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WO2013086415A1 - Composés antimicrobiens - Google Patents

Composés antimicrobiens Download PDF

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Publication number
WO2013086415A1
WO2013086415A1 PCT/US2012/068570 US2012068570W WO2013086415A1 WO 2013086415 A1 WO2013086415 A1 WO 2013086415A1 US 2012068570 W US2012068570 W US 2012068570W WO 2013086415 A1 WO2013086415 A1 WO 2013086415A1
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WIPO (PCT)
Prior art keywords
compound
rna polymerase
rifalazil
resistant
rif
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PCT/US2012/068570
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English (en)
Inventor
Hollis D. Showalter
George A. GARCIA
Hao Xu
Sumandeep K. ATWAL
Irosha NAWARATHNE
Paul D. KIRCHHOFF
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The Regents Of The University Of Michigan
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Publication of WO2013086415A1 publication Critical patent/WO2013086415A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems

Definitions

  • Tuberculosis is a contagious and deadly disease that has reached pandemic proportions.
  • WHO World Health Organization
  • 8 to 10 million new cases of TB are diagnosed each year and 2 to 3 million people die from the disease each year. Consequently, the causative agent of TB, Mycobacterium tuberculosis, is a leading cause of adult deaths from infectious disease.
  • a high proportion of these newly diagnosed cases and deaths occurs in HIV-positive people, and a significant number of AIDS deaths in Africa is attributable to TB infections.
  • Global population growth is increasing the disease burden and, in turn, posing a continuing health and financial burden in various parts of the world, particularly in Asia and Africa.
  • MTB Mycobacterium tuberculosis
  • MTB Mycobacterium tuberculosis
  • the chemical composition of its cell wall includes peptidoglycans and complex lipids, in particular mycolic acids, which are a significant determinant of its virulence.
  • the unique structure of the cell wall of MTB allows it to lie dormant for many years as a latent infection, particularly as it can grow readily inside macrophages, hiding it from the host's immune system.
  • MDR-TB Mycobacterium tuberculosis
  • rifamycins are the most commonly used drugs for TB, and semisynthetic derivatives have been reported that show improved antimycobacterial activities. These include rifampin (RMP, see Figure l), which is the cornerstone of current short-term TB treatment. Amongst newer derivatives, rifalazil (RLZ, see Figure l) has proved most interesting because of its potency and relative lack of toxicity in early rodent studies. RLZ is an exceedingly potent rifamycin derivative that is 16 - 256 times more potent than RMP and is particularly effective against many of the RMP-resistant strains of MTB.
  • RMP rifampin
  • RLZ rifalazil
  • Streptococcus pyogenes Chlamydia trachomatis, and Chlamydia pneumonia.
  • RMP including having activity against some RMP-resistant strains.
  • RLZ has shown a high volume of distribution and produced tissue levels in rats up to 200 times those in plasma. It displayed a very long half-life (60-100 hours) in human trials.
  • antimicrobial e.g., anti- mycobacterial, anti-TB
  • rifalazil having increased inhibition of RNA polymerase (e.g., through an interaction with a sigma factor or, e.g., through an interaction with another subunit of the RNA polymerase).
  • the compounds also demonstrate decreased induction of human cytochrome P450.
  • R is an independent chemical moiety that increases an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil and causes a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor and wherein A is H— or is an independent chemical moiety that increases an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil and causes a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor.
  • A is H- and R is Et2NCH2CH2-, in some embodiments A is H- and R is
  • A is H- and R is
  • A is selected from the group consisting of H- and .
  • R comprises
  • R' is selected from the roup consisting of H, methyl, eth l, i ' -Pr, and i ' -Bu? R"
  • the compounds have an increased affinity for a bacterial RNA polymerase (e.g., a MTB RNA polymerase) and a decreased affinity for a human pregnane X receptor.
  • a bacterial RNA polymerase e.g., a MTB RNA polymerase
  • the steric clash of A or R with residues in the binding pocket of the human pregnane X receptor reduces an affinity of the compound for the human pregnane X receptor. Consequently, in some embodiments the steric clash thus reduces the induction (e.g., an activity) of a cytochrome P450 and/or other related proteins.
  • some embodiments provide that the A moiety or the R moiety interacts with a sigma factor of the RNA polymerase.
  • the A or the R interacts with a beta region of the RNA polymerase and in some embodiments the A or the R interacts with a beta prime region of the RNA polymerase.
  • the A or R group adds steric volume to the compound, and thus in some embodiments the steric clash comprises an interaction of the A or the R with an amino acid of the human pregnane X receptor at a position in the primary sequence selected from the group consisting of 237, 238, 239, 240, 241, 242, and 243.
  • the steric clash comprises an interaction of the A or the R with an amino acid in the human pregnane X receptor selected from the group consisting of Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243 (see, e.g., UniProt accession number 075469 or PDB accession number lskx).
  • the compounds exhibit increased potency against Mycobacterium
  • the compound when compared to conventional compounds. Accordingly, in some embodiments the compound has a MIC90 against Mycobacterium tuberculosis that is less than the MIC90 of rifampin against Mycobacterium tuberculosis. For instance, in some embodiments the MIC90 of the compounds encompassed by the present technology against a Mycobacterium tuberculosis is approximately 0.02 - 0.08 micromolar. Furthermore, in some embodiments the compound inhibits a rif- resistant RNA polymerase at a concentration that is lower than the concentration at which rifalazil inhibits the rif-resistant RNA polymerase.
  • the concentration at which the compound inhibits a rif-resistant RNA polymerase is approximately one-half to one-fifth of the concentration at which rifalazil inhibits the rif-resistant RNA polymerase. In some embodiments the concentration at which the compound inhibits a rif-resistant RNA polymerase is approximately one-fifth to one-fiftieth of the concentration at which rifalazil inhibits the rif-resistant RNA polymerase.
  • RNA-resistant bacteria e.g., a drug-resistant Mycobacterium tuberculosis, e.g., having a mutant RNA polymerase.
  • the compounds are effective against a rif-resistant RNA polymerase that is a Mycobacterium tuberculosis rif-resistant RNA polymerase.
  • the compounds are effective against a Mycobacterium
  • tuberculosis rif-resistant RNA polymerase comprising a S450L substitution (e.g., a substitution of a leucine for the serine at position 450 of the native primary amino acid sequence).
  • a S450L substitution e.g., a substitution of a leucine for the serine at position 450 of the native primary amino acid sequence.
  • the compounds are effective against a S450L substitution.
  • Mycobacterium tuberculosis rif-resistant RNA polymerase comprising a D435V substitution (e.g., a substitution of a valine for the aspartate at position 435 of the native primary amino acid sequence).
  • the technology provides methods of manufacturing antimicrobial compounds.
  • provided herein are embodiments of technology providing methods of manufacturing a compound having the formula
  • the technology provides a compound having the formula
  • some embodiments provide a compound having the formula Me Me
  • the compounds find use in treating a wide range of bacterial infections (e.g., caused by different organisms and characterized by different clinical descriptions).
  • the bacterial infection is caused by a rif-resistant organism.
  • the compound finds use in treating tuberculosis.
  • the compound finds use in treating a bacterial infection that is an infection with Mycobacterium tuberculosis.
  • the bacterial infection is caused by a multidrug-resistant strain of a bacterium and in some embodiments the bacterial infection is caused by an extensively drug-resistant bacterium.
  • the compounds find use in the treatment of subjects that have tuberculosis in combination with another disease or malady.
  • the technology is related to compounds having the formula
  • the compound for use as a medicament for a treatment of a subject infected with HIV, and in some embodiments the compound is used to treat a subject that has AIDS. Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.
  • Figure 1 shows the chemical structures of rifampin (RMP, Figure 1A) and related compounds including rifalazil (RZL, Figure IB).
  • FIG. 2a shows a schematic of a synthesis known as Scheme 1 for producing embodiments of the compounds provided herein.
  • Figure 2b shows a schematic of a synthesis for producing embodiments of the compounds provided herein.
  • Figure 2c shows a schematic of a synthesis for producing embodiments of the compounds provided herein.
  • Figure 2d shows a schematic of a synthesis for producing embodiments of the compounds provided herein.
  • Figure 2e shows a schematic of a synthesis for producing embodiments of the compounds provided herein.
  • Figure 3 shows data plots for experiments testing the activation of hPXR by embodiments of the compounds provided herein.
  • Figure 4a shows plots of the linear plasma mean concentration versus time for analog 2b in single dose studies.
  • Figure 4b shows plots of the linear plasma mean concentration versus time for analog 2b in multiple dose studies.
  • Figure 5 shows a plot of the linear lung tissue mean concentration versus time for analog 2b in a multiple dose study.
  • Figure 6 shows a schematic of a synthesis for producing embodiments of the compounds provided herein.
  • the term "effective amount” refers to the amount of a compound required to treat or prevent an infection.
  • the effective amount of an active compound used for therapeutic or prophylactic treatment of conditions caused by or contributed to by a microbial infection varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • administering refers to a method of giving a composition to a patient, by a route such as inhalation, ocular administration, nasal instillation, parenteral administration, dermal
  • parenteral administration includes intrathecal, intraarticular, intravenous, intraperitoneal, subcutaneous, and intramuscular administration.
  • the optimal method of administration of a drug or drug combination to treat a particular disease varies depending on various factors, e.g., the oral bioavailability of the drug(s), the anatomical location of the disease tissue, and the severity of disease.
  • co-administration refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co ⁇ administration of two or more agents or therapies is concurrent. In other embodiments, a first agent or therapy is administered prior to a second agent or therapy.
  • a first agent or therapy is administered prior to a second agent or therapy.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co- administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for therapeutic use.
  • compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
  • terapéuticaally effective dose refers to an amount of a therapeutic agent sufficient to bring about a beneficial or desired clinical effect. Said dose can be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired (e.g., aggressive versus conventional treatment).
  • treat refers to administering a pharmaceutical composition for prophylactic or therapeutic purposes, wherein the growth of bacteria is prevented, stabilized, or inhibited; or wherein bacteria are killed.
  • treating refers to administering or prescribing a
  • prevent disease refers to prophylactic treatment of a patient who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease.
  • treat disease or use for “therapeutic treatment” refers to administering treatment to a patient already suffering from a disease to improve the patient's condition.
  • treating is the administration to an animal either for therapeutic or prophylactic purposes.
  • animal specifically include mammals, such as a human, as well as cattle, horses, dogs, cats, and birds, but also can include many other species to which a drug is administered.
  • alkyl and the prefix “alk-” are inclusive of both straight chain and branched chain saturated or unsaturated groups, and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups.
  • acyclic alkyl groups are from 1 to 6 carbons.
  • Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms.
  • Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl groups.
  • Alkyl groups may be substituted with one or more substituents or unsubstituted.
  • substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halogen, alkylsilyl, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
  • alk the number of carbons contained in the alkyl chain is given by the range that directly precedes this term, with the number of carbons contained in the remainder of the group that includes this prefix defined elsewhere herein.
  • C1-C4 alkaryl exemplifies an aryl group of from 6 to 18 carbons (e.g., see below) attached to an alkyl group of from 1 to 4 carbons.
  • aryl refers to a carbocyclic aromatic ring or ring system. Unless otherwise specified, aryl groups are from 6 to 18 carbons. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl groups.
  • heteroaryl refers to an aromatic ring or ring system that contains at least one ring heteroatom (e.g., 0, S, Se, N, or P). Unless otherwise specified, heteroaryl groups are from 1 to 9 carbons.
  • Heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole, indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl, purinyl, and carbazolyl groups.
  • heterocycle refers to a non- aromatic ring or ring system that contains at least one ring heteroatom (e.g., 0, S, Se, N, or P). Unless otherwise specified, heterocyclic groups are from 2 to 9 carbons. Heterocyclic groups include, for example, dihydropyrrolyl, tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl,
  • Aryl, heteroaryl, or heterocyclic groups may be unsubstituted or substituted by one or more substituents selected from the group consisting of Ci-6 alkyl, hydroxy, halo, nitro, Ci-6 alkoxy, Ci-6 alkylthio, trifluoromethyl, Ci-6 acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, Ci-6 alkoxycarbonyl, alkaryl (where the alkyl group has from 1 to 4 carbon atoms), and alkheteroaryl (where the alkyl group has from 1 to 4 carbon atoms).
  • alkoxy refers to a chemical substituent of the formula -OR, where R is an alkyl group.
  • aryloxy is meant a chemical substituent of the formula -OR', where R' is an aryl group.
  • C x - y alkaryl refers to a chemical substituent of formula -RR', where R is an alkyl group of x to y carbons and R' is an aryl group as defined elsewhere herein.
  • C x - y alkheteraryl refers to a chemical substituent of formula RR", where R is an alkyl group of x to y carbons and R" is a heteroaryl group as defined elsewhere herein.
  • halide or "halogen” or “halo” refers to bromine, chlorine, iodine, or fluorine.
  • non- vicinal 0, S, or N refers to an oxygen, sulfur, or nitrogen heteroatom substituent in a linkage, where the heteroatom substituent does not form a bond to a saturated carbon that is bonded to another heteroatom.
  • bacteria and “bacterium” refer to prokaryotic organisms of the domain Bacteria in the three-domain system (see, e.g., Woese CR, et al., Proc Natl Acad Sci USA 1990, 87: 4576 - 79). It is intended that the terms encompass all microorganisms considered to be bacteria including Mycobacterium, Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. In some embodiments, bacteria are capable of causing disease and product degradation or spoilage.
  • a "pathogen” is a bacterium that is capable of causing a disease.
  • "Strain” as used herein in reference to a microorganism describes an isolate of a microorganism (e.g., bacteria, virus, fungus, parasite) considered to be of the same species but with a unique genome and, if nucleotide changes are non- synonymous, a unique proteome differing from other strains of the same organism. Strains may differ in their non-chromosomal genetic complement. Typically, strains are the result of isolation from a different host or at a different location and time, but multiple strains of the same organism may be isolated from the same host.
  • bacterial infection refers to the invasion of a host animal by pathogenic bacteria.
  • the infection may include the excessive growth of bacteria that are normally present in or on the body of an animal or growth of bacteria that are not normally present in or on the animal.
  • a bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to a host animal.
  • an animal is "suffering" from a bacterial infection when an excessive amount of a bacterial population is present in or on the animal's body, or when the presence of a bacterial population(s) is damaging the cells or other tissue of the animal.
  • Persistent bacterial infection refers to an infection that is not completely eradicated through standard treatment regimens using anti ⁇ bacterial agents. Persistent bacterial infections are caused by bacteria capable of establishing a cryptic or latent phase of infection and may be classified as such by culturing the bacteria from a patient and demonstrating bacterial survival in vitro in the presence of anti-bacterial agents or by determination of anti-bacterial treatment failure in a patient. An in vivo persistent infection can be identified through the use of a reverse transcriptase polymerase chain reaction (RT-PCR) to demonstrate the presence of 16S rRNA transcripts in bacterially infected cells after treatment with anti-bacterial agents (see, e.g., Antimicrob. Agents Chemother. 12- 3288-97, 2000).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • rifampin and "rifampicin” are interchangeable.
  • rif-resistant and “rif-resistance” and “RifR” are meant to describe a bacterium, bacteria, or an RNA polymerases that are not readily inhibited by a rifampicin or a related derivative or analog drug in the rifampicin family, including but not limited to rifampicin, rifamycin, rifapentine, rifabutin, and rifalazil.
  • MIC90 or “minimum inhibitory concentration, 90%” is used to refer to the lowest concentration of a compound that causes >90% growth inhibition (e.g., growth is ⁇ 10% the growth in the absence of the compound). MIC90 values are used to characterize the potency of a compound and a lower MIC90 value indicates a more potent compound.
  • IC50 or “inhibition constant, 50%” refers to the lowest concentration of a compound that causes the activity of a biological molecule (e.g., an enzyme) to decrease to 50% of the activity of the biological molecule in the absence of the compound. IC50 values are used to characterize the potency of a compound and a lower IC50 value indicates a more potent compound.
  • variant and mutant when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties.
  • conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine? a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine?
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are : valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. More rarely, a variant may have "non- conservative" changes (e.g., replacement of a glycine with a tryptophan).
  • Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have fewer than 10%, and preferably fewer than 5%, and still more preferably fewer than 2% of the amino acids being changed (whether substitutions, deletions, and so on).
  • nucleic acids or proteins specifies the type of mutation and base or amino acid changes.
  • a nucleotide substitution e.g., 76A>T
  • the number is the position of the nucleotide from the 5' end
  • the first letter represents the wild type nucleotide
  • the second letter represents the nucleotide which replaced the wild type.
  • the adenine at the 76th position was replaced by a thymine. If it becomes necessary to differentiate between mutations in genomic DNA, mitochondrial DNA,
  • cDNA complementary DNA
  • RNA RNA
  • cDNA complementary DNA
  • RNA RNA
  • a simple convention is used. For example, if the 100th base of a nucleotide sequence is mutated from G to C, then it would be written as g.l00G>C if the mutation occurred in genomic DNA, m.l00G>C if the mutation occurred in mitochondrial DNA, c.l00G>C if the mutation occurred in cDNA, or r.l00g>c if the mutation occurred in RNA.
  • amino acid substitution e.g., D111E
  • the first letter is the one letter code of the wild type amino acid
  • the number is the position of the amino acid from the N-terminus
  • the second letter is the one letter code of the amino acid present in the mutation.
  • Nonsense mutations are represented with an X for the second amino acid (e.g. D111X).
  • X for amino acid deletions (e.g. AF508, F508del)
  • the Greek letter ⁇ (delta) or the letters "del” indicate a deletion.
  • the letter refers to the amino acid present in the wild type and the number is the position from the N terminus of the amino acid where it is present in the wild type.
  • domain when used in reference to a polypeptide refers to a subsection of the polypeptide that possesses a unique structural and/or functional characteristic; typically, this characteristic is similar across diverse polypeptides.
  • the subsection typically comprises contiguous amino acids, although it may also comprise amino acids that are far apart in the primary sequence but that act in concert or that are in close proximity in the tertiary or quaternary structure due to folding or other configurations.
  • the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or a polypeptide or its precursor (e.g., proinsulin).
  • a functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained.
  • portion when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide comprising at least a portion of a gene” may comprise fragments of the gene or the entire gene.
  • the term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • the sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' non-translated sequences.
  • the term “gene” encompasses both cDNA and genomic forms of a gene.
  • Genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript).
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene.
  • the 3' flanking region may contain sequences that direct the termination of transcription or posttranscriptional cleavage.
  • wild-type when made in reference to a gene refers to a gene that has the characteristics of a gene isolated from a naturally occurring source.
  • wild-type when made in reference to a gene product refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source.
  • naturally-occurring as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally-occurring.
  • a wild-type gene is frequently that gene which is most frequently observed in a population and is thus arbitrarily designated the "normal” or "wild-type” form of the gene.
  • Strains of an organism may comprise naturally- occurring variants that differ from the wild-type form.
  • the term "modified” or “mutant” when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product that displays modifications in sequence or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • sample is used in its broadest sense. In one sense it can refer to an animal cell or tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • in vitro environments may include, but are not limited to, test tubes and cell cultures.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.
  • the modeling was based on the 2.5 A resolution structure of rifabutin (RFB) in complex with the Thermus thermophilus RNAP holoenzyme (PDB ID: 2a68) (Artsimovitch, I. et al. Cell 2005, 122 (3): 351-63).
  • RRB rifabutin
  • PDB ID: 2a68 Thermus thermophilus RNAP holoenzyme
  • the rifamycin-binding site is highly conserved among bacteria; therefore this structure provides a basis for understanding how rifamycin analogs interact with the MTB RNA polymerase.
  • the crystal structure (PDB accession number 2a68) contains two complete complexes. Observed differences between these two complexes and what is seen in the related complexes present in a structure of rifapentine in complex with the T. thermophilus RNAP holoenzyme (PDB accession number 2a69) suggest that, at least in the free holoenzyme, the sigma factor hairpin loop exists in two distinct physiologically relevant conformations.
  • the modeling studies described herein indicate that there are a number of analogs with a range of size, flexibility, and spatial variation that interact with the sigma hairpin loop and other portions of the RNAP complex. These variations are further extended by the differences in attachment points on the planned subclasses. These analogs were chosen to increase drug potency by doing one or more of the following: (a) making additional contacts with the sigma factor, beta, or beta prime regions of the RNAP and/or (b) interfering with the binding of the sigma factor or further occluding the channel.
  • Modeling the RLZ/RNAP complex displays the presence of interaction surfaces at 4.5 A between the analog tail and residues of the surrounding RNAP.
  • the models show that these analogs have the potential to interact with different regions of RNAP and can participate in additional contacts with RNAP sidechains that may have different potency profiles.
  • RMP is the largest activator of hPXR currently known and is a potent activator of hPXR (Chrencik, J. E. et al. Mol Endocrinol 2005, 19: 1125-34). RMP fills the ligand-binding pocket very well. Differences in RMP activation of the human versus the mouse PXR are interpreted in light of structural information. For example, Leu308 in the human PXR is replaced by Phe in the mouse and Ser247 is replaced by Trp in the mouse. These changes help explain the differential RMP activation of the human and mouse PXRs, e.g., due to impaired binding of RMP to the mouse PXR.
  • the relative potency of CYP3A4 induction is RMP > rifapentine > rifabutin > RLZ.
  • Modeling the hPXR binding site with these four rifamycins in spatial relation to resolved hPXR residues shows that there are seven hPXR residues, Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243, in very close proximity to the synthetic branch point for the embodiments of the analogs designed herein. These residues are resolved in each of the five hPXR structures and, other than one of the Phe-237 rings, have fairly conserved relative coordinates. This, along with the presence of Pro-241, would suggest that these residues are located in a more rigid region of the hPXR ligand-binding pocket.
  • the hPXR ligand-binding pocket is large, flexible, and capable of adapting itself to bind a large variety of ligands.
  • the modeling suggests that the proposed analogs designed to target RNAP sigma factors have the added benefit of overwhelming the normally promiscuous hPXR binding pocket. It appears that the tails of the designed analogs prevent binding of the compounds to hPXR by projecting into rigid, sterically encumbered regions of hPXR, although an understanding of the mechanism is not needed to practice the technology described herein, nor is the technology limited by any particular mechanism of action.
  • Impeded binding of compounds to hPXR is contemplated to reduce induction of CYP450.
  • the analog is a compound having the formula
  • A is H- and R is Et2NCH2CH2-, in some embodiments A is H- and R is in some embodiments, A is H- and R is
  • the A or R group adds steric volume to the compound, and thus in some embodiments the steric clash comprises an interaction of the A or the R with an amino acid of the human pregnane X receptor at a position in the primary sequence selected from the group consisting of 237, 238, 239, 240, 241, 242, and 243. In some embodiments the steric clash comprises an interaction of the A or the R with an amino acid in the human pregnane X receptor selected from the group consisting of Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243 (see, e.g., UniProt accession number 075469 or PDB accession number lskx).
  • R is a moiety defined as one of the following
  • R is a moiet defined as one of the following:
  • n 1-3.
  • the compounds according to the technology have a structure according to one of the following:
  • the present technology provides a fully tethered 2-aminoresorcinol monoether that is annulated onto the rifamycin S framework in a single step.
  • This approach allowed the design of a wide range of tethers off the "southeastern" part of the rifalazil-type template and, more importantly, minimized difficult synthetic transformations and product
  • Each 2- aminoresorcinol ether (7, 11a, lib) was then annulated onto rifamycin S (12) to provide target compounds 2b - 2d in 35 - 74% yields following a two- stage purification utilizing medium pressure and then preparative plate silica gel chromatography.
  • compounds are contemplated that are synthesized according to a schema such as
  • step of reacting with Et 3 N/HC0 2 H, Pd(OAc) 2 , and PPh 3 in DMF is performed in some embodiments according to the method of Cabri, et al. as described in J. Org. Chem. 55: 350-353 (1990), incorporated herein by reference.
  • These compounds are contemplated to have activities within the scope of the technology and find use in applications as described herein.
  • a pharmaceutically effective amount varies depending on the therapeutic agent used, the subject's age, condition, and sex, and on the extent of the disease in the subject. Generally, the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. The dosage can also be adjusted by the individual physician or veterinarian to achieve the desired therapeutic goal.
  • “pharmaceutically effective amount” will depend on the route of administration, the type of subject being treated, and the physical characteristics of the specific subject under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical, veterinary, and other related arts. This amount and the method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication, and other factors that those skilled in the art will recognize.
  • a rifalazil or rifampin analog according to the technology provided herein, a derivative thereof, or a pharmaceutically acceptable salt thereof is administered in a pharmaceutically effective amount.
  • the dosage amount and frequency are selected to create an effective level of the compound without substantially harmful effects.
  • the dosage of a rifampin analog or related compounds will generally range from 0.001 to 10,000 mg/kg/day or dose (e.g., 0.01 to 1000 mg/kg/day or dose? 0.1 to 100 mg/kg/day or dose).
  • Methods of administering a pharmaceutically effective amount include, without limitation, administration in parenteral, oral, intraperitoneal, intranasal, topical, sublingual, rectal, and vaginal forms.
  • Parenteral routes of administration include, for example, subcutaneous, intravenous, intramuscular, intrastemal injection, and infusion routes.
  • a rifampin analog, a derivative thereof, or a pharmaceutically acceptable salt thereof is administered orally.
  • compositions preferably comprise one or more compounds of the present invention associated with one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • Pharmaceutically acceptable carriers are known in the art such as those described in, for example, Remingtons Pharmaceutical
  • a single dose of a rifalazil or rifampin analog or related compounds is administered to a subject.
  • multiple doses are administered over two or more time points, separated by hours, days, weeks, etc.
  • compounds are administered over a long period of time (e.g., chronically), for example, for a period of months or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months or years? e.g., for the lifetime of the subject).
  • compounds may be taken on a regular scheduled basis (e.g., daily, weekly, etc.) for the duration of the extended period.
  • a rifalazil or rifampin analog or a related compound is co -administered with another compound or more than one other compound (e.g., 2 or 3 or more other compounds).
  • the rifalazil or rifampin analog or related compound is co-administered with an antibiotic such as, e.g., isoniazid, pyrazinamide, ethambutol, or streptomycin.
  • the antibiotic co-administered with the rifampicin or rifampin analog is an antibiotic such as, e.g., isoniazid, pyrazinamide, ethambutol, or streptomycin.
  • the antibiotic co-administered with the rifampicin or rifampin analog is an antibiotic such as, e.g., isoniazid, pyrazinamide, ethambutol, or streptomycin.
  • aminoglycoside e.g., amikacin, kanamycin
  • a polypeptide e.g., capreomycin, viomycin, enviomycin
  • a fluoroquinolone e.g., ciprofloxacin, levofloxacin
  • a thioamide e.g., ethionamide, prothionamide, tiocarlide
  • the antibiotic co ⁇ administered with a rifalazil or rifampin analog is rifabutin, a macrolide (e.g., clarithromycin), linezolid, thioacetazone, thioridazine, arginine, vitamin D, or R207910.
  • a steroid e.g., a corticosteroid (e.g., prednisolone or dexamethasone) is co-administered with the rifalazil or rifampin analog.
  • Thalidomide is co- administered with a rifalazil or rifampin analog in some embodiments.
  • interferon- ⁇ is co-administered; in some embodiments, meropenem, morinamide, terizidone, or clavulanic acid is co ⁇ administered.
  • co-amoxiclav, clofazimine, prochlorperazine, metronidazole, or PA-824 is co-administered with a rifalazil or rifampin analog or related compound.
  • Dzherelo, Anemin, Svitanok, Lizorm, Immunoxel, or Immunitor is co-administered with a rifalazil or rifampin analog.
  • kits for use in the instant methods comprise one or more containers comprising rifalazil or rifampin, a derivative thereof, or a pharmaceutically acceptable salt thereof, and/or a second agent, and in some variations further comprise instructions for use in accordance with any of the methods provided herein.
  • the kit may further comprise a description of selecting an individual suitable for treatment.
  • Instructions supplied in the kits of the technology are typically written instructions on a label or package insert (e.g., a paper insert included with the kit), but machine -readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also contemplated. Examples
  • RFB drifted approximately 1 A toward the cleft of the complex from its
  • Hydrogen atoms, modified portions of RFB and protein side chains within ⁇ 16 A of the modified portions were allowed to move during the conformational search and energy minimizations.
  • Generated poses were ranked by interaction energies and duplicate poses based on a RMSD cutoff removed.
  • RNAP complex soaked with water to a surrounding distance of 6 A.
  • a series of energy minimizations was then conducted using the MMFF94x force field to relax the complex.
  • hydrogen atoms and then the water molecules were allowed to relax while the entire analog and all of the RNAP atoms were held fixed.
  • the modified portions of RFB and side chains of RNAP within 16 A were also allowed to relax with the water molecules and hydrogen atoms.
  • the entire analog molecule, residues of RNAP within 16 A, and the water molecules and hydrogen atoms were allowed to relax.
  • the relaxed complexes were then examined to determine how the proposed analog may interact with the sigma factor or other portions of RNAP (specifically the beta and beta prime subunits).
  • 3D structures were generated using the 2.5 A resolution crystal structure for Thermus thermophilus RNAP in complex with rifabutin (RFB) obtained from the Protein Data Bank (PDB accession number 2a68). As described above, coordinates for RFB were relaxed in the presence of the RNAP using a series of energy minimizations with decreasing constraints on the surrounding protein atoms and water molecules. Coordinates for RMP, rifapentine, and RLZ were created and relaxed in the same fashion after modifying RFB. The 2.8 A resolution crystal structure of hPXR in complex with rifampin was retrieved from the Protein Data Bank (PDB accession number lskx).
  • the l-amino-4- methylpiperazine tail of RMP and three hPXR loops adjacent to the binding pocket are disordered and unresolved in the structure.
  • the naphthalene portions of the four rifamycins generated from the 2a68 structure were overlayed onto the naphthalene portion of RMP in complex with hPXR structure lskx.
  • four other relatively complete hPXR structures are available.
  • the hPXR apo structure (PDB accession number lilg) and hPXR complexes with SR12813 (PDB accession number lilh), hyperforin (PDB accession number lml3), and colupulone (PDB accession number 2qnv) were obtained from the Protein Data Bank. These four hPXR complexes were superimposed onto the hPXR structure of lskx containing the four modeled-in rifamycins. Coordinates were not relaxed with energy minimization due to many missing residues.
  • the mobile phase was a 15 min binary gradient of acetonitrile (containing 0.1 % TFA) and water (20-90%).
  • Thin-layer chromatography (TLC) was performed on silica gel GHLF plates (250 microns) purchased from Analtech. Extraction solutions were dried over MgSC prior to concentration.
  • 2-Amino-3-(2-(diethylamino)ethoxy)phenol (7) 2-(Diethylamino)ethyl ether 6 (1.8 g, 5.2 mmol) was dissolved in 10% acetic acid in methanol (50 mL) in a 250 mL Parr hydrogenation bottle. Catalyst (20% Pd(OH) 2 /C, 0.1 g) was added and the mixture was hydrogenated at 40 psi H 2 for ⁇ 20 h. The reaction mixture was rapidly filtered over Celite®, and the filtrate was concentrated and diluted with ethyl acetate.
  • Benzoxazinorifamycin (2b) A mixture of aminophenol 7 (0.336 g, 1.5 mmol), rifamycin S (12; 2.085 g, 3 mmol) and 1,4-dioxane (20 mL) was stirred at room temperature overnight. The mixture was then concentrated to a black solid that was dissolved in 20 mL of methanol and treated with MnC>2 (0.3 g, 3.45 mmol). The mixture was stirred at room temperature for 30 min, filtered over Celite® and the filtrate concentrated to a dark residue that was purified by flash silica gel chromatography eluting with dichloromethane : methanol (95 : 5 to 90 : 10).
  • Benzoxazinorifamycin (2c) A mixture of aminophenol 11a (80 mg, 0.22 mmol), rifamycin S (12; 220 mg, 0.32 mmol) and 1,2-dichloroethane (10 mL) was stirred at room temperature for 16 h. The reaction mixture was then concentrated to a black solid that was dissolved in 10 mL of methanol and treated with MnC>2 (80 mg, 0.92 mmol). The mixture was stirred at room temperature for 30 min, filtered over Celite®, and concentrated to a dark residue that was purified by flash silica gel chromatography eluting with dichloromethane : methanol : NH4OH (94 : 6 : 0.5).
  • the Low Oxygen Recovery (LORA) in vitro assay was designed to detect compounds which may have the potential for shortening the duration of therapy through (more) efficient killing of the non-replicating persistor (NRP) population.
  • the assay involves l) adaptation of MTB to low oxygen through gradual, monitored, self-depletion of oxygen during culture in a sealed fermenter, 2) exposure for 10 days of the low-oxygen adapted culture to test compounds in microplates that are maintained under an anaerobic environment, thus precluding growth, and 3) subsequent evaluation of MTB viability as determined by the ability to recover.
  • Recovery/viability is determined either a) by (aerobic) subculture onto solid, drug- free media and determination of colony forming units or b) by the extent to which a luciferase-expressing strain can recover the ability to produce luminescence.
  • the wild-type MTB RNAP and the RifR mutants were prepared as previously described with minor alterations. See, e.g., Gill, S. K. & Garcia, G. A. Tuberculosis 2011, 91 : 361-9.
  • the sonication method was preferred over the freeze/thaw method.
  • the protocol outlined by Gill and Garcia was followed.
  • the pAvitag vector (modified pMSCG7 vector with an Avitag introduced between Bglll and Kpnl sites) was linearized with Sspl at 37°C for 1 h and the reaction product was purified using the Qiagen PCR kit.
  • the linearized pAvitag vector (1.6-2.0 ⁇ g) was treated with T4 DNA polymerase in 10x T4 polymerase buffer, 5 mM DTT, and 4 mM dGTP in a final reaction volume of 60 ⁇ iL. The reaction was incubated for 30 min at 22°C and then for 20 min at 75°C before being stored at -20°C.
  • PCR primers were designed to amplify the Rv2703/sigA gene encoding SigA from pSROl.
  • the primers included an overhang sequence that complemented the vector Ligation Independent Cloning (LIC) overhangs.
  • the sigA gene was purified via Qiagen PCR kit.
  • the purified PCR product (0.2 pmol) was incubated with T4 DNA polymerase, 5 mM DTT, 4 mM dCTP, 10x T4 DNA polymerase in a final reaction volume of 20 iL. The reaction was incubated for 30 min at 22°C and then for 20 min at 75°C and stored at -20°C.
  • the treated sigA was incubated with treated pAvitag vector (-0.2 pmol) for 10 min at 22°C. Then 6.25 mM EDTA was added followed by incubation at 22°C for 5 min before reducing the temperature to 4°C.
  • the annealed pAvitag vector containing sigA was transformed into BL21(DE3) CodonPlus RIPL cells.
  • SigA protein in BL2l(DE3) CodonPlus RIPL cells, the cells were grown in 500 mL of 2x TY liquid cultures containing 100 ⁇ g/mL
  • Ni 2+ -NTA wash buffer 300 mM NaCl, 50 mM NaH 2 P0 4 , 20 mM imidazole, pH 8.0
  • the protein was then eluted in 6 mL of Ni 2+ -NTA elute buffer (300 mM NaCl, 50 mM NaH 2 P0 4 , 250 mM imidazole, pH 8.0).
  • the protein was concentrated to a final volume of -500iL and then sterile-filtered with 0.22-micromolar syringe before being applied to a HiPrep 16/60 Sephacryl S-200 HR (GE Healthcare) column using RNAP storage buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 0.1 mM DTT, 0.1 M NaCl) as the running buffer.
  • RNAP storage buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 0.1 mM DTT, 0.1 M NaCl
  • concentration ranges used for MTB RNAP (S450L) with SigA were as follows: for 2a (8.2-2000 micromolar); for 2b (3.3-800 micromolar); for 2c and 2d (1.64-400 micromolar); for 2e (6.55-1600 micromolar).
  • the final concentration of the wild-type MTB RNAP was 10 nM and the final concentrations of the mutant RNAPs were 100 nM.
  • RNAP and SigA were incubated for 30 min on ice in lx RNAP reaction buffer (40 mM Tris-HCl (pH 8.0), 50 mM KC1, 10 mM MgCl 2 , 0.01% Triton X-100) before adding the analog and DNA nanocircle template (80 nM). Each reaction was initiated upon the addition of NTP solution (500 micromolar of each NTP). The IC50 values were determined via non-linear regression to a modified four parameter logistic equation.
  • hPXR activation assay system from Puracyp, Inc. was used. The manufacturer's protocol was followed for the 96 well plate assay. Briefly, the DPX2 cells were thawed in a 37°C water bath and mixed thoroughly with culture media. Then 100 ⁇ of cell mixture was transferred into each well and the plate was incubated overnight in a 5% CO2 incubator at 37°C. The following day, the dosing media was thawed in a 37"C water bath. The dilutions of RLZ and analogs (2a-2e) and RMP (l, positive control) were prepared as described in the manual. The 96 well plate was removed from the incubator and liquid from each well was discarded before adding 100 ⁇ of the dilutions to the specific wells. Each dilution of the rifamycin derivative was tested in duplicate. The plate was placed in the 5%
  • Equation 1 y 1 + [(M3 - 1)/(1 + 10K Mi - M ° )*M2] )] (l)
  • M3 is the ECMAX
  • 1 is the lower limit of the assay
  • MO is the log of the rifamycin concentration
  • Ml is the log of the EC50
  • M2 is the Hill slope.
  • the data were normalized such that the lower limit was set to 1.
  • Ml, M2, and M3 were fit by the regression.
  • Test compound stock solutions were prepared at 200 micromolar in
  • venous blood was collected via retro-orbital bleeding in BD Vacutainer® spray-coated K2EDTA tubes at 0.5, 1, 2, 4, 8, and 24 hours post- dose. Tubes were inverted several times and kept on ice. Blood was transferred to polypropylene tubes and centrifuged at 4,000 x gior 30 minutes at 4°C. The harvested plasma was transferred to new polypropylene tubes and stored at -80°C until analysis. To each sample, a 3x volume of chilled acetonitrile was added containing 0.2 micromolar internal standard (IS). The solution was vortexed and then subsequently centrifuged at 10,000 x gior 15 minutes.
  • IS micromolar internal standard
  • Calibration standard samples were prepared by spiking the stock solution of analog 2b in acetonitrile into mouse plasma to yield the following concentrations ⁇ 0.097656, 0.195313, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, and 50 micromolar.
  • mice were dosed once daily for 5 consecutive days by oral gavage. Blood samples were collected at time points of 0.5, 1, 2, 4, 8, and 24 hours and analyzed in the same way as in the single-dose study. After collecting the blood, the mice were sacrificed by carbon dioxide asphyxiation. Lung tissue was aseptically removed, rinsed in 3 ml PBS, air dried on sterilized gauze pads, weighed, and suspended in 4 x (w/v solvent/tissue) PBS buffer. Lung tissue was homogenized, mixed, and extracted with 3 x acetonitrile containing internal standard at 0.2 micromolar and centrifuged at 10,000 x gior 15 minutes at 4°C. The supernatant was collected for LC-MS/MS analysis.
  • Calibration standard lung samples were prepared by spiking the stock solution of compound (in MeOH or MeCN) into homogenized mouse lungs and extracting with a 3 x volume of acetonitrile to yield the following concentrations: 0.024, 0.049, 0.098, 0.195, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 micromolar.
  • a blank blank lung tissue extracted with a 3 x volume of IS
  • a double blank blank lung tissue extracted with a 3 x volume of pure acetonitrile
  • the compounds were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic conditions (Table l). Briefly, the 8 _ day microplate- based assay using Alamar blue reagent (added on day 7) for determination of growth (MABA) (see, e.g., Collins, L. & Franzblau, S. Antimicrob. Agents
  • MIC90 Minimum inhibitory concentration
  • LORA activity for RLZ analog 2e is strikingly poor being essentially inactive (MIC90 > 6.72 micromolar).
  • the addition of the RLZ side chain into the 5' position of 2d introduces another basic moiety, which may impede transport across the cell membrane of the non-replicating bacterial strain of the LORA.
  • inhibition constants (IC50) for RLZ and the analogs against the wild-type MTB RNAP and three rif-resistant (RifR) MTB RNAP mutants were determined via dose- response assays. Each compound was tested in duplicate dilution series ranging over the concentrations as specified below. The data were plotted as the logarithm of the rifamycin or analog concentration versus the activity relative to the activity without the RLZ or analog (expressed as a percentage). The data were then fit by nonlinear regression. The log IC50 values and the standard errors of the fit are reported in Table 2. The log IC50 values are such that the IC50 values are in units of micromolar.
  • Negative log IC50 values reflect IC50 values less than in the micromolar range (e.g., in the nM range). Values were fit to a four-parameter logistic regression model with the top and bottom limits set at 100 and 0 respectively. The average Hill slope is 1.02. Table 2. Log IC 50 values and standard errors for rifalazil & analogs against RNAP
  • the MTB D435V mutant was inhibited at 5" to 50-fold lower concentrations of 2b, 2c, 2d & 2e relative to RLZ. Interestingly, the H445Y mutant appears to retain resistance to all tested rifamycins.
  • Analog 2d was fit to a dose-response curve that revealed a 6-fold maximal activation of hPXR and an EC50 of ⁇ 6 micromolar (Figure 3). This analog also starts to exhibit loss of cell viability at 25 micromolar (Table 4), such that the 100 micromolar data point was not used in the dose-response curve fit. Analog 2e shows hPXR activity very similar to that of RLZ, with no activation nor loss of cell viability below 25 micromolar and ⁇ 3-fold activation and -25% loss of cell viability at 100 micromolar.
  • rifalazil and analog 2b were evaluated for metabolic stability in human microsomes.
  • the data produced by the experiments demonstrated that each is relatively stable with estimated half-lives of 65 and 54 minutes, respectively (Table 5).
  • the estimated half- life of rifalazil in mouse microsomes is 53 minutes while that of the analog 2b is 141 minutes.
  • rifalazil compounds were synthesized comprising a dansyl moiety.
  • all reagents were commercially available and used without further purification. Routine ⁇ and 13 C NMR spectra were obtained on a Varian
  • reaction mixture was rapidly filtered over Celite®, and the filtrate was concentrated and diluted with ethyl acetate.
  • the solution was washed with 5% aqueous sodium carbonate, dried, and concentrated to an oil that was purified by silica gel fast flash chromatography eluting with hexane/ethyl acetate (l ; l).
  • Benzoxazinorifamycin dansyl probe 43b A mixture of dansyl aminophenol 42b (0.24 g, 0.541 mmol), rifamycin S (12; 0.75 g, 1.1 mmol), and 1,2-dichloroethane (20 mL) was stirred at room temperature overnight. The mixture was then concentrated to a black solid that was dissolved in 20 mL of methanol and treated with MnC>2 (0.3 g, 3.5 mmol).
  • Ni-NTA His»Bind ® resin was from Novagen (San Diego, CA).
  • the nucleotide triphosphates (NTPs) were from Roche Applied Science (Indianapolis, IN). PhastGel Precast Gels and SDS Buffer Strips were from VWR (Arlington Heights, IL).
  • the BkrRad Protein Assay kit was from BkrRad (Hercules, CA).
  • the EC2880 strain permeable strain with toJC ⁇ and imp- mutations was a generous gift from Dr. Michael Hubband (Pfizer Scientific).
  • Rifamycin S was from AAPharmaSyn LLC.
  • Rifampin was from Roche Scientific. Dansyl chloride and dansyl amide were from Sigma-Aldrich.
  • MABA Microplate Alamar Blue Assay
  • LORA Low Oxygen Recovery Assay
  • MIC90 values were determined for E. coliby the microdilution method described previously (Wiegand et al. (2008) "Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances” Nature Protocols 3(2): 163-175; Gill, S. K. and G. A. Garcia (2011) “Rifamycin inhibition of WT and Rif-resistant Mycobacterium tuberculosis and Escherichia coli RNA polymerases in vitro" Tuberculosis 91(5): 361-369).
  • the MIC90 values are also reported against the MTB virulent strain (H37RV) and the E coli strains (Table 7). 43b inhibited the MTB with MIC90 values 0.009 ⁇ and 0.93 ⁇ for MABA and LORA, respectively. The MIC90 values were higher for E. coli strains including the fo/Cknockout strain (EC2880).

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Abstract

La présente invention concerne la technologie relative aux composés antimicrobiens et en particulier, mais non exclusivement, aux analogues de rifalazil ayant une inhibition accrue de l'ARN polymérase et une induction diminuée du cytochrome P450 humain. Les composés ont une affinité accrue pour une ARN polymérase bactérienne (par exemple, une MBT ARN polymérase) et une affinité diminuée pour un récepteur du prégnane X humain. Ainsi, dans certaines formes de réalisation, le choc stérique de A ou R avec des résidus dans la poche de liaison du récepteur du prégnane X humain réduit une affinité du composé pour le récepteur du prégnane X humain. Par conséquent, dans certaines formes de réalisation, le choc stérique réduit ainsi l'induction (par exemple, une activité) d'un cytochrome P450 et/ou d'autres protéines apparentées.
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US10214536B2 (en) 2016-01-29 2019-02-26 The Regents Of The University Of Michigan Amlexanox analogs
US10245255B2 (en) 2011-02-14 2019-04-02 The Regents Of The University Of Michigan Compositions and methods for the treatment of obesity and related disorders

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US10245255B2 (en) 2011-02-14 2019-04-02 The Regents Of The University Of Michigan Compositions and methods for the treatment of obesity and related disorders
US9944652B2 (en) 2013-05-02 2018-04-17 The Regents Of The University Of Michigan Deuterated amlexanox
US10590142B2 (en) 2013-05-02 2020-03-17 The Regents Of The University Of Michigan Deuterated amlexanox
US10214536B2 (en) 2016-01-29 2019-02-26 The Regents Of The University Of Michigan Amlexanox analogs

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