WO2024258613A9 - Tétrachlorovancomycine et dérivés - Google Patents

Tétrachlorovancomycine et dérivés Download PDF

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WO2024258613A9
WO2024258613A9 PCT/US2024/031453 US2024031453W WO2024258613A9 WO 2024258613 A9 WO2024258613 A9 WO 2024258613A9 US 2024031453 W US2024031453 W US 2024031453W WO 2024258613 A9 WO2024258613 A9 WO 2024258613A9
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compound
equiv
pharmaceutically acceptable
vancomycin
acceptable salt
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WO2024258613A3 (fr
WO2024258613A2 (fr
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Dale L. Boger
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Scripps Research Institute
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Scripps Research Institute
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a remaining challenge enroute to their translation to the clinic is their accessibility, presently requiring total syntheses to obtain the targeted glycopeptide analogues.
  • 5 A new class of structurally simplified synthetic glycopeptide antibiotics is disclosed that is now easily accessible by total synthesis and directly addresses this challenge. The class retains all the intricate vancomycin structural features that contribute to its target binding affinity and selectivity, maintains the potent antimicrobial activity of vancomycin, and achieves this simplification by an unusual addition, not removal, of benign substituents to the core structure.
  • this simplification allows full control of all stereochemical features, results in a technically straightforward total synthesis with reduction in the step count [15 steps in longest linear sequence (LLS), 15% overall yield], improves the CD/DE macrocyclization rates and efficiencies that are now run concurrently, and provides a synthetic glycopeptide antibiotic that maintains the ligand binding and antimicrobial activity of the natural product.
  • the class of compounds retains all the intricate vancomycin structural features that contribute to its target binding affinity and selectivity, maintains the potent antimicrobial activity of vancomycin, and achieves this simplification by an unusual addition, not removal, of benign substituents to the core structure.
  • the present invention contemplates a new, biologically active vancomydn analogue called tetrachlorovancomydn and its derivatives that are produced, except for glycosylation, solely by synthetic organic chemistry.
  • Key elements of the synthetic approach indude a catalyst-controlled diastereoselective formation of the AB biaryl axis of chirality (>30:1 dr), an instantaneous macrolactamization of the AB ring system free of competitive epimerization (>30:1 dr), an epimerization free coupling of the E ring tetrapeptide, the room temperature dual CD/DE ring system SNAT cydizations, a highly refined 4-step conversion of the product to the aglycon, and a protecting group free one-pot enzymatic glycosylation for disaccharide introduction.
  • the above two compounds surprisingly exhibit activity against methicillin-resistant S. aureus at about a factor of 10 or less than the activity of vancomycin against vancomycin-sensitive and vancomycin-resistant bacteria.
  • their derivatives substituted similarly to some of the most active vancomycin derivatives show almost the same activities. Being chemically prepared in relatively high yield provides a route to less expensive very active antibiotics.
  • R 1 is selected from the group consisting of hydrido (hydrogen), (C 1 -C 16 )hydrocarbyl, aryl(C 1 -C 6 )-hydrocarbyldiyl l heteroaryl-(C1- C 6 )hydrocarbyldiyl, (C 1 -C 6 )hydrocarbyldiylheteroaryl, halo(C1-C12)- hydrocarbyldiyl, and (C 1 -C 16 )amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of
  • Circle A is a linking moiety having the length of a saturated chain of 2 carbon atoms and less than a saturated chain of about 12 carbon atoms
  • the “X” moiety above can be H,H making the carbon to which the two hydrogens are bonded a methylene group.
  • “X” is 0 (oxygen) double bonded to the depicted carbon atom as the carbonyl group of an amide.
  • X can also be S (sulfur) double-bonded to the depicted carbon, making that carbon a thiocarbonyl moiety and thereby, the thiocarbonyl bonded to the -NH- group form a thioamide linkage.
  • a compound where “X” is “S” is usually used as an intermediate to the preparation of a compound of Formula I, II and III in which “X” is “H,H” forming a methylene group as above, or is “NH”, forming an amidine linkage.
  • R 1 substituents other than H those hydrophobic materials are present and discussed in one of the inventors’ U.S. Patents No.9,879,049, No. 10,577,395, No. 10,934,326, well as to U.S. Patent Publication 2023/0146239, and the papers dted therein. Many of these substituents are present in commercially available in semisynthetic derivatives of vancomydn and similar glycopeptide antibiotics such as teicoplanin A2, oritavandn, dalbavandn and telavandn.
  • Hydrophobic R1 substituents that are presently preferred are the benzyl, 4-chlorobenzyl, (biphenyl)methyl, (4- chlorobiphenyl)methyl [CBP], 4-fluorobenzyl, and (4- fluorobiphenyl)methyl substituent groups.
  • Each of these six substituents can be added to the tetrachlorovancosaminyl amino group by NaCNBH4 reduction of the corresponding aldehyde as is shown in Scheme 5 hereinafter.
  • the R2 substituents contain at least two nitrogen atoms separated by a linker group referred to as Cirde A and depicted as wherein the remaining valence of the nitrogen in the depicted "-HN-" group bonds to carboxyl group of the tetrachlorovancomycinyl portion of the molecule to form an amido group.
  • the R 3 contains at least a second nitrogen atom bonded directly to the Circle A linker.
  • the second nitrogen of Circle A is the nitrogen of a tertiary amine or a quaternary ammonium group, as noted above.
  • R 3 is a quaternary ammonium group
  • an optional anion, Y ⁇ that is preferably pharmaceutically acceptable is also present to balance the charge.
  • R 3 is a tertiary amine or guanidinyl group, both of which are typically basic, a compound containing such a group can also be present as a salt with an acid.
  • the add of such an add salt is a pharmaceutically acceptable add, that provides the optional anion, Y ⁇ .
  • a pharmaceutical composition containing an anti- bacterially effective amount a before-described tetrachlorovancomydn or derivative, or a pharmaceutically acceptable salt dissolved or dispersed in a pharmaceutically (physiologically) diluent acceptable diluent is also contemplated.
  • Such a composition can be in solid, liquid, gel or other appropriate form.
  • a method of treating a bacterial infection, particularly from Gram positive bacteria, is also contemplated.
  • Fig. 1 shows a comparison of vancomycin and tetrachlorovancomydn that highlights the structural and synthetic simplification and atropisomerism elimination achieved by adding two benign chlorine substituents;
  • Fig. 2 is a schematic representation of key elements of a retrosynthetic analysis for tetrachlorovancomydn;
  • Fig. 3 illustrates reaction Scheme 4 that illustrates a direct synthetic route from Compound 27 to Compound 29 in 56% yield and five steps followed by the one-pot two-step enzymatic glycosylation of tetrachlorovancomydn aglycon (29) to form tetrachlorovancomydn (Compound 1) that proceeded in high yield (82%) for installation of both sugar residues despite the added 2 e and 6e aryl chlorides;
  • Fig. 4 outlines a synthetic pathway by which a tetrachlorovancomydn derivative of Formula III where can be prepared
  • Fig. 5 shows a reaction scheme whereby the 4-thioamide derivative, Compound 41, can be prepared from Compound 38;
  • Fig. 6 shows two reaction schemes by which Compounds 40 and 39 can be prepared from Compound 38;
  • FIG. 7 in two panels, as Fig.7A that illustrates two reaction schemes by which Compounds 42, 43, and 44 can be prepared from Compound 41, and in which Compounds 46 and 45 can also be prepared from Compound 44, and Fig. 7B in which Compound 44 is used to prepare Compound 47, that in turn is used to prepare Compounds 48 and 49;
  • FIG. 8 in two panels, as Fig. 8A that illustrates tetrachlorovancomycin, Compound 1, and its three 4-position differently substituted analogues, Compounds 41, 42 and 43, titration binding study results of with model ligands A and B, and similar studies with vancomycin itself and its three similarly substituted analogues in Fig. 8B;
  • Fig. 9 in two panels as Figs. 9A and 9B, are tables showing minimum inhibitory concentrations (MIC values) for tetrachlorovancomycin analogue Compounds 41, 44, 47, 48 and 49 against bacteria that are vancomycin-sensitive (Fig. 9A) and vancomycin-resistant (Fig. 9B); data for G3.CBP-vancomydn 44 ;
  • Figs. 10 through 15 provide illustrative tetrachlorovancomycin derivative compounds with varying 4-position substituents, as well as various substituents bonded to the vancosaminyl nitrogen, and also still further substituents bonded to the vancomycin usually unsubstituted (C-14) carboxyl group.
  • 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 are grouped as an "aryl " group.
  • Exemplary hydrocarbyl groups contain a chain of 2 to about?? carbon atoms, and preferably 2 to about 6 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 1-methyl-2-propynyl.
  • hydrocarbyl ether is referred to as a "hydrocarbyloxy” group 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 relative ease and enhanced yield of synthetically-prepared tetrachlorovancomycin and derivatives as compared to vancomycin itself when synthetically prepared, and also when compared to vancomycin preparation by fermentation using bacteria whose 4-postiion derivatives are very difficult prepare.
  • a salient advantage of the invention is that the antibacterial activity of tetrachloro-vancomydn compared to that of vancomydn itself is almost identical.
  • Another benefit of the invention is that the activity of the herein discussed derivatized tetrachloro-vancomycins against both vancomycin-resistant bacteria (VRE) and those bacteria that are not vancomycin-resistant compared to the activities of identically derivatized vancomycin are also almost identical.
  • the present invention contemplates a new, biologically active vancomycin derivative called tetrachlorovancomydn that is produced, except for glycosylation, solely by synthetic organic chemistry.
  • tetrachlrovancomydn The structural formula for tetrachlrovancomydn itself is shown below as Formula I.
  • the above two compounds surprisingly exhibit activity against methicillin-resistant S. aureus and about a factor of 10 or less the activity of vancomycin against vancomycin-sensitive and vancomycin-resistant bacteria.
  • their derivatives substituted similarly to the most active vancomycin derivatives show almost the same activities as those similarly substituted vancomycins. Being chemically prepared in relatively high yield provide a route to less expensive very active antibiotics.
  • R1 is selected from the group consisting of hydrido (hydrogen), (C 1 -C 16 )hydrocarbyl, aryl(C 1 -C 6 )-hydrocartoyldiyl, heteroaryl-(C1- C6)hydrocarbyldiyl, (C 1 -C 6 )hydrocarbyldiylheteroaryl, halo(C2-C-12)- hydrocarbyldiyl, and (C 1 -C 16 )amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of
  • Circle A is a linking moiety having the length of a saturated chain of 2 carbon atoms and less than a saturated chain of about 12 cartoon atoms
  • the compound above is tetrachloro-vancomycin. When one or both of R 1 and R 2 are other than H and OH, respectively, a derivative of tetra- chlorovancomydn is being contemplate.
  • the “X” moiety above can be H,H making the carbon to which the two hydrogens are bonded a methylene group.
  • “X” is 0 (oxygen) double bonded to the depicted carbon atom as the carbonyl group of an amide.
  • X can also be S (sulfur) double-bonded to the depicted carbon, making that carbon a thiocarbonyl moiety and thereby, the thiocarbonyl bonded to the -NH- group form a thioamide linkage.
  • a compound where “X” is S is usually used as an intermediate to the preparation of a compound of Formula I, II and III in which “X” is H,H forming a methylene group as above, or is NH, forming an amidine linkage.
  • R1 substituents other than H those hydrophobic materials are present and discussed in the inventor’s U.S. Patents No.9,879,049, No. 10,577,395, No. 10,934,326, well as to U.S. Patent Publication 2023/0146239, and the papers cited therein. Many of these substituents are present in commercially available derivatives of vancomycin and similar glycopeptide antibiotics such as teicoplanin A2, oritavancin, dalbavandn and telavandn.
  • Hydrophobic R 1 substituents that are presently preferred are the benzyl, 4-chlorobenzyl, (biphenyl)methyl, (4- chlorobiphenyl)methyl [CBP], 4-fluorobenzyl, and (4- fluorobiphenyl)methyl substituent groups.
  • Each of these four substituents can be added to the vancosaminyl amino group by NaCNBH4 reduction of the corresponding aldehyde as is shown in Scheme 5 hereinafter.
  • the R 2 substituents, other than H, contain at least two nitrogen atoms separated by a divalent linker group referred to as Cirde A and depicted as wherein the remaining valence of the nitrogen in the depicted " HN " group bonds to carboxyl group of the tetrachlorovancomycinyl portion of the molecule to form an amido group, and R3 contains at least a second nitrogen atom.
  • the second nitrogen of Circle A is the nitrogen of a tertiary amine or a quaternary ammonium group.
  • the chain lengths herein are measured along the longest linear atom chain in the radical between the amido nitrogen and the first nitrogen atom of a guanidinyl group or the nitrogen of a tertiary amine or a quaternary ammonium group.
  • Each atom in the chain is presumed to be carbon for ease in calculation.
  • the lengths are thus recited in terms of carbon atoms.
  • Such lengths can be readily determined by using published bond angles, bond lengths and atomic radii, as needed, to draw and measure a staggered chain, or by building models using commercially available kits whose bond angles, lengths and atomic radii are in accord with accepted, published values.
  • a 1 ,4-bonded 6-membered aromatic ring group (phenyl) not part of a fused ring system has a length of about a butyl group.
  • a 1 ,2- or 1 ,3-bonded 6-ring has a length of a 2- or 3-carbon chain, respectively, as the shortest path around the ring between the two bonding position regardless of formal naming criteria. Where a 5- membered ring is present, length is calculated as the length of a 2-carbon chain.
  • length is calculated as the shortest path around the rings between the two bonding positions to the amido and guanidinyl, quaternary ammonium or tertiary amine nitrogen atoms of a compound of Formula III regardless of formal naming criteria.
  • Radical lengths can also be determined somewhat less exactly by assuming that all atoms have bond lengths of saturated C-C bonds, that unsaturated bonds have the same lengths as saturated bonds, and that bond angles for unsaturated bonds are the same as those for saturated C-C bonds (108°) , although the above-mentioned modes of measurement are preferred. Both methods produce similar results within one or two carbon atoms, and thus the use of "about".
  • a contemplated linker moiety Circle A can also be a hydrocarbyl chain of two to about 12 saturated carbon atoms, or preferably two to about ten saturated carbon atoms.
  • a more preferred linking Circle A group contains a chain of atoms that is equal to or greater than the length of two saturated carbons and is shorter than about a saturated ten carbon (decyl) chain.
  • the hydrocarbyl chain has a chain length of two saturated carbon atoms to about eight saturated carbon (octyl) atoms.
  • the length is simply the length of the longest chain of atoms linking those two nitrogens.
  • Circle A hydrocarbyl linker groups it is noted that such groups can contain a substituent that is pendant from the chain of atoms that link the amido and second nitrogens (e.g. , guanidinyl) shown in Formula III.
  • Such a substituent are selected from amino acid side chain substituents other than those containing a carboxyl group, a sulfhydryl group (-SH) or a substituent that provides a negative charge in an aqueous solution at physiological pH values, e.g., pH 7.2-7.4.
  • Additional pendant substituents include 2-hydroxyethyl and 2-hydroxypropyl, C-
  • "Co" is intended to indicate that the carbonyl carbon is bonded directly to an atom of the Circle A linking chain.
  • a contemplated divalent Circle A linker moiety also can comprise a ring system that can be carbocyclic or heterocyclic as discussed below.
  • a single 5- or 6-membered ring optionally contains one or two ring hetero atoms that can independently be nitrogen, oxygen or sulfur.
  • Individual rings can be aliphatic or aromatic, including heteroaromatic, and also be aralkyl as in a benzyl group.
  • exemplary divalent aromatic carbocyclic ring moieties include phenyl and naphthyl groups.
  • exemplary divalent heteroaryl groups include 6-membered ring substituents such as pyridyl, pyrazyl, pyrimidinyl, and pyridazinyl; 5-membered ring substituents such as 1,3,5-, 1,2,4- or 1 ,2,3-triazinyl, imidazyl, furanyl, thiophenyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, 1 ,2,3-, 1,2,4-, 1,2,5-, or 1 ,3,4-oxadiazolyl and isothiazolyl groups.
  • Aliphatic 5- and 6-membered carbocyclic rings are contemplated such as cydohexyl and cyclopentyl, as well as their mono- and d iethy lenically unsaturated derivatives, using monovalent names for convenience.
  • divalent aliphatic 5- and 6-membered heterocyclic rings include, piperidinyl, piperazinyl, imidazolinyl, imidazolidinyl, pyrrolinyl, pyrrolidinyl, pyrazolidinyl, pyrazolinyl, pyranyl, morpholinyl, oxazinyl, isooxazinyl, and oxathiolyl.
  • a further aspect of the invention is a method of treating a mammal infected with a microbial infection such as a bacterial infection, typically either a Gram-positive infection or a Gram-negative bacterium; i.e., an infection caused by Gram-positive or Gram-negative bacteria, and the infected mammal is in need of antimicrobial (antibacterial) treatment.
  • a microbial infection such as a bacterial infection
  • a Gram-positive infection or a Gram-negative bacterium i.e., an infection caused by Gram-positive or Gram-negative bacteria
  • an antimicrobial (antibacterial) treatment treatment of Gram-positive bacteria are typically more successful that treatment of Gram-negative bacteria.
  • an antibacterial-effective amount of one or more compounds of Formula III 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, as a liquid formulation, as a thickened preparation e.g., as a gel, as for topical use, and is preferably administered via a pharmaceutical composition discussed hereinafter. That administration can also be oral or parenteral, as are also discussed further hereinafter.
  • mammals are infected with bacteria and other microbes.
  • the present invention methods of treatment is intended for use against infections of pathogenic bacteria that cause illness in the mammal to be treated.
  • Illustrative pathogenic microbes include S. aureus, methidlin-resistant S. aureus (MRSA), VanA strains of E. faecalis and E. feadum, as well as VanB strains of E. faecalis.
  • MRSA methidlin-resistant S. aureus
  • VanA strains of E. faecalis and E. feadum 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 III or its pharmaceutically acceptable salt to 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 contemplated compound is active in in vitro assay studies at less than 1 pg/mL amounts, which corresponds to a molar concentration of about 1 to about 100 nanomolar (nM), using the molecular weight of G3-CBP- tetrachlorovancomycin (Compound 31).
  • 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 ⁇ M to contact microbes to be assayed.
  • a contemplated pharmaceutical composition contains an effective antibiotic (or antimicrobial) amount of a Compound of Formula III 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 following: sulfate, hydrochloride, hydro bromides, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate, persulfate, 3-
  • 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.
  • the 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 parenterally, in a formulation containing conventional nontoxic physiologically acceptable carrier or diluent, adjuvant, and vehicle as desired.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania; 1975 and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.
  • a contemplated pharmaceutical composition is preferably adapted for parenteral administration.
  • a pharmaceutical composition is preferably in liquid form when administered, and most preferably, 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 physiologically 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, isotonic sodium chloride solution, and phosphate-buffered saline.
  • liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration.
  • Sterile water solutions of a Compound of Formula III or its salt or sterile solution of a Compound of Formula III in a solvent comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration.
  • a contemplated Compound of Formula III 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 add find use in the preparation of an injectable composition.
  • Dimethyl acetamide, surfactants including ionic and non- ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful.
  • 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 III such as Compounds 48 or 49 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 add, magnesium stearate, magnesium oxide, sodium and caldum salts of phosphoric and sulfuric adds, gelatin, acada gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.
  • Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.
  • the dosage forms can also comprise buffering agents such as sodium citrate, magnesium or calcium carbonate or bicarbonate. 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 two added chlorine substituents need to be benign and not have a significant effect on the target D-Ala- D-Ala binding and resulting antimicrobial activity.
  • they would need to be compatible with the enzymatic glycosylations used to introduce the disaccharide. The latter could only be established experimentally as little is known about the stringency of the glycopeptide substrate requirements for the native glycosyltransferases, especially what might be accommodated on the proximal D-ring phenol by GtfE in the initial glycosylation reaction.
  • Table A aRef 9. b Methicillin-sensitive S. aureus (ATCC 29213). Methicillin- resistant S. aureus (MMX 2002). Vancomycin-sensitive E. faecalis (MMX 101). e Ref 8. f Ref 10. 9 ATCC 25923.
  • the C-ring chloride In addition to its stabilizing hydrophobic interaction with the ligand terminal D-Ala methyl group, the C-ring chloride also provides a cap to the binding pocket, which provides selectivity for D-Ala-D-Ala binding by restricting the size of peptide substituent that it can accommodate (Me > H » all others). 11 Thus, a vancomycin structural simplification achieved through removal of both aryl chlorides may be too detrimental to be useful.
  • glycosyltransferases GtfE and GtfD would recognize it as a substrate for the disaccharide introduction.
  • These native glycosyltransferases 14 " 17 were instrumental to our total synthesis of vancomycin, 6 - 18 allowing direct aglycon glycosylation without the need for protecting groups and avoiding the less efficient chemical glycosylation methods.
  • 19-21 The success of the enzymatic glycosylations of tetrachlorovancomycin, as established herein, is key to direct synthetic access to not only Compound 1 , but also future pocket-modified analogues.
  • Saponification of the isopropyl ester Compound 10 was surprisingly dean (Me3SnOH, 26 CICH2CH2CI, 96%), providing carboxylic add Compound 11 without detectable epimerization of the ⁇ -stereocenter.
  • use of even carefully controlled aqueous saponification conditions (3 equiv LiOH, 2:1 t-BuOH-H2O, 0 °C, 1 h) led to significant C ⁇ epimerization (4:1 dr).
  • Macrolactam ization of Compound 24 under simulated high- dilution conditions promoted by 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methyl-morpholinium hexafluorophosphate 34 provided the AB macrocycle Compound 26 in superb yield (83%/2 steps).
  • the cyclization reaction proceeds essentially instantaneously upon dropwise addition of Compound 24 to a solution containing DMTMMH without trace of epimerization (>30:1 dr) and benefits from the modulated nucleophilicity of the reacting amine that precludes its competitive addition to the coupling reagent. 6
  • Boc deprotection of Compound 26 was accomplished under conditions that may allow reversible deprotection of the slightly acid-labile f-butyl ester 36 (8 equiv H2SO4, f-BuOAc, 0 to 23 'C, 2 h), providing Compound 26 (82%) that serves as the common precursor to tetrachlorovancomycin (Compound 1) as well as the subsequent binding pocket-modified analogues. Strikingly, NOESY studies of the AB macrocycle Compound 26, bearing the free amine, revealed exclusive adoption of the 5,6-ds amide conformation.
  • Highlights in this sequence include not only the mild room temperature double SnAr cyclization of Compound 27 ( ⁇ 4 h, 95%), but also the clean Fe-mediated dual nitro group reduction with avoidance of hydroxylamine byproducts, 7 a highly refined two-fold Sandmeyer substitution reaction with Lewis add-mediated diazonium salt formation 7 and deuterated solvent suppression 6 of competitive reduction, a remarkably effective TFA-mediated nitrile hydration, 40 and a scalable AIBr3/EtSH-mediated global deprotection. 7
  • amidine pocket modified tetrachlorovancomycin binds both ligands with affinities roughly only two- fold lower than the affinity of tetrachlorovancomycin for Ac 2 -L-Lys-D-Ala- D-Ala (A).
  • these affinities proved to be only 3-5-fold lower than those of the corresponding vancomycin pocket modified analogues.
  • the lower antimicrobial potency of these latter two pocket modified tetrachlorovancomycin analogues, like that of tetrachlorovancomycin itself, that smoothly follow the ligand binding affinity differences proved to incrementally diminish and ultimately disappear with each subsequent peripheral modification (CBP, G3).
  • peripheral modifications which sequentially introduce two additional independent mechanisms of action that do not rely on ligand binding, 59 not only synergistically improve potency but also largely eliminate potential small distinguishing activity differences due to relative ligand binding affinities.
  • Tetrachlorovancomydn (Compound 1) and its aglycon Compound 29 were found to be slightly less potent than vancomycin and its aglycon (about 4-fold) consistent with their relative ligand binding affinities toward Ac 2 -L-Lys-D-Ala-D-Ala.
  • CBP- tetrachlorovanoomycin (Compound 30) and CBP-vancomycin were indistinguishable in sensitive strains.
  • Compound 30 displays activity against the VRE strain comparable to that of CBP-vancomycin, which is derived from direct competitive inhibition of transglycosylase (a second independent mechanism of action) that does not require AoL-Lys-D-Ala-D-Ala binding. 49 - 50 This activity of CBP-tetrachloro-vancomycin (Compound 30) is improved about 100-fold against the sensitive strains due to the expression of two independent and synergistic mechanisms of action.
  • the increase in potency attributable to G3 is derived from a newly added mechanism of action, permeabilization of the cell envelop without membrane disruption or lysis, 58 that is independent of both the CBP-mediated transglycosylase competitive inhibition and pocket-derived ligand binding and transpeptidase inhibition. 58 Notably, Compound 47 expresses this activity now through two synergistic and independent mechanisms of action, neither of which require D-Ala-D-Ala or D-Ala-D-Lac binding.
  • This increased activity can be ascribed to the pocket dual ligand binding where that to D-Ala-D-Ala is operative and that to D-Ala-D-Lac is newly installed, inhibiting transpeptidase-catalyzed cell wall cross-linking and maturation in both vancomydn-sensitive and vancomydn-resistant bacteria.
  • Such analogues which we have come to refer to as maxamydns, display their activity through three independent and synergistic mechanisms of action of which only one requires D-Ala-D-Ala/D-Lac binding.
  • synergistic antimicrobial activity observed with the combined peripheral and pocket modifications within tetrachlorovancomycin analogues likely requires their incorporation in a single molecule as we have demonstrated with vancomycin and its pocket-modified analogues. This atypical behavior further suggests that the spatial and temporal localization of the individual effects is needed to provide the observed synergistic activity.
  • peripherally-modified pocket analogues of tetrachlorovancomycin that act by three independent mechanisms of action against both vancomycin-sensitive and vancomycin-resistant bacteria are unlikely to raise resistance and represent the newest members of what can be expected to be an unusually durable antibiotic class we refer to as maxamydns.
  • Additional key elements of the approach indude a catalyst- controlled diastereoselective formation of the AB biaryl axis of chirality (>30:1 dr), an instantaneous macrolactamization of the AB ring system free of competitive epimerization (>30:1 dr), an epimerization free coupling of the E ring tetrapeptide, the room temperature dual CD/DE ring system SnAr cyclizations, a refined 4-step conversion of the product to the aglycon, and a one-pot enzymatic glycosylation for disaccharide introduction.
  • results of the study not only highlight the key role of the natural product chloride substituents, improving target ligand binding affinity and selectivity, but also help define the glycopeptide substrate tolerance of the native glycosyltransferases enlisted to enzymatically introduce the disaccharide for which little is known.
  • this includes the preparation of binding pocket-modified analogues 4 of tetrachlorovancomycin to reinstate binding to the altered target D-Ala-D- Lac of vancomycin-resistant bacteria while maintaining binding for the unaltered target D-Ala-D-Ala found in sensitive bacteria as well as extension to their even more potent and durable peripherally-modified derivatives.
  • reaction mixture was poured into aqueous 1 M MCI (12 mL) and stirred for 5 min, then extracted with CH2CI2 (3 x 50 mL).
  • CH2CI2 3 x 50 mL
  • the combined organic layers were dried with MgSO4 concentrated under reduced pressure, and the residue was purified by chromatography (SiO2, wet load 50% CH2CI2- hexanes, 50-100% CH2CI2-hexanes, rapid elution) to provide 4 (872 mg, 65%) as a yellow foam and recycled 2.
  • reaction mixture was cooled to 23 °C, concentrated under reduced pressure and the residue was purified by chromatography (SiO2, 20- 60% EtOAc-hexanes + 1% EbN, rapid elution) to provide 7 (3.84 g, 89%) as a moisture-sensitive yellow oil.
  • the workup is ideally performed immediately upon completion of the hydrolysis. Degradation of 9 is observed after prolonged exposure to the reaction conditions. Free base 9 is unstable at 23 "C and should either be stored cold (5 -20 °C), or preferably used immediately in the following step.
  • the semi-pure carboxylic add S4 from the previous step (2.0 mmol) was coevaporated with PhMe (2 x 50 mL), dissolved in CH2CI2 (2 mL), and diluted with cyclohexane (6 mL).
  • a solution of f-butyl trichloroacetimidate (3.6 mL, 20 mmol, 10 equiv) in cydohexane (3.6 mL) was added to the reaction mixture over 7 h by syringe pump.
  • the reaction mixture was stirred at 23 "C for an additional 9 h, filtered, and the filter cake was washed with minimal 30% CH 2 Cl 2 -hexanes (3 x 5 mL).
  • the combined filtrate was concentrated under reduced pressure and purified by column chromatography (50 g SiO2, wet-load CH2CI2, washed with 100% CH2CI2 (1.5 L) to remove trichloroacetamide, then eluted with 0-10% acetone-CH 2 Cl 2 over 500 mL) to provide 23 (1.87 g, 90%) as a tan solid.
  • Trituration of this sample of 25 with Et2O (4 mL) afforded analytically pure 25 (217 mg, 83%/2 steps) as a light tan solid.
  • the 1 H- 1 H NOESY spectrum of 26 (600 MHz, CDaOD) displayed the following diagnostic nOe cross-peaks: 8.26/5.31 (6B/Z6), 8.26/4.34 (6B/X6), 8.06/5.31 (6F/Z6), 8.06/4.34 (6F/X6), 7.14/5.05 (5B/X5), 7.14/4.34 (5B/X6), 5.05/4.34 (X5/X6).
  • the latter correlation is indicative of a 5,6-ds amide conformation. 35
  • the light yellow-green reaction mixture was stirred at 0 °C for 30 min, cooled to -35 "C, and stirred vigorously as a chilled (0 °C) suspension of CuCI (320 mg, 3.2 mmol, 250 equiv) and CuCb (520 mg, 3.9 mmol, 300 equiv) in 50% CD3CN-H 2 O (1.6 mL) was added by syringe.
  • the reaction mixture was slowly warmed to 5 °C over 2 h, added to saturated aqueous NH4CI (100 mL), adjusted to pH 9 with the addition of concentrated NH 4 OH, and extracted with EtOAc (100 mL).
  • the reaction mixture was warmed to 37 'C for 17 h, cooled to 23 "C, and treated with additional TCEP-HCI (10.5 mg, 35 ⁇ mol, 6 equiv), 750 mM tridne-NaOH (pH 9, 1 mL), the azide precursor to UDP- vancosamine 41 (45 ⁇ mol, 8 equiv), and GtfD 41 (65 pM, 0.92 mL, 1 mol %).
  • the reaction mixture was warmed to 37 "C for 16 h, cooled to 23 °C, diluted with 50% MeOH-MeCN (32 mL), and filtered through a 0.22 pm PES membrane, rinsing with MeOH.
  • Amine 36A is unstable, especially in its free base form, and could not be isolated without decomposition.
  • reaction was quenched by transferring the mixture into a saturated solution of EDTA in H 2 O-MeOH (1:1, 10 mL), and the resulting mixture was stirred at 23 "C for 1 h with the color changing from dark to light blue.
  • reaction was quenched by addition of cold H 2 O (0 °C, 0.07% TFA, 5 mL) and transferred into a saturated EDTA H 2 O-MeOH (1 :1 , 5 mL), and the resulting mixture was stirred at 23 "C for 1 h with the color changing from dark to light blue.
  • tetrachlorovancomycin (1) like vancomycin, fails to bind to an appreciable extent the model ligand of the peptidoglycan precursor found in vancomycin-resistant organisms, Ac 2 - L-Lys-D-Ala-D-Lac (Compound 33). 48 Finally, and although not examined herein, it has been shown elsewhere that addition of the peripheral 4-chlorobiphenylmethyl (CBP) group to vancomycin and related structures does not impact (increase) the solution phase binding affinity for model ligands.
  • CBP peripheral 4-chlorobiphenylmethyl
  • ITC measurements were carried out in a MicroCaiTM Auto- iTC200 system with 400 mL of antibiotic solution as the cell sample and 120 mL of ligand solution as syringe sample (2.5 mL of each injection volume). Control titration runs were conducted by using blank buffer solution against blank buffer solution, each antibiotic, and the two ligands, respectively to show no heat contribution from the individual binding components. The titration data were processed by using OriginLab software (for ITC) and “one set of sites” fitting model for curve fitting.
  • UV spectra were recorded after each addition of a solution of N,N - Ac 2 -Lys- D-Ala-D-Ala (A) or N.N- Ac 2 -Lys-D-Ala-D-Lac (B) in 20 mM sodium citrate buffer to each cell from 0.1 to up to 60.0 equiv for the weaker binding partners.
  • the absorbance value at the ⁇ max was recorded, measuring the running change in absorbance.
  • VSSA strain ATCC 25923 vancomycin-sensitive Staphlococcus aureus
  • MRSA strain ATCC 43300 vancomycin-resistant Enterococcus faecium
  • vancomycin-resistant Enterococcus faecalis vancomycin-resistant Enterococcus faecalis
  • vancomydn- sensitive Enterococcus faecium unknown origin
  • vancomycin- sensitive Enterococcus feecaiis unknown origin
  • This diluted bacterial stock solution was then inoculated in a 96-well U- shaped glass coated microtiter plate, supplemented with serial diluted aliquots of the antibiotic solution in DMSO (4 ⁇ L), to achieve a total assay volume of 0.1 mL.
  • the plate was then incubated at 37 °C for 18 h, 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 pg/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.
  • the initial fresh cultures were grown in the presence of vancomycin (1 pg/mL) for resistant strains and chloramphenicol for sensitive strains (1 pg/mL).
  • the incorporation of the D ring amino add into tetrapeptide 17 also avoids the challenging and epimerization prone coupling of the corresponding E ring tripeptide with the D ring amine when embedded in the ABCD ring system (see cites 6 and 7).
  • T etrachlorovancomydn (1 ) like vancomydn, fails to bind Ac 2 -L-Lys-D-Ala-D-Lac (33) to an appredable extent (K a ⁇ 470 M' 1 ) being 1000-fold less effective than its binding with Ac 2 -L-Lys-D-Ala-D- Ala.

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Abstract

Une synthèse totale d'une nouvelle classe d'analogues de vancomycine de complexité synthétique réduite a été développée. La synthèse, réalisée par l'ajout de deux substituants du chlorure d'aryle pour proposer un aglycone de tétrachlorovancomycine (Composé II), de la tétrachlorovancomycine (Composé I), et leurs dérivés, permet une synthèse totale rationalisée de la nouvelle classe d'antibiotiques glycopeptidiques par élimination de la commande stéréochimique atropisomère et a permis l'activation simultanée et supplémentaire de macrocyclisations SNAr qui établissent le squelette tricyclique du Composé I. En plus de l'évaluation antimicrobienne de la tétrachlorovancomycine (Composé I), la préparation de la poche de liaison clé et les dérivés à modification périphérique, qui surmontent la résistance à la vancomycine et introduisent des mécanismes d'action indépendants et synergiques, ont révélé leur puissance antimicrobienne exceptionnelle et proposent le fondement pour l'utilisation de cette nouvelle classe d'analogues de glycopeptides synthétiques. Sont également divulgués une composition pharmaceutique contenant une quantité bactéricide de tétrachlorovancomycine, un dérivé de celle-ci ou un sel de celui-ci dissous ou dispersé dans un diluant pharmaceutiquement acceptable.
PCT/US2024/031453 2023-06-16 2024-05-29 Tétrachlorovancomycine et dérivés Ceased WO2024258613A2 (fr)

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