 36 CI, respectively.
• Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts of said compounds and of said prodrugs that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
• Certain isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3 H and 1 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 1 C, isotopes are particularly preferred for their ease of preparation and detectability.
• isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out known or referenced procedures and by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
• Stereoisomers refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.
• a person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.
• “Patient” refers to human and non-human animals, especially mammals.
• “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, phosphate, sulfate and the like.
• “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder.
• Synergy or “synergistic” as used herein means the combined effect of the compounds when used in combination is greater than the additive effects of the compounds when used individually.
• Synergism can be defined quantitatively as a fractional inhibitory concentration index (FICI) of ⁇ 0.5, where FICI is defined as the sum of the fractional inhibitory concentrations (FICs) of the individual components in a combination of two compounds, and the FIC is defined as the ratio of the minimal inhibitory concentration (MIC) of the compound in the combination divided by the MIC of the compound alone:
• “synergism,” more particularly “in vivo synergism,” can be defined quantitatively as an at least two-fold decrease in the static dose of the agents used in combination as compared to the LpxC inhibitor or second antibacterial agent alone. In certain cases one agent alone may never reach a static dose. In such cases, a combination is synergistic if bacterial growth can be halted (CFU load at 24 hours that is identical to that measured at 0 hours post infection) by combined administration with two compounds that alone cannot achieve stasis.
• Co-administration can be in the form of a single formulation (combining, for example, a compound of the present invention and a second antibacterial agent with pharmaceutically acceptable excipients, optionally segregating the two active ingredients in different excipient mixtures designed to independently control their respective release rates and durations) or by independent administration of separate formulations containing the active agents.
• Co-administration further includes concurrent administration (administration of a compound of the present invention and a second antibacterial agent at the same time) and time varied administration (administration of a compound of the present invention at a time different from that of the second antibacterial agent), as long as both the compound of the present invention and the second antibacterial agent are present in the body in therapeutically effective concentrations during at least partially overlapping times.
• antibacterial agent refers to agents that have either bactericidal or bacteriostatic activity.
• inhibiting the growth indicates that the rate of increase in the numbers of a population of a particular bacterium is reduced. Thus, the term includes situations in which the bacterial population increases but at a reduced rate, as well as situations whene the growth of the population is stopped, as well as situations where the numbers of the bacteria in the population are reduced or the population even eliminated. If an enzyme activity assay is used to screen for inhibitors, one can make modifications in uptake/efflux, solubility, half-life, etc. to compounds in order to correlate enzyme inhibition with growth inhibition.
• the activity of antibacterial agents is not necessarily limited to bacteria but may also encompass activity against parasites, virus, and fungi.
• the present invention provides compounds of Formula I:
• A is a substituted C 3 -C 6 cycloalkyl, wherein at least one substituent is a C C 3 primary alcohol;
• R 1 , R 2 and R 3 independently are selected from hydrogen and substituted or unsubstituted C C 3 alkyl, or R 1 and R 2 , together with the carbon atom to which they are attached, form an unsubstituted C 3 -C 6 cycloalkyl group, or R 2 and R 3 , together with the carbon atom and Q to which they are attached, form a substituted or unsubstituted heterocyclic ring, having from 5 to 8 ring atoms
• Q is NR, and in some such embodiments Q is NH or NCH 3 .
• R 1 , R 2 , and R 3 independently are selected from hydrogen and substituted or unsubstituted C C 3 alkyl, and in some such embodiments are selected from hydrogen and unsubstituted C C 3 alkyl.
• R 1 and R 2 independently are unsubstituted d-C 3 alkyl.
• A is a C 3 - C 6 cycloalkyl substituted with hydroxymethyl.
• both B and C are a -C ⁇ C-.
• One aspect of the invention provides compounds of Formula l-A:
• A is a substituted C 3 -C 6 cycloalkyl, wherein at least one substituent is a C C 3 primary alcohol
• R 1 , R 2 and R 3 independently are selected from hydrogen and substituted or unsubstituted C C 3 alkyl, or R 1 and R 2 , together with the carbon atom to which they are attached, form a C 3 -C 6 cycloalkyl group, or R 2 and R 3 , together with the carbon atom and nitrogen to which they are attached, form a substituted or unsubstituted heterocyclic ring, having from 5 to 8 ring atoms, wherein 1 -2 ring atoms of the heterocyclic ring are selected from N, O and S; and R is hydrogen or an unsubstituted C C 3 alkyl.
• R 1 , R 2 and R 3 independently are selected from hydrogen and substituted or unsubstituted C C 3 alkyl, and in some such embodiments are selected from hydrogen and unsubstituted CrC 3 alkyl.
• A is a C 3 -C 6 cycloalkyl, mono-substituted with a Ci-C 3 primary alcohol.
• Compounds of the present invention include those listed in Table I . Certain of the compounds illustrated in Table 1 represent mixtures of two diastereomers. In such a case, the notation “*” indicates that the two moieties attached to the cyclic moiety are trans to one another. The notation “#” indicates that the two moieties attached to the cyclic moiety are cis to one another.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
• the invention provides a method of inhibiting a deacetylase enzyme in a gram-negative bacteria, thereby affecting bacterial growth, comprising administering to a patient in need of such inhibition a compound of Formula I or a stereoisomer or pharmaceutically acceptable salt thereof.
• the invention provides a method of inhibiting LpxC in a gram-negative bacteria, thereby modulating the virulence of a bacterial infection, comprising administering to a patient in need of such inhibition a compound of Formula I or a stereoisomer or pharmaceutically acceptable salt thereof.
• the IC 50 value of the compound is less than or equal to 10 ⁇ with respect to LpxC.
• the IC 50 value is less than or equal to 1 ⁇ , is less than or equal to 0.1 ⁇ , is less than or equal to 0.050 ⁇ , is less than or equal to 0.030 ⁇ , is less than or equal to 0.025 ⁇ , or is less than or equal to 0.010 ⁇ .
• the invention provides a method for treating a subject having a gram-negative bacterial infection comprising administering to the subject in need thereof an antibacterially effective amount of a compound of Formula I or a stereoisomer or pharmaceutically acceptable salt thereof.
• the bacteria is Pseudomonas aeruginosa, Burkholderia (e.g., Burkholderia cepacia), Enterobacteriaceae, Franciscellaceae (e.g., Franciscella tularensis), Serratia, Proteus, Klebsiella, Enterobacter, Citrobacter, Salmonella, Providencia, Yersinia (e.g., Yersinia pestis), Morganella or Escherichia coli.
• the bacteria is Pseudomonas aeruginosa, Burkholderia, Franciscellaceae, Enterobacter, Yersinia or Escherichia coli. In one such embodiment the bacteria is Pseudomonas aeruginosa. In another such embodiment the bacteria is Escherichia coli. In another embodiment the bacteria is Stenotrophomonas maltophila, Alcaligenes xylosoxidans, Haemophilus, Neisseria species, Cedecea or Edwardsiella species.
• the subject may be a mammal, and in some embodiments, a human.
• Bacterial infections susceptible to treatment according to the present invention include primary infections and co-infections caused by a species of bacteria and one or more additional infectious agents such as, for example, bacteria, virus, parasite and fungus.
• Compounds of the invention can be used for treating conditions caused by the bacterial production of endotoxin and, in particular, by gram-negative bacteria and bacteria that use LpxC in the biosynthesis of lipopolysaccharide (LPS) or endotoxin.
• LPS lipopolysaccharide
• Compounds of the invention also are useful in treating conditions that are caused or exacerbated by the bacterial production of lipid A and LPS or endotoxin, such as sepsis, septic shock, systemic inflammation, localized inflammation, chronic obstructive pulmonary disease (COPD) and acute exacerbations of chronic bronchitis (AECB).
• treatment includes the administration of a compound of the invention, or a combination of compounds of the invention, optionally with a second agent wherein the second agent is a second antibacterial agent or a non-antibacterial agent.
• non-antibacterial agents include antiendotoxins including endotoxin receptor-binding antibodies, endotoxin-binding antibodies, anti- CD14-binding protein antibodies, anti-lipopolysaccharide-binding protein antibodies and tyrosine kinase inhibitors.
• compounds of the present invention may also be used with non-antibacterial agents administered via inhalation.
• Representative non-antibacterial agents used in this treatment include anti-inflammatory steroids, non-steroidal anti-inflammatory agents, bronchodilators, mucolytics, anti-asthma therapeutics and lung fluid surfactants.
• the non-antibacterial agent may be albuterol, salbuterol, budesonide, beclomethasone, dexamethasone, nedocromil, beclomethasone, fluticasone, flunisolide, triamcinolone, ibuprofin, rofecoxib, naproxen, celecoxib, nedocromil, ipratropium, metaproterenol, pirbuterol, salmeterol, formoterol, indacaterol, bronchodilators, mucolytics, calfactant, beractant, poractant alfa, surfaxin or pulmozyme (also called domase alfa).
• Compounds of the invention can be used alone or in combination with a second antibacterial agent for the treatment of a serious or chronic respiratory tract infection including serious lung and nosocomial infections such as those caused by Enterobacter aerogenes, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Serratia marcescens, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Burkholderia cepacia, Alcaligenes xylosoxidans, Flavobacterium meningosepticum, Providencia stuartii and Citrobacter freundi, community lung infections such as those caused by Haemophilus Influenzae, Legionella species, Moraxella catarrhalis, Branhamella catarrhalis, Enterobacter species, Klebsiella species, and Proteus species, infections caused by other bacterial species such as Neisser
• compounds of the present invention When used for treating subjects infected with gram-negative bacterial infections, compounds of the present invention can be used to sensitize gram-negative bacteria to the effects of a second antibacterial agent.
• the present invention provides novel combinations of compounds including a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, as well as methods for treating subjects infected with gram-negative bacteria.
• novel combinations provided herein can be formulated into pharmaceutical formulations and medicaments that are useful in the methods of the invention.
• the invention also provides for the use of the novel combinations in preparing medicaments and pharmaceutical formulations, for use of the combinations in treating bacterial infections in a patient.
• checkerboard assay One classic method for assessing synergy, referred to as the checkerboard assay, is used to predict the efficacy of antibacterial agents, and is described by Scribner et. al., (1982, Antimicrobial Agents and Chemotherapy 21 (6):939-943) and in Goodman & Gilman (1980, The Pharmacological Basis of Therapeutics, Sixth Edition, pp. 1097-1098).
• the checkerboard assay involves serial two-fold dilutions of the antibiotics individually and in combination in broth, which is then inoculated with the microorganism to be tested.
• the minimum inhibitory concentration (MIC) of each drug used individually and in combination is determined (N.B., the MIC is the lowest concentration of the drug that inhibits growth in the medium).
• Synergism is indicated by a decrease in the MIC of each drug when used in combination.
• Antagonism is indicated by an increase in the MIC of either or both drugs when used in combination.
• LpxC an essential gene in gram-negative bacteria, encodes the enzyme uridyldiphospho-3-0-(R-hydroxydecanoyl)-N-acetylglucosamine deacetylase. This enzyme catalyzes an early committed step in the bio-synthesis of lipid A, the lipid moiety of lipopolysaccharide that is an essential component of all gram-negative bacteria. Above the MIC, an LpxC inhibitor is expected to disrupt the outer membrane, thus permitting other antibacterial compounds to penetrate the outer membrane.
• these agents may affect periplasmic targets as is the case with vancomycin, or they may then diffuse across the inner membrane to interact with an intracellular target such as the ribosome (Erythromycin) or RNA polymerase (Rifampin).
• an intracellular target such as the ribosome (Erythromycin) or RNA polymerase (Rifampin).
• Erythromycin ribosome
• Rifampin RNA polymerase
• the biochemical mechanism that may underlie the observed synergy is the enhanced permeability of the outer membrane to agents such as vancomycin when combined with LpxC inhibitors.
• the second antibacterial agent used in combination with a compound of Formula I, or stereoisomer or pharmaceutically acceptable salt thereof is vancomycin, linezolid , azithromycin, imipenem, teicoplanin, daptomycin, clindamycin, rifampin, cefotaxime, gentamicin, novobiocin or telavancin.
• the second antibacterial agent is vancomycin, teicoplanin, rifampin, azithromycin, telavancin or novobiocin.
• the second antibacterial agent is vancomycin or rifampin.
• the second antibacterial agent and/or the compound of Formula I, or stereoisomer or pharmaceutically acceptable salt thereof is administered at a sub-therapeutic dose, wherein a subtherapeutic dose is a dose that would be insufficient to treat bacterial infections, if administered alone.
• compositions of the present invention comprise a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, formulated together with one or more pharmaceutically acceptable carriers or diluents.
• pharmaceutically acceptable carrier means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulf
• compositions of this invention can be administered to humans and other animals orally, rectally, parenterally (as by intravenous, intramuscular or subcutaneous injection), intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), bucally, or as an oral or nasal spray, or a liquid aerosol or dry powder formulation for inhalation.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• the oral compositions can also include adjuvants such as wetting agents,
• sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3-butanediol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution, 1 % lidocaine, U.S. P. and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid are used in the preparation of injectables.
• the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
• the rate of drug release can be controlled.
• biodegradable polymers include poly(orthoesters) and poly(anhydrides).
• Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
• compositions for rectal or vaginal administration are preferably suppositories that can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
• the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and
• compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
• compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
• the antibacterial compounds can also be in micro-encapsulated form with one or more excipients as noted above.
• the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
• the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
• Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
• the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
• buffering agents include polymeric substances and waxes.
• Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
• the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
• Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of this invention.
• the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• compositions of the invention may also be formulated for delivery as a liquid aerosol or inhalable dry powder.
• Liquid aerosol formulations may be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles where bacteria reside in patients with bronchial infections, such as chronic bronchitis and pneumonia.
• Pathogenic bacteria are commonly present throughout airways down to bronchi, bronchioli and lung parenchema, particularly in terminal and respiratory bronchioles. During exacerbation of infection, bacteria can also be present in alveoli.
• Liquid aerosol and inhalable dry powder formulations are preferably delivered throughout the endobronchial tree to the terminal bronchioles and eventually to the parenchymal tissue.
• Aerosolized formulations of the invention may be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of an aerosol particles having with a mass medium average diameter predominantly between 1 to 5 ⁇ .
• the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosolizable volume able to deliver effective dose of the compounds of the invention to the site of the infection.
• the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.
• Aerosolization devices suitable for administration of aerosol formulations of the invention include, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the invention into aerosol particle size predominantly in the size range from 1 -5 pm. Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are 1 to 5 ⁇ range.
• a jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate.
• An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets.
• suitable devices including, for example, AeroNeb and AeroDose vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, Calif.), Sidestream7 nebulizers (Medic-Aid Ltd., West Wales, England), Pari LC7 and Pari LC Star7 jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Va.), and Aerosonic (DeVilbiss Medizinische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffische Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffo Kunststoffotechnik (Deutschland) GmbH, Heiden, Germany) and UltraAire7 (Omron Healthcare, Inc., Vernon Hills, III.) ultrasonic nebulizers.
• Compounds of the invention may also be formulated for use as topical powders and sprays that can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
• Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
• Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
• dosage forms can be made by dissolving or dispensing the compound in the proper medium.
• Absorption enhancers can also be used to increase the flux of the compound across the skin.
• the rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
• bacterial infections are treated or prevented in a patient such as a human or lower mammal by administering to the patient a therapeutically effective amount of a compound of Formula I, or a stereoisomer or pharmaceutically acceptable salt thereof, in such amounts and for such time as is necessary to achieve the desired result.
• a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat bacterial infections, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
• the total daily dose of the compounds of this invention administered to a human or other mammal in single or in divided doses can be in amounts, for example, from 0.01 to 200 mg/kg body weight or more usually from 0.1 to 50 mg/kg body weight. In certain embodiments, the total daily dose administered to a human or other mammal is from 1.0 to 100 mg/kg body weight or from 5.0 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
• treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 15 g of the compound(s) of this invention per day in single or multiple doses, more usually, from 100 mg to 5 g, and even more usually from 250 mg to 1 g per day in single or multiple doses.
• compositions for use in the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.
• a "kit” as used in the instant application includes a container for containing the pharmaceutical compositions and may also include divided containers such as a divided bottle or a divided foil packet.
• the container can be in any conventional shape or form as known in the art that is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a resealable bag (for example, to hold a "refill” of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule.
• the container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle that is in turn contained within a box.
• Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil that is opposite from the direction in which the recesses were formed.
• the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses bet een the plastic foil and the sheet.
• the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.
• the written memory aid is of the type containing information and/or instructions for the physician, pharmacist or other health care provider, or subject, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen that the tablets or capsules so specified should be ingested or a card that contains the same type of information.
• a memory aid is a calendar printed on the card e.g., as follows "First Week, Monday, Tuesday,". . . etc . . . "Second Week, Monday, Tuesday, . . .” etc.
• Other variations of memory aids will be readily apparent.
• a “daily dose” can be a single tablet or capsule or several tablets or capsules to be taken on a given day.
• a daily dose of one or more compositions of the kit can consist of one tablet or capsule while a daily dose of another one or more compositions of the kit can consist of several tablets or capsules.
• kits are a dispenser designed to dispense the daily doses one at a time in the order of their intended use.
• the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen.
• a memory-aid is a mechanical counter that indicates the number of daily doses that has been dispensed.
• a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal that, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.
• kits of the present invention may also include, in addition to a compound of the present invention, one or more additional pharmaceutically active compounds.
• the additional compound second antibacterial.
• the additional compounds may be administered in the same dosage form as the compound of the present invention or in a different dosage form. Likewise, the additional compounds can be administered at the same time as the compound of the present invention or at different times.
• compositions of the present compounds may also be used in combination with other known antibacterial agents of similar spectrum to (1 ) enhance treatment of severe gram-negative infections covered by the spectrum of this compound or (2) add coverage in severe infections in which multiple organisms are suspected in which another agent of a different spectrum may be required in addition to this compound.
• Potential agents include members of the aminoglycosides, penicillins, cephalosporins, fluoroquinolones, macrolides, glycopeptides, lipopeptides and oxazolidinones.
• the treatment can involve administering a composition having both a compound of the present invention and a second antibacterial compound or administration of a compound of the present inventive compounds followed by or preceded by administration of a second antibacterial agent.
• TLC thin layer chromatography
• glass or plastic backed silica gel plates such as, for example, Baker-Flex Silica Gel 1 B2-F flexible sheets.
• TLC results were readily detected visually under ultraviolet light, or by employing well known iodine vapor and other various staining techniques.
• Mass spectrometric analysis was performed on one of two LCMS instruments: a Waters System. (Alliance HT HPLC and a Micromass ZQ mass spectrometer; Column: Eclipse XDB-C-18, 2.1 x50 mm; solvent system: 5-95% (or 35- 95%, or 65-95% or 95-95%) acetonitrile in water with 0.05%TFA; flow rate 0.8 mL/min; molecular weight range 500-1500; cone Voltage 20 V; column temperature 40° C.) or a Hewlett Packard System (Series 1 100 HPLC; Column: Eclipse XDB-C18, 2.1 x50 mm; solvent system: 1-95% acetonitrile in water with 0.05% TFA; flow rate 0.4 mL/min; molecular weight range 150-850; cone Voltage 50 V; column temperature 30° C). All masses are reported as those of the protonated parent ions.
• GCMS analysis was performed on a Hewlet Packard instrument (HP6890 Series gas chromatograph with a Mass Selective Detector 5973; injector volume: 1 ⁇ _; initial column temperature: 50° C; final column temperature: 250C; ramp time: 20 minutes; gas flow rate: 1 mL/min; column: 5% phenyl methyl siloxane, Model #HP 190915-443, dimensions: 30.0 mx25 mx0.25 m).
• Nuclear magnetic resonance (NMR) analysis was performed with a Varian 300 MHz NMR (Palo Alto, Calif.). The spectral reference was either TMS or the known chemical shift of the solvent. Some compound samples were run at elevated temperatures (e.g. 75° C.) to promote increased sample solubility.
• Preparative separations were carried out using a Flash 40 chromatography system and KP-Sil, 60A (Biotage, Charlottesville, Va.), or by flash column chromatography using silica gel (230-400 mesh) packing material, or by HPLC using a C-18 reversed phase column.
• Typical solvents employed for the Flash 40 Biotage system and flash column chromatography were dichloromethane, methanol, ethyl acetate, hexane, acetone, aqueous hydroxylamine and triethyl amine.
• Typical solvents employed for the reverse phase HPLC were varying concentrations of acetonitrile and water with 0.1 % trifluoroacetic acid.
• Methyl 4-(((1 R,2R)-2-(acetoxymethyl)cyclopropyl)buta-1 ,3- diynyl)benzoate 10 (1.92g, 6.5mmol) was dissolved in THF (40 mL), then added a solution of sodium hydroxide (2.60g, 65 mmol) in 10mL water. The mixture was kept stirring at r.t. for 8 hours.
• the crude material was purified by reverse-phase HPLC (gradient of 0-
• Methyl 4'-(((1 ,2-£rans)-2-(hydroxymethyl)cyclopropyl)ethynyl)biphenyl-4- carboxylate (900 mgs, 2.94 mmol, 1.0 equiv) was dissolved in methanol (5 mL), DMF (2 mL), and THF (5 mL). At room temp was added 1.0M NaOH (4.41 mL, 4.407 mmol, 1.5 equiv). The reaction stirred for 4 days. Reaction concentrated, to remove MeOH and THF, acidify to pH ⁇ 3 with 6N HCI ( ⁇ 5mL). Extract with EtOAc (3x50 mL), combine organic layers, wash with sat.
• N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)-4-((2- (hydroxymethyl)cyclopropyl)buta-1 ,3-diynyl)benzamide (300mg, 0.81 mmol) was dissolved in A/,A/-dimethylformamide (10mL) and treated with paraformaldehyde (732mg, 8.1 mmol, 10eq) and A/,A/-diisopropylethylamine (0.56mL, 3.3mmol, 4eq) at room temperature for 16 hours. Trifluoroacetic acid (1.3mL, 16.2mmol, 20eq) and sodium cyanoborohydride (101 mg, 1.6mmol, 2 eq) were introduced.
• reaction mixture was diluted with water, then extracted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, removed the solvent and the residue was purified with silica gel chromatography. (28.73g, 95%)
• reaction mixture was diluted with NH 4 Cl aq solution and extracted with ethyl acetate.
• the combined ethyl acetate layer was washed with water, brine and dried.
• reaction mixture was diluted with NH Cl aq solution and extracted with ethyl acetate.
• the combined ethyl acetate layer was washed with water, brine and dried.
• the flask was placed at 4°C for 96h. Then the reaction was concentrated (keeping reaction at 0°C) to a gummy mass. To this was added water (3 mL) and ACN (0.5 mL). The solution was acidified at 0°C using TFA (3 mL). Additional water (1 mL) and ACN (1 mL) was added. The compound was purified by RP HPLC (2" column, 50mL/min, 0.1 % TFA in water/ACN, equil @ 5%B); loaded onto 2" column (10mL/min, 5%B) using syringe filter (2X7 mL); ramped to 50mL/min over 1 minute and 5-35%B over 55min.
• target compound 2 (7.5 g, 46 %) as a white solid without purification for next step.
• Glacial acetic acid (3.18 g, 0.0530 mol) was added rapidly to quench the reaction, and the reaction mixture was allowed to warm to r.t., stirred for 3h, finally diluted with DCM (200mL x 2) and washed with saturated NaHC0 3 (100mL), brine (100mL) and water (100mL).
• the reaction was concentrated, (at 0°C) to remove the isopropyl alcohol, and then was acidified at 0°C using TFA (3 mL). Additional water (15 mL) and ACN (3 mL) were added.
• the product was purified by reverse phase HPLC (2" column,50mL/min, 0.1 % TFA in water/ACN, equil @ 2%B). The column was loaded at 10mL/min, 2%B using syringe filter (24 mL) and then ramped to 50mL/min over 1 minute. A gradient from 2%B to 95%B was run over 73 min. Desired fractions were combined, frozen and placed on a lyophilizer.
• Bacterial isolates were cultivated from -70° C. frozen stocks by overnight passages at 35° C in ambient air on Mueller-Hinton agar (Beckton Dickinson, Franklin Lakes, NJ). Clinical isolates tested were obtained from various geographically diverse hospitals in the US and abroad (Focus Diagnostics, Herndon, VA and J Ml, North Liberty, IA). Quality control strains were from the American Type Culture Collection (ATCC; Rockville, Md.). Susceptibility Testing
• MICs Minimum Inhibitory Concentrations
• CFU colony-forming units
• Drug dilutions and inocula were made in sterile, cation adjusted Mueller-Hinton Broth (Beckton Dickinson). An inoculum volume of 100 ⁇ - was added to wells containing 100 ⁇ . of broth with 2-fold serial dilutions of drug. All inoculated microdilution trays were incubated in ambient air at 35° C for 18-24 h.
• MICs Minimum Inhibitory Concentrations
• AECO001 is E. coli ATCC25922; APAE001 is Pseudomonas aeruginosa ATCC27853; AKPN001 is Klebsiella pneumoniae ATCC43816; APAE002 is a clinical isolate of Pseudomonas aeruginosa expressing a normal level of efflux activity.
• mice were administered in mice via subcutaneous injection.
• the mice were group-housed (five per cage) at 18-28°C and -50% humidity, and fed with standard rodent chow. Water and food were given ad libitum. Mice were given subcutaneous doses of no more than 20 ml_ per kg of body weight.
• mice Groups of three mice were dosed at 50, 100, 200, 400 or 600 mg/kg/day as a single dose of the test compound by subcutaneous injection in a formulation consisting of 15% Captisol in 20 mM acetate buffer, pH 5. Subcutaneous injections were done on the back in the interscapular area. Volumes of 10-20 mL/kg were injected into this area. The needle was inserted parallel to the surface of the skin to such a depth that the point of the needle lay within the subcutaneous pocket. A gentle but firm pressure on the plunger was used to expel the contents of the syringe.
• the minimum time range to monitor the mice was 30 minutes post-dose if the animals looked alert, normal, and responsive. If animals showed toxicity effects, they were monitored closely until they showed recovery signs to near baseline, or up to 4 hr post-dose, whichever came first. Animals were kept for 72 hours after dosing for clinical observation including monitoring of survival and activity level.
• Observations considered symptoms of central nervous system distress such as seizures, lethargy, recumbency, motionlessness, hyperactivity
• neuromuscular abnormalities such as ataxia, twitching, convulsions, splayed limbs, jumping or kicking
• autonomic symptoms such as salivation, lacrimation, urination, defecation, piloerection or squinting
• respiratory distress such as labored or rapid breathing, depression, panting or gasping
• stereotypic behaviors such as repetitive chewing, circling, pacing, grooming, sniffing, head movements or hunched stance
• abnormal behaviors such as escape behavior or wet dog shake.
• No_alc the synthesis and activity of which is disclosed in International PCT Publication No. 2008/06676; compound 91 -12
• Compound 1-1 of the present invention which differs from this known compound, simply by a hydroxymethyl substitution of the cyclopropyl group, has antimicrobial activity comparable to that of No_alc but is substantially better tolerated in mammals (maximum tolerated dose of approximately 200 mg/kg when subcutaneously injected into mice).
• Table III provides the results of observations of the subcutaneously dosed mice. Tolerability was characterized by clinical observation. Class A was assigned to animals with no to little toxicity symptoms such as occasional short pausing when moving, slight labored breathing, or slight lethargy with a quick recovery (e.g., within 10 min). Class B was assigned to animals showing some toxicity symptoms such as longer pausing when moving, slight lethargy with a longer recovery time (up to 1 hour) during which animals are still able to move around. Class C was assigned to animals with moderate to severe toxicity symptoms such as lethargy, recumbence, lethargy with squinting and labored breathing, severe twitching (jumping, kicking), or escape behavior. Finally, Class D was assigned when any lethal effects (including moribund state requiring euthanasia) occurred within the clinical observation time (up to 72 hour post-dose).
• Bacterial isolates were cultivated from -70°C frozen stocks of K. pneumoniae (ATCC 43816) by overnight passages at 35° C. in ambient air on Mueller- Hinton agar (Beckton Dickinson, Franklin Lakes, NJ). Minimum Inhibitory Concentrations (MICs) were determined by the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. In brief, organism suspensions were adjusted to a 0.5 McFarland standard to yield a final inoculum between 3x 10 5 and 7x10 5 colony-forming units (CFU)/mL. Drug dilutions and inocula were made in sterile, cation adjusted Mueller-Hinton Broth (Beckton Dickinson).
• Standard checkerboard assays were performed with a combination of the indicated agents and N-((S)-3-amino-1 -(hydroxyamino)-3-methyl-1 -oxobutan-2-yl)- 4-(((1 ,2-irans)-2-(hydroxymethyl)cyclopropyl)buta-1 ,3-diynyl)benzamide (1-1).
• Table IV provides the FICI calculated according to standard techniques.
• Compound 1-1 was synergistic in vitro with vancomycin, teicoplanin, erythromycin, azithromycin, rifampin and novobiocin.
• mice were rendered neutropenic prior to infection with 2 doses of cyclophosphamide, and then infected intramuscularly in the thigh with inocula of 10 3 - 10 5 CFU of K. pneumo. (ATCC 43816).
• Antibiotics or vehicle alone as a negative control were administered twice at 2 hrs and 14 hrs post-infection.
• the animals were kept neutropenic for the duration of the experiment in order to minimize the effect of white blood cells on the infection such that the microbiological readout measures the in vivo interaction of drugs and bacteria.
• thighs were harvested, homogenized, and plated to measure the number of CFUs surviving per thigh.
• Thighs from a subset of animals were also harvested 2 hrs post-infection to record the CFUs present just prior to the first antibiotic treatment (pre-treatment).
• the static dose defined as the dose required to result in a CFU load at 24 hours that is identical to that measured at 0 hours post infection, was calculated by standard methods in Prizm (GraphPad Software) from a dose response curve.
• Vancomycin exhibits significant in vivo synergy with LpxC inhibitors for treatment of infections with K. pneumo. ATCC43816. As indicated in Figure 1 , treatment of infected mice with vancomycin alone at 220 mg/kg/day results in no significant reduction in CFU. However, when co-dosed with compound 1-1 the static doses of the LpxC inhibitors is reduced significantly ( Figure 1 & Table V).
• Vancomycin q6hr 220 n.d. 10.21 -3.97 -0.26
• organic compounds according to the invention may exhibit the phenomenon of tautomerism.
• chemical structures within this specification can only represent one of the possible tautomeric forms, it should be understood that the invention encompasses any tautomeric form of the drawn structure.

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  • 2011-11-04 BR BR112013011693A patent/BR112013011693A2/pt not_active IP Right Cessation
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  • 2011-11-04 AU AU2011367819A patent/AU2011367819A1/en not_active Abandoned
  • 2011-11-04 AR ARP110104117A patent/AR083760A1/es not_active Application Discontinuation
  • 2011-11-04 EP EP11849908.6A patent/EP2638006A1/en not_active Withdrawn
• 2013
  • 2013-05-09 IL IL226270A patent/IL226270A0/en unknown

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