Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
  • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
  • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a vancomycin analog molecule that is modified in three ways in three different portions of the molecule.
• the resulting modified vancomycin compounds are many times more potent than vancomycin itself in both vancomycin-sensitive and vancomycin-resistant bacteria.
• a pharmaceutical composition containing a modified vancomycin and a method of treating a bacterially-infected mammal using a modified vancomycin.
• durable antibiotics are created by deliberate design that may directly address such evolutionary forces.
• the glycopeptide antibiotics were identified as an antibiotic class already endowed with features that avoid many mechanisms of resistance.
• peripheral structural changes were created and examined in the molecules that provide them with additional and now multiple synergistic mechanisms of action, thereby not only increasing their potency but also creating durable antibiotics.
• MRSA methicillin-resistant Staphylococcus aureus
• Vancomycin ( 1 , below) [Harris et al., J. Am. Chem. Soc. 1983 , 105:6915-6922] was disclosed in 1956 and introduced into the clinic in 1958 [McCormick et al., Antibiot. Annu. 1955-1956 , 606-611]. Even after nearly 60 years of clinical use and even with the past widespread use of glycopeptide antibiotics for agricultural livestock (avoparcin) , vancomycin- resistant pathogens have only slowly emerged and vancomycin remains an integral and increasingly important antibiotic today. Clinical resistance was initially observed with vancomycin-resistant Enterococci (VRE, 1987) detected only after 30 years of clinical use
• the primary biological target for vancomycin and the glycopeptide antibiotics is bacterial cell wall precursors containing the dipeptide D-Ala-D-Ala, binding to which results in inhibition of cell wall maturation [Perkins,
• selectivity of the antibiotic class for bacteria versus their mammalian hosts is also an atypical biological target, being a substrate for an enzymatic reaction and a precursor to a structural component of the bacterial cell wall. It is not a protein or nucleic acid biological target subject to changes by a single genetic mutation to the target that can result in resistance.
• the primary mechanism of action of vancomycin involves sequestration of this substrate (D-Ala-D-Ala) for a late-stage enzyme-catalyzed (aPBP or bPBP transpeptidase) reaction used for cell wall cross-linking [Kahne, Chem. Rev. 2005 , 105(2): 425- 448] .
• aPBP or bPBP transpeptidase enzyme-catalyzed
• Vancomycin is also thought to inhibit the preceding step in the cell wall biosynthesis, the aPBP transglycosylase-catalyzed incorporation of lipid intermediate II into the repeating
• vancomycin Further contributing to the durability of vancomycin is the site of action at the bacterial cell wall surface. Cell wall penetration or import is not needed and this permits vancomycin to avoid the common resistance mechanisms mediated by
• organisms initiate a late stage remodeling of their peptidoglycan termini from D-Ala-D-Ala to D-Ala-D-Lac to avoid the action of the antibiotic.
• vancomycin analogs were reported that contain changes at a key single atom site in its target binding pocket (residue 4 carbonyl 0 —> S, NH, 3 ⁇ 4) , the latter two of which were designed to directly address this underlying molecular basis of resistance to
• aMIC Minimum inhibitory concentration. b ATCC 25923.
• VSSA vancomycin-sensitive and vancomycin-resistant bacteria
• vancomycin analogs with another, different second mechanism of action that is also independent of D- Ala-D-Ala/D-Ala-D-Lac binding. This modification also provides synergistic and similarly spectacular improvements in antimicrobial potencies against vancomycin-resistant bacteria (VRE) .
• VRE vancomycin-resistant bacteria
• peripheral modifications are shown to be combinable with the pocket-modified vancomycins to provide even more potent antimicrobial agents whose activity can be attributed to three independent and synergistic mechanisms of action, only one of which requires D- Ala-D-Ala/D-Ala-D-Lac binding.
• Such antibiotics display durable antimicrobial activity that is not prone to rapidly acquired clinical resistance.
• glycopeptide antibiotics including vancomycin have been designed that not only directly overcome the molecular basis of existing vancomycin resistance, but that contain two added peripheral modifications that endow them with two additional independent mechanisms of actions not found in the parent antibiotics . It is shown hereinafter that such peripherally- and binding pocket-modified vancomycin analogs described herein display little propensity for acquired resistance by vancomycin-resistant Enterococci (VRE) , and that both their antimicrobial potency and durability against such challenges follow trends (3 > 2 > 1 mechanisms of action) that are now predictable .
• VRE vancomycin-resistant Enterococci
• the present invention contemplates a compound, a pharmaceutical composition containing the compound and a method of treatment using the
• a contemplated compound corresponds in structure to that shown in Formula I or its
• X is H2, 0 or NH; and n is 0, 1 or 2.
• R is a substituent group that is selected from the group consisting of N, N- (di-C ⁇ -Cg- hydrocarbyl) amino, ⁇ , ⁇ , ⁇ - ( tri-C ⁇ -Cg-hydrocarbyl ) - ammonium, N- (C]_o ⁇ C]_g-hydrocarbyl) -N,N- (di-C]_-Cg- hydrocarbyl) ammonium, and N- (C ⁇ -Cg-hydrocarbyl) -N-
• X is preferably H2 or NH, and n is individually, preferably 1.
• R-L is other than hydrido when X is 0.
• a compound corresponds in structure to
• a contemplated compound corresponds in structure to Formula lb, in which R is CBP, and n and R are as defined above
• a contemplated compound corresponds in structure to Formula Ic , in which R1 is CBP, and n and R are as defined above
• n be 1, so that the linker between the amido nitrogen and R groups is a propan-1, 3-diyl (-CH2CH2CH2-) group .
• a particularly preferred compound corresponds in structure to Formula II
• R is selected from the group consisting of N,N- (di-C ] _-Cg-hydrocarbyl) amino, ⁇ , ⁇ , ⁇ - ( tri-C_-Cg-hydrocarbyl ) ammonium, N- (CIQ-CIQ- hydrocarbyl) -N,N- (di-C ] _-Cg-hydrocarbyl) ammonium, and N- (C ] _-Cg-hydrocarbyl) -N- (C5-C7-cyclohydrocarbyl) - ammonium.
• R is an ammonium compound, an appropriate anion is also present.
• anion is a pharmaceutically acceptable anion such as a halide like chloride, bromide, or iodide, a carbonate, bicarbonate, sulfate, bisulfate, benzene sulfonate, or a methane sulfonate.
• a pharmaceutically acceptable anion such as a halide like chloride, bromide, or iodide, a carbonate, bicarbonate, sulfate, bisulfate, benzene sulfonate, or a methane sulfonate.
• compositions that comprises a pharmaceutically acceptable diluent (carrier) in which is dispersed or dissolved an anti-microbial amount of a compound of Formula I or II as discussed above.
• an element means one element or more than one element.
• hydrocarbyl is used herein as a short hand term for a non-aromatic group that includes straight and branched chain aliphatic as well as alicyclic groups or radicals that contain only carbon and hydrogen.
• alkyl, alkenyl and alkynyl groups are contemplated, whereas aromatic hydrocarbons such as phenyl .
• hydrocarbyl groups contain a chain of 1 to 4 carbon atoms, and preferably 1 or 2 carbon atoms.
• hydrocarbyl group is an alkyl group.
• a generalized, but more preferred substituent can be recited by replacing the descriptor "hydrocarbyl” with “alkyl” in any of the substituent groups enumerated herein.
• alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
• suitable alkenyl radicals include ethenyl (vinyl), 2-propenyl, 3- propenyl, 1 , 4-butadienyl, 1-butenyl, 2-butenyl, and 3-butenyl.
• alkynyl radicals include ethynyl, 2-propynyl, 1-propynyl, 1-butynyl, 2- butynyl, 3-butynyl, and l-methyl-2-propynyl .
• hydrocarbyl ether is referred to as a
• hydrocarbyloxy rather than a “hydrocarboxy” group as may possibly be more proper when following the usual rules of chemical nomenclature.
• Illustrative hydrocarbyloxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, allyloxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy groups.
• the present invention has several benefits and advantages.
• One salient benefit of the invention is the enhanced potency of the contemplated C-terminus- modified vancomycin compounds against both
• VRE vancomycin-resistant bacteria
• a salient advantage of the invention is that the C-terminal-modification provides synergistic potency enhancements to those provided by the incorporation of a ( -chlorobiphenyl ) methyl (CBP) addition to the vancomycin disaccharide .
• CBP -chlorobiphenyl methyl
• a further salient benefit of the invention is that the contemplated C-terminus-modified vancomycin compounds function by three independent, synergistic mechanisms, only one of which requires D-Ala-D-Ala/D- Ala-D-Lac binding.
• a further salient advantage of the invention is a contemplated modified vancomycin displays little propensity for acquired resistance through serial exposure of vancomycin-resistant Enterococci (VRE) and that their durability against such challenges as well as their antimicrobial potency follow predicable trends (3 > 2 > 1 mechanisms of action) .
• VRE vancomycin-resistant Enterococci
• Such antibiotics display durable antimicrobial activity not prone to rapidly acquired clinical resistance.
• Fig. 1 is a graph whose bar heights illustrate inhibition of bacterial cell synthesis in VRE, VanA, E. faecalis ATCC BM4166 as quantitated by accumulation of the peptidoglycan precursor
• Fig. 2 is a graph whose bar heights illustrate inhibition of bacterial cell synthesis in VRE, VanA, ATCC BAA-2317 as quantitated by
• Fig. 3 is a graph whose plots illustrate cell wall permeability induced by compounds 1 -8 (10 ⁇ added at 5 minutes) in VanA VRE [E . faecium ATCC BAA-2317) ;
• Fig. 4 is a graph whose plots illustrate cell wall permeability induced by compounds 9- 13 (10 ⁇ added at 5 minutes) in VanA VRE (E. faecium ATCC BAA-2317) ;
• Fig. 5 is a graph whose plots illustrate cell wall permeability induced by compounds 14- 18 (10 ⁇ added at 5 minutes) in VanA VRE (E. faecium ATCC BAA-2317) ;
• Fig 6 is a table that provides a summary of distinct mechanisms of action of preferred modified vancomycin analogs and their individual and
• Fig 7 is a table that provides a summary of the minimum inhibitory concentrations (MICs) of various of the modified vancomycins disclosed herein that illustrate the varying potencies provided by the modifications ;
• Fig. 8 and Fig. 9 are graphs showing increases in MIC with increasing passaging of modified vancomycins disclosed herein and commercial anti-bacterials daptomycin, tigecycline and linezolid against VRE, VanA E. faecium (ATCC BAA 2317) (Fig. 8) and VRE, VanA E. faececalis (BM 4166) (Fig. 9);
• Fig. 10 is a table showing a summary of resistance development study against VRE, VanA E. faecium (ATCC BAA 2317) resulting from serial passaging (up to 50 passages) in the presence of 0.5 x MIC levels of vancomycin analogs.
• One of two replicate studies is presented; and
• Fig. 11 and Fig. 12 are graphs illustrating cell wall permeability similar to those of Figs. 4 and 5, but induced by Compounds 1, 12, 15 and 18 (10 mM added at 5 minutes) in VanA VRE (E. faecalis BM 4166; Fig. 11) and induced by those same Compounds (10 mM added at 5 minutes) in VanA VRE ⁇ E. faecium ATCC BAA-2317; Fig. 12) .
• a compound, a pharmaceutical composition containing the compound and a method of treatment using the compound are contemplated herein.
• a contemplated compound corresponds in structure to that shown in Formula I or its pharmaceutically acceptable salt
• X is Hj, 0 or NH.
• X is Hj, 0 or NH.
• similar compounds in which X is ]3 ⁇ 4 or NH are more potent than are those in which X is 0.
• X is
• R is a substituent group that contains a tertiary amine or a quaternary amine group.
• An R substituent is selected from the group consisting of N, N- (di-C ] _-Cg-hydrocarbyl) amino, N, N, N- (tri-C]_-Cg- hydrocarbyl) ammonium, N- (C ] _ Q -C]_8-hydrocarbyl) -N, N- (di-C ] _-Cg-hydrocarbyl ) ammonium, and N-(C]_-Cg- hydrocarbyl) -N- ( C5-C -cyclohydrocarbyl ) ammonium.
• a preferred C ] _-Cg-hydrocarbyl group is a C]_ (methyl) group, so that a preferred N, N- (di-C ⁇ -Cg- hydrocarbyl ) amino group is a N, N-dimethylamino group and a preferred N, N, N- (tri-C ] _-Cg-hydrocarbyl ) ammonium group is a N, N, N- (tri-methylammonium) substituent.
• a C]_o-C]_g-hydrocarbyl group is preferably a straight chain alkyl or alkenyl group such as a lauryl, myristyl, palmityl, stearyl, oleyl or palmitoleyl group. When R is an ammonium group, as is preferred, an appropriate, preferably a pharmaceutically acceptable, anion is also present.
• the linker chain between the amido nitrogen atom and the nitrogen atom of the R group can contain 2, 3, or 4 carbon atoms such that n is 0, 1 or 2. It is preferred that n be 1 so that three carbons are in the chain of the linker.
• a R1 substituent is H (hydrido) or halo(C]_- C ] _2 ) -hydrocarbyldiyl . is other than hydrido when
• a preferred halo (C]_-C]_2 ) -hydrocarbyldiyl substituent is a 4- ( ' -chlorophenyl ) -phenylmethyldiyl group, below, that can also be named
• CBP 4- ( 4' -chlorobiphenyl) methyl group
• CBP 4- (4' -chlorophenyl) benzyl group
• X is preferably H2 or NH, and n is individually, preferably 1.
• a compound corresponds in structure to
• a contemplated compound corresponds in structure to Formula lb, in which R is CBP, and n and R are as defined above
• a contemplated compound corresponds in structure to Formula Ic, in which RI is CBP, and n and R are as defined above
• n be 1, so that the linker between the amido nitrogen and R groups contains a chain of 3 carbon atoms, and is therefore a propan-1, 3-diyl ( -CH2CH2CH2- ) group.
• a particularly preferred compound corresponds in structure to Formula II
• R is an ammonium compound
• an appropriate anion is also present.
• that anion is a pharmaceutically acceptable anion such as a halide like chloride, bromide, or iodide, a carbonate, bicarbonate, sulfate, bisulfate, benzene sulfonate, or a methane sulfonate.
• a pharmaceutically acceptable anion such as a halide like chloride, bromide, or iodide, a carbonate, bicarbonate, sulfate, bisulfate, benzene sulfonate, or a methane sulfonate.
• Appropriate anions are discussed further hereinafter.
• compositions that comprises a pharmaceutically acceptable diluent (carrier) in which is dispersed or dissolved an anti-microbial amount of a compound of Formula I or II as discussed above.
• a further aspect of the invention is a method of treating a mammal infected with a microbial infection such as a bacterial infection, typically a Gram-positive infection; i.e., an infection caused by Gram-positive bacteria, and in need of antimicrobial (antibacterial) treatment.
• a microbial infection such as a bacterial infection, typically a Gram-positive infection; i.e., an infection caused by Gram-positive bacteria, and in need of antimicrobial (antibacterial) treatment.
• an antibacterial-effective amount of one or more compounds of Formula I or I I or a pharmaceutically acceptable salt of such a compound is administered to an infected mammal in need.
• the compound can be administered as a solid or as a liquid formulation, and is preferably administered via a pharmaceutical composition discussed hereinafter. That administration can also be oral or parenteral, as are also discussed further hereinafter .
• viable mammals are infected with bacteria and other microbes.
• the present invention's method of treatment is intended for use against infections of pathogenic microbes that cause illness in the mammal to be treated.
• Illustrative pathogenic microbes include S. aureus, methicilin-resistant S. aureus (MRSA) , VanA strains of E. faecalis and E. feacium, as well as VanB strains of E. faecalis.
• MRSA methicilin-resistant S. aureus
• VanA strains of E. faecalis and E. feacium as well as VanB strains of E. faecalis.
• Evidence of the presence of infection by pathogenic microbes is typically understood by physicians and other skilled medical workers .
• a mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a Compound of Formula I or II , or its
• a pharmaceutically acceptable salt can be administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.
• a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.
• a contemplated compound is typically present in the composition in an amount that is sufficient to provide a concentration of about 0.1 nM to about 1 ⁇ to contact microbes to be assayed.
• antibiotic or antibacterial or antimicrobial
• that amount is not an amount that is effective to kill all of the pathogenic bacteria or other microbes present in an infected mammal in one administration. Rather, that amount is effective to kill some of the pathogenic organisms present without also killing the mammal to which it is administered, or otherwise harming the recipient mammal as is well known in the art.
• a contemplated pharmaceutical composition contains an effective antibiotic (or antimicrobial) amount of a Compound of Formula I or II or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically (pharmaceutically) acceptable diluent or carrier.
• An effective antibiotic amount depends on several factors as is well known in the art. However, based upon the relative potency of a contemplated compound relative to that of vancomycin itself for a susceptible strain of S. aureus shown hereinafter, and the relative potencies of vancomycin and a contemplated compound against the VanA E. faecalis and E. faecium strains, a skilled worker can readily determine an appropriate dosage amount .
• Exemplary salts useful for a contemplated compound include but are not limited to the
• ethanesulfonate glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide , hydroiodide, 2-hydroxy- ethanesulfonate , lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,
• palmoate palmoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate.
• the salts can also be used as an aid in the isolation, purification or
• the salt prepared need not be
• a contemplated composition is typically administered repeatedly in vivo to a mammal in need thereof until the infection is diminished to a desired extent, such as cannot be detected.
• administration to a mammal in need can occur a plurality of times within one day, daily, weekly, monthly or over a period of several months to several years as directed by the treating physician. More usually, a contemplated composition is administered a plurality of times over a course of treatment until a desired effect is achieved, typically until the bacterial infection to be treated has ceased to be evident.
• a contemplated pharmaceutical composition can be administered orally (perorally) or
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
• a contemplated pharmaceutical composition is preferably adapted for parenteral administration.
• a pharmaceutical composition is preferably in liquid form when
• the liquid is an aqueous liquid, although other liquids are contemplated as discussed below, and a presently most preferred
• composition is an injectable preparation.
• injectable preparations for example, sterile injectable aqueous or oleaginous solutions or suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 , 3-butanediol .
• acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline.
• liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration.
• Sterile water solutions of a Compound of Formula I or II or its salt or sterile solution of a Compound of Formula I or II in a solvent comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
• a contemplated Compound of Formula I or II is provided as a dry powder that is to be dissolved in an appropriate liquid medium such as sodium chloride for injection prior to use.
• 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 find use in the preparation of an injectable composition.
• a sterile solution can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
• Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules.
• the amount of a contemplated Compound or salt of Formula I or II such as Compound 18 in a solid dosage form is as discussed previously, an amount sufficient to provide an effective antibiotic (or antimicrobial) amount.
• a solid dosage form can also be administered a plurality of times during a one week time period.
• a compound of this invention is ordinarily admixed as a solution or suspension in one or more diluents appropriate to the indicated route of administration.
• the compounds can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,
• polyvinylpyrrolidone and/or polyvinyl alcohol, and then tableted or encapsulated for convenient
• Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in
• the dosage forms can also comprise buffering agents such as sodium
• citrate magnesium or calcium carbonate
• Tablets and pills can additionally be prepared with enteric coatings.
• a sample to be assayed such as cells and tissue can be used.
• These in vitro compositions typically contain water, sodium or potassium chloride, and one or more buffer salts such as and acetate and phosphate salts, Hepes or the like, a metal ion chelator such as EDTA that are buffered to a desired pH value such as pH 4.0 -8.5, preferably about pH 7.2-7.4, depending on the assay to be performed, as is well known.
• the pharmaceutical composition is in unit dosage form.
• the composition is divided into unit doses containing appropriate quantities of the active compound.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, in vials or ampules .
• the second alternative, peripheral modification examined was C-terminus amide
• VanA VRE VanA vancomycin-resistant organisms
• 'MIC Minimum inhibitory concentration. b BM 4166. C ATCC BAA-2317.
• 'MIC Minimum inhibitory concentration. b BM 4166. C ATCC BAA-2317.
• This vancomycin analog is >10-fold more potent than the CBP derivative 8 , >1, 000-fold more potent than the pocket analog 4 , and a stunning >10, 000-fold more potent than vancomycin itself. It is also >25-100-fold more potent than its comparison Cl/CBP-vancomycin derivative Compound 15 and >250- fold more active than either CBP-vancomycin ( 5 ) or C14-vancomycin ( 12 ) .
• VanA VRE vancomycin-resistant organisms
• a larger scale isolation and full characterization of 19 provided the material needed to establish a linear calibration curve .
• test compounds were incubated with tetracycline pretreated VanA VRE for 30 minutes (37 °C) before the cultured bacteria was collected by centrifugation, washed, and resuspended in pH 7.2 buffer (5 mM HEPES and 5 mM glucose, 1:1) .
• pH 7.2 buffer 5 mM HEPES and 5 mM glucose, 1:1
• the bacterial suspension was heated at 100 °C for 15 minutes to release cytosolic 19 .
• the entire test compounds were incubated with tetracycline pretreated VanA VRE for 30 minutes (37 °C) before the cultured bacteria was collected by centrifugation, washed, and resuspended in pH 7.2 buffer (5 mM HEPES and 5 mM glucose, 1:1) .
• the bacterial suspension was heated at 100 °C for 15 minutes to release cytosolic 19 .
• the entire test compounds were incubated with tetracycline pretreated VanA VRE for 30 minutes (37
• the effect of the compounds on cell membrane permeability was examined by measuring cytoplasmic membrane uptake of the fluorescent probe propidium iodide in the same VanA vancomycin- resistant E. faecalis (BM 4166) and E. faecium (ATCC BAA-2317) strains.
• This fluorescent probe only enters cells with permeabilized cell membranes and is detected by the emission of fluorescence upon intracellular nucleic acid binding [Boulos et al., J Microbiol Methods 1999 , 37 (1) : 77-86 ] .
• fluorescence intensity are observed immediately upon addition of test compounds that induce bacterial cell membrane permeability.
• peripheral CBP modification to the vancomycin disaccharide found in 5-8. This is followed by the discussion of the results from the examination of the peripheral quaternary ammonium salt modifications found in 9-13, including their effects on both unmodified and pocket modified vancomycin analogs. Finally, the effects of the two combined peripheral modifications in 14-18 are detailed.
• vancomycin (1) and the thioamide pocket modified vancomycin analog 2 do not effectively inhibit bacterial cell wall synthesis in VanA VRE and do not result in the significant accumulation of 19 in the assay (Figs. 1 and 2) .
• the pocket-modified analog Compound 4 designed for dual D-Ala-D-Ala/D-Ala-D-Lac binding inhibits bacterial cell wall biosynthesis, resulting in the buildup of the precursor 19 in the assay at levels consistent with its relative model ligand binding affinities and antimicrobial activity.
• Antimicrobial activity of Compound 4 correlates directly with its expected impact on bacterial cell wall biosynthesis, binding D-Ala-D-Lac and inhibiting cell wall maturation. Incorporation of the
• peripheral CBP modification inhibits cell wall synthesis more effectively than 4 , lacking the CBP modification, and more potently than either Compounds 5 or 6 , lacking a productive pocket modification. This is the result of the combined effects of the two independent mechanisms of action, both of which impact cell wall biosynthesis but only one of which depends on D-Ala-D-Ala/D-Ala-D-Lac binding.
• the antimicrobial activity against VanA VRE can be attributed to a mechanism independent of cell wall biosynthesis and independent of D-Ala-D- Ala/D-Ala-D-Lac binding.
• the antimicrobial activity correlates with disruption of the cell wall
• compound 13 represents a pocket modified vancomycin analog that displays potent and further improved activity against VanA VRE derived from two independent and synergic mechanisms of action.
• One mechanism relies on the dual D-Ala-D- Ala/D-Ala-D-Lac binding like 4 and results in effective cell wall synthesis inhibition.
• the second mechanism is independent of this ligand binding property and is derived from induced cell wall permeabilit .
• VanA VRE vancomycin analogs with zero ( 1 ) , one ( 4 ) , two ( 8 and 13 ) , and three ( 18 ) distinct and synergistic
• each structural modification and mechanism of action independently expresses its functional activity at the level of 2- 30 ⁇ g/mL (1-15 ⁇ ) in both the antimicrobial and mechanistic assays, but each provides synergistic improvements in the functional antimicrobial activity when combined.
• peripherally C14-modified vancomycin analog 12 , 1 mechanism of action
• peripherally CBP-modified pocket analog Compound 8 (2 mechanisms of action)
• peripherally C14-modified pocket analog Compound 13 (2 mechanisms of action)
• pocket analog Compound 18 that contains the two complementary peripheral CI and CBP-modifications (3 mechanisms of action) were examined for their susceptibility to acquired resistance upon sub-lethal (0.5 x MIC) serial exposure to the same two VRE bacterial strains, monitoring MICs daily (Fig. 8 and Fig. 9) .
• the antibiotic susceptibility profiles of the VanA VREs used herein indicate they are resistant to a number of additional classes of antibiotics and are on the verge of being classified as multi-drug resistant VRE, indicating they have already assimilated a number of common resistance mechanisms.
• the magnitude of the changes in the MICs for the compounds acting by two or more mechanisms is sufficiently small to indicate none experience a full loss of one of the contributing mechanisms.
• each mechanism is rendered more robust when combined with structural modifications that provide one or two additional mechanisms of action.
• CBP-vancomycin (5) is representative of the expected behavior of the clinically approved semisynthetic vancomycin analogs.
• the key compounds in the series were examined for in vitro toxicity that might result from the combined mechanisms of action, especially the introduction of structural modifications (quaternary ammonium salt) that might impact host as well as bacterial cell wall integrity.
• the compounds were examined for red blood cell hemolytic activity, resulting from membrane lysis. No compound in the series, including Compound 18, exhibits any hemolytic activity even at concentrations >1000-fold above their MICs.
• NIH/3T3 ATCC CRL-1658, mouse embryonic fibroblast
• HepG2 ATCC HB-8065, human liver cancer cell line
• No growth inhibition (cytotoxic activity) was observed up to 100 ⁇ , the highest dose tested. They were also found to be inactive (>100 ⁇ , highest dose tested) against HCT116 (human colon cancer cell line) .
• antibiotics that display additional drug-target binding contacts to enhance the robustness of targe engagement and decrease resistance susceptibility [Silver, Nat Rev 2007, 6 (1) : 41-55 ( 57 ) .
• VRE are already vancomycin resistant and because many have already reached a point where they are no longer susceptible to most other antibiotic classes, the CDC recently placed VRE on its serious threat list and the WHO placed it fourth on its list of drug-resistant bacteria that pose the greatest threat to human health.
• the glycopeptide antibiotics constitute an antibiotic class already endowed with features that avoid many mechanisms of resistance [James et al . , ACS Chem Biol 2012 , 7(5) .-797-804] .
• binding pocket modifications designed for dual ligand binding reinstated binding to the altered target D-Ala-D-Lac and maintained binding affinity for the unaltered target D-Ala-D-Ala were developed. These modifications were found to reinstate antimicrobial activity against vancomycin- resistant organisms that employ the altered D-Ala-D- Lac peptidoglycan precursor targets and remain active against vancomycin-sensitive bacteria that employ only D-Ala-D-Ala precursors.
• vancomycin in which the residue 4 amide carbonyl was removed A more potent pocket modified vancomycin analog is the residue 4 amidine (Compound 3 vs 4) , which exhibits antimicrobial activity against both vancomycin-resistant and vancomycin-sensitive bacteria equipotent with the activity vancomycin displays against vancomycin-sensitive bacteria.
• the reaction mixture was stirred at 25 °C for 5 minutes and quenched with the addition of 50% MeOH in 3 ⁇ 40 (0.5 mL) at 25 °C.
• VSSA strain ATCC 25923 vancomycin-sensitive Staphlococcus aureus
• MRSA strain ATCC 43300 vancomycin-resistant Enterococcus faecalis
• Enterococcus faecium vancomycin-resistant Enterococcus faecalis
• VanB VRE vancomycin-resistant Enterococcus faecalis
• baumannii ATCC BAA-1710
• Pseudomonas aeruginosa ATCC 15442
• Klebsiella pneumoniae ATCC 700603
• VSSA Mueller-Hinton broth
• VanA VRE 100% brain-heart infusion broth
• Luria broth E. coli and P. aeruginosa
• the bacterial stock solutions were serially diluted with the culture medium (10% Mueller-Hinton broth for VSSA, MRSA and VanB VRE or 10% brain-heart infusion broth for VanA VRE A.
• baumannii and K. pneumoniae or 10% Luria Broth for E. coli and P. aeruginosa) to achieve a turbidity equivalent to a 1:100 dilution of a 0.5 M McFarland solution.
• This diluted bacterial stock solution was then inoculated in a 96-well V-shaped glass coated microtiter plate, supplemented with serial diluted aliquots of the antibiotic solution in DMSO (4 ⁇ ,) , to achieve a total assay volume of 0.1 mL.
• the plate was then incubated at 37 °C for 18 hours, after which minimal inhibitory concentrations (MICs) were determined by monitoring the cell growth (observed as a pellet) in the wells.
• MICs minimal inhibitory concentrations
• the lowest concentration of antibiotic (in ⁇ g/mL) capable of eliminating cell growth in the wells is the reported MIC value.
• the reported MIC values for the vancomycin analogues were determined against vancomycin as a standard in the first well.
• VanA E. faecalis (VanA VRE, BM 4166) : resistant to erythromycin, gentamicin,
• VanA E. faecium (VanA VRE, ATCC BAA-2317) :
• vancomycin and teicoplanin insensitive to linezolid; sensitive to tigecycline and dalfopristine .
• VanA E. faecalis (VanA VRE, BM 4166) : resistant to erythromycin, gentamicin,
• VanA E. faecium (VanA VRE, ATCC BAA-2317) :
• vancomycin and teicoplanin insensitive to linezolid; sensitive to tigecycline and dalfopristine .
• This bacterial suspension (130 ⁇ .) was charged into a 96-well black plate with a clear bottom (Corning 3650) .
• the propidium iodide dye (10 ⁇ , 150 ⁇ DMSO solution) was added to the above suspension and the fluorescence was monitored at 25 °C for 5 minutes at 30 second intervals using a microplate reader (Molecular Devices ® , Max Gemini EX) at an excitation wavelength of 535 nm and an emission wavelength of 617 nm.
• the test compound (150 ⁇ , 10 ⁇ ) was added to the cell suspension and the
• Vancomycin (1), C14-vancomycin (12), CBP Cl- vancomycin (15) , and CBP Cl-aminomethylene vancomycin (18) displayed similar induced permeabilities against both vancomycin-resistant Enterococcus faecalis (VanA VRE, BM4166) and Enterococcus faecium (VanA VRE, ATCC BAA-2317) .
• VanA VRE vancomycin-resistant Enterococcus faecalis
• BM4166 vancomycin-resistant Enterococcus faecium
• the dye (DiSC3(5) : 3 , 3 ' -Dipropylthiadi- carbocyanine iodide, 150 ⁇ DMSO solution, 2.5 ⁇ .) was added to the above suspension and the dye (DiSC3(5) : 3 , 3 ' -Dipropylthiadi- carbocyanine iodide, 150 ⁇ DMSO solution, 2.5 ⁇ .) was added to the above suspension and the dye (DiSC3(5) : 3 , 3 ' -Dipropylthiadi- carbocyanine iodide, 150 ⁇ DMSO solution, 2.5 ⁇ .) was added to the above suspension and the dye (DiSC3(5) : 3 , 3 ' -Dipropylthiadi- carbocyanine iodide, 150 ⁇ DMSO solution, 2.5 ⁇ .) was added to the above suspension and the dye (DiSC3(5) : 3 , 3 ' -Dipropy
• the MICs of the vancomycin analogues against vancomycin-resistant Enterococcus faecalis (VanA VRE, BM4166) and Enterococcus faecium (VanA VRE, ATCC BAA-2317) were determined.
• the bacterial suspension (40 ⁇ .) in the 96-well plate at sub-MIC concentration (MICs/2) was inoculated with 100% brain-heart infusion broth and the bacteria were grown in an orbital shaker at 37 °C for 6 hours until the value of OD 6 oo became 0.6.
• a new MIC assay was performed with the same protocol. This process was repeated for 50 passages, and the fold increase in MIC was determined at each passage. [****Pollard et al., J. Antimicrob. Chemother. 2012, 57:2665-2672.]
• Enterococcus faecium (VanA VRE, ATCC BAA-2317) were inoculated and grown in an orbital shaker at 37 °C in 100% brain-heart infusion broth for 12 hours.
• the above bacterial solution was subjected to a
• Vancomycin analogues were added and the mixture was incubated at 37 °C for a further 30 minutes.
• the blood cells in pig whole blood (2 mL, Pel-Free Biologicals, non-sterile, sodium citrate) were harvested (3000 rpm, 4 °C, 20 minutes), and the red blood precipitate was washed and resuspended in phosphate buffered saline (pH 7.4) .
• This diluted red blood cell stock solution (384 ⁇ ) was incubated with the antibiotic solution in DMSO (16 ⁇ .) in a 1 rtiL microtube to achieve the final concentration of the test compounds. The mixture was then incubated at 37 °C for 1 hour.
• the solution was diluted with phosphate buffer saline (pH 7.4, 200 ⁇ ,) at 25 °C and
• red blood cells mammalian cell membranes
• the standard red blood cell hemolysis assay was conducted and measures the extent of red blood cell lysis after 1 hour exposure to candidate compounds (pH 7.4, PBS, 37 °C, 1 hour) . No compound in the series, including Compound 18 , exhibited any hemolytic activity even at
• control daptomycin that acts by permeabilizing (not lysing) the bacterial cell membrane .

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